A functional–structural model for radiata pine (Pinus radiata) focusing on tree architecture and wood quality

PubMed Central

Fernández, M. Paulina; Norero, Aldo; Vera, Jorge R.; Pérez, Eduardo

2011-01-01

Backgrounds and Aims Functional–structural models are interesting tools to relate environmental and management conditions with forest growth. Their three-dimensional images can reveal important characteristics of wood used for industrial products. Like virtual laboratories, they can be used to evaluate relationships among species, sites and management, and to support silvicultural design and decision processes. Our aim was to develop a functional–structural model for radiata pine (Pinus radiata) given its economic importance in many countries. Methods The plant model uses the L-system language. The structure of the model is based on operational units, which obey particular rules, and execute photosynthesis, respiration and morphogenesis, according to their particular characteristics. Plant allometry is adhered to so that harmonic growth and plant development are achieved. Environmental signals for morphogenesis are used. Dynamic turnover guides the normal evolution of the tree. Monthly steps allow for detailed information of wood characteristics. The model is independent of traditional forest inventory relationships and is conceived as a mechanistic model. For model parameterization, three databases which generated new information relating to P. radiata were analysed and incorporated. Key Results Simulations under different and contrasting environmental and management conditions were run and statistically tested. The model was validated against forest inventory data for the same sites and times and against true crown architectural data. The performance of the model for 6-year-old trees was encouraging. Total height, diameter and lengths of growth units were adequately estimated. Branch diameters were slightly overestimated. Wood density values were not satisfactory, but the cyclical pattern and increase of growth rings were reasonably well modelled. Conclusions The model was able to reproduce the development and growth of the species based on mechanistic

Immunolocalization of IAA and ABA in roots and needles of radiata pine (Pinus radiata) during drought and rewatering.

PubMed

De Diego, N; Rodríguez, J L; Dodd, I C; Pérez-Alfocea, F; Moncaleán, P; Lacuesta, M

2013-05-01

Anatomical, physiological and phytohormonal changes involved in drought tolerance were examined in different Pinus radiata D. Don breeds subjected to soil drying and rewatering. Breeds with the smallest stomatal chamber size had the lowest transpiration rate and the highest intrinsic water-use efficiency. Xylem cell size was positively correlated with leaf hydraulic conductance and needle indole-3-acetic acid (IAA) concentrations, whereas transpiration rate was negatively correlated with needle abscisic acid (ABA) levels. Since these two phytohormones seem important in regulating the P. radiata drought response, they were simultaneously immunolocalized in roots and needles of the most tolerant breed (P. radiata var. radiata × var. cedrosensis) during two sequential drought cycles and after rewatering. During drought, IAA was unequally distributed into the pointed area of the needle cross-section and mainly located in mesophyll and vascular tissue cells of needles, possibly inducing needle epinasty, whereas ABA was principally located in guard cells, presumably to elicit stomata closure. In the roots, at the end of the first drought cycle, while strong IAA accumulation was observed in the cortex, ABA levels decreased probably due to translocation to the leaves. Rewatering modified the distribution of both IAA and ABA in the needles, causing an accumulation principally in vascular tissue, with residual concentrations in mesophyll, likely favouring the acclimatization of the plants for further drought cycles. Contrarily, in the roots IAA and ABA were located in the exodermis, a natural barrier that regulates the phytohormone translocation to other plant tissues and hormone losses to the soil solution after rewatering. These results confirm that immunolocalization is an efficient tool to understand the translocation of IAA and ABA in plants subjected to different water stress situations, and clarify their role in regulating physiological responses such as stomata

Mechanism of Resistance in Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae).

PubMed

War, Abdul R; Murugesan, Surya; Boddepalli, Venkata N; Srinivasan, Ramasamy; Nair, Ramakrishnan M

2017-01-01

Mungbean [ Vigna radiata (L.) R. Wilczek var. radiata ] is an important pulse crop in Asia, and is consumed as dry seeds and as bean sprouts. It is an excellent source of digestible protein. Bruchids [ Callosobruchus chinensis (L.) and Callosobruchus maculatus (F.)] are the important pests of mungbean and cause damage in the field and in storage. Bruchid infestation reduces the nutritional and market value of the grain and renders seeds unfit for human consumption, agricultural and commercial uses. These pests are controlled mainly by fumigation with highly toxic chemicals such as carbon disulfide, phosphene, and methyl bromide, or by dusting with several other insecticides, which leave residues on the grain, thus, threatening food safety. Some plant-based extracts have been found useful in controlling bruchids, but are not fully successful due to their short-term activity, rapid degradability, and potentially negative effect on seed germination. Although some wild sources of bruchid resistance in mungbean have been reported, which have been used to develop bruchid- resistant lines, undesirable genetic linkages threaten the proper exploitation of genetic diversity from wild germplasm into commercial cultivars. Further, biotype variation in bruchids has rendered some mungbean lines susceptible that otherwise would have been resistant to the pest. Host plant resistance is a cost-effective and a safe alternative to control bruchids in mungbean and is associated with morphological, biochemical, and molecular traits. These traits affect insect growth and development, thereby, reduce the yield losses by the pests. Understanding the defense mechanisms against insect pests could be utilized in exploiting these traits in crop breeding. This review discusses different traits in mungbean involved in defense against bruchids and their utility in pest management. We also highlight the breeding constraints for developing bruchid-resistant mungbean and how can these constraints

Mechanism of Resistance in Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae)

PubMed Central

War, Abdul R.; Murugesan, Surya; Boddepalli, Venkata N.; Srinivasan, Ramasamy; Nair, Ramakrishnan M.

2017-01-01

Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is an important pulse crop in Asia, and is consumed as dry seeds and as bean sprouts. It is an excellent source of digestible protein. Bruchids [Callosobruchus chinensis (L.) and Callosobruchus maculatus (F.)] are the important pests of mungbean and cause damage in the field and in storage. Bruchid infestation reduces the nutritional and market value of the grain and renders seeds unfit for human consumption, agricultural and commercial uses. These pests are controlled mainly by fumigation with highly toxic chemicals such as carbon disulfide, phosphene, and methyl bromide, or by dusting with several other insecticides, which leave residues on the grain, thus, threatening food safety. Some plant-based extracts have been found useful in controlling bruchids, but are not fully successful due to their short-term activity, rapid degradability, and potentially negative effect on seed germination. Although some wild sources of bruchid resistance in mungbean have been reported, which have been used to develop bruchid- resistant lines, undesirable genetic linkages threaten the proper exploitation of genetic diversity from wild germplasm into commercial cultivars. Further, biotype variation in bruchids has rendered some mungbean lines susceptible that otherwise would have been resistant to the pest. Host plant resistance is a cost-effective and a safe alternative to control bruchids in mungbean and is associated with morphological, biochemical, and molecular traits. These traits affect insect growth and development, thereby, reduce the yield losses by the pests. Understanding the defense mechanisms against insect pests could be utilized in exploiting these traits in crop breeding. This review discusses different traits in mungbean involved in defense against bruchids and their utility in pest management. We also highlight the breeding constraints for developing bruchid-resistant mungbean and how can these constraints be

Salt and drought stress and ABA responses related to bZIP genes from V. radiata and V. angularis.

PubMed

Wang, Lanfen; Zhu, Jifeng; Li, Xiaoming; Wang, Shumin; Wu, Jing

2018-04-20

Mung bean and adzuki bean are warm-season legumes widely cultivated in China. However, bean production in major producing regions is limited by biotic and abiotic stress, such as drought and salt stress. Basic leucine zipper (bZIP) genes play key roles in responses to various biotic and abiotic stresses. However, only several bZIP genes involved in drought and salt stress in legumes, especially Vigna radiata and Vigna angularis, have been identified. In this study, we identified 54 and 50 bZIP proteins from whole-genome sequences of V. radiata and V. angularis, respectively. First, we comprehensively surveyed the characteristics of all bZIP genes, including their gene structure, chromosome distribution and motif composition. Phylogenetic trees showed that VrbZIP and VabZIP proteins were divided into ten clades comprising nine known and one unknown subgroup. The results of the nucleotide substitution rate of the orthologous gene pairs showed that bZIP proteins have undergone strong purifying selection: V. radiata and V. angularis diverged 1.25 million years ago (mya) to 9.20 mya (average of 4.95 mya). We also found that many cis-acting regulatory elements (CAREs) involved in abiotic stress and plant hormone responses were detected in the putative promoter regions of the bZIP genes. Finally, using the quantitative real-time PCR (qRT-PCR) method, we performed expression profiling of the bZIP genes in response to drought, salt and abscisic acid (ABA). We identified several bZIP genes that may be involved in drought and salt responses. Generally, our results provided useful and rich resources of VrbZIP and VabZIP genes for the functional characterization and understanding of bZIP transcription factors (TFs) in warm-season legumes. In addition, our results revealed important and interesting data - a subset of VrbZIP and VabZIP gene expression profiles in response to drought, salt and ABA stress. These results provide gene expression evidence for the selection of

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

PubMed Central

Zimmerman, Shawn W.; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K.; Sutovsky, Miriam; Odhiambo, John F.; Powell, Michael D.; Miller, David J.; Sutovsky, Peter

2011-01-01

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

PubMed

De Silva, Matharage S I; Dayton, Adam W; Rhoten, Lance R; Mallett, John W; Reese, Jared C; Squires, Mathieu D; Dalley, Andrew P; Porter, James P; Judd, Allan M

2018-06-01

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1. Copyright © 2018 Elsevier Inc. All rights reserved.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

PubMed

Louros, Nikolaos N; Chrysina, Evangelia D; Baltatzis, Georgios E; Patsouris, Efstratios S; Hamodrakas, Stavros J; Iconomidou, Vassiliki A

2016-03-01

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP-N and ZP-C. Here, we have synthesized six 'aggregation-prone' peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy indicate that these peptides self-assemble forming fibrils with distinct amyloid-like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self-assembly mechanism of human ZP proteins. © 2016 Federation of European Biochemical Societies.

Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.

PubMed

Chamorro, David; Alarcón, Lourdes; Ponce, Arturo; Tapia, Rocio; González-Aguilar, Héctor; Robles-Flores, Martha; Mejía-Castillo, Teresa; Segovia, José; Bandala, Yamir; Juaristi, Eusebio; González-Mariscal, Lorenza

2009-09-01

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

The practical side of immunocontraception: zona proteins and wildlife.

PubMed

Kirkpatrick, J F; Rowan, A; Lamberski, N; Wallace, R; Frank, K; Lyda, R

2009-12-01

With shrinking habitat, the humane control of certain wildlife populations is relevant. The contraceptive vaccine based on native porcine zona pellucida (PZP) has been applied to various wildlife populations for 20 years. Prominent efforts include wild horses, urban deer, zoo animals and African elephants, among others. This approach has been successful in managing entire populations and to date, no significant debilitating short- or long-term health effects have been documented.

Physiological response to drought in radiata pine: phytohormone implication at leaf level.

PubMed

De Diego, N; Pérez-Alfocea, F; Cantero, E; Lacuesta, M; Moncaleán, P

2012-04-01

Pinus radiata D. Don is one of the most abundant species in the north of Spain. Knowledge of drought response mechanisms is essential to guarantee plantation survival under reduced water supply as predicted in the future. Tolerance mechanisms are being studied in breeding programs, because information on such mechanisms can be used for genotype selection. In this paper, we analyze the changes of leaf water potential, hydraulic conductance (K(leaf)), stomatal conductance and phytohormones under drought in P. radiata breeds (O1, O2, O3, O4, O5 and O6) from different climatology areas, hypothesizing that they could show variable drought tolerance. As a primary signal, drought decreased cytokinin (zeatin and zeatin riboside-Z + ZR) levels in needles parallel to K(leaf) and gas exchange. When Z + ZR decreased by 65%, indole-3-acetic acid (IAA) and abscisic acid (ABA) accumulation started as a second signal and increments were higher for IAA than for ABA. When plants decreased by 80%, Z + ZR and K(leaf) doubled their ABA and IAA levels, the photosystem II yield decreased and the electrolyte leakage increased. At the end of the drought period, less tolerant breeds increased IAA over 10-fold compared with controls. External damage also induced jasmonic acid accumulation in all breeds except in O5 (P. radiata var. radiata × var. cedrosensis), which accumulated salicylic acid as a defense mechanism. After rewatering, only the most tolerant plants recovered their K(leaf,) perhaps due to an IAA decrease and 1-aminocyclopropane-1-carboxylic acid maintenance. From all phytohormones, IAA was the most representative 'water deficit signal' in P. radiata.

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

PubMed Central

Baibakov, Boris; Boggs, Nathan A.; Yauger, Belinda; Baibakov, Galina

2012-01-01

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1–3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm–egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy. PMID:22734000

Structural Analysis of Peptide-Analogues of Human Zona Pellucida ZP1 Protein with Amyloidogenic Properties: Insights into Mammalian Zona Pellucida Formation

PubMed Central

Louros, Nikolaos N.; Iconomidou, Vassiliki A.; Giannelou, Polina; Hamodrakas, Stavros J.

2013-01-01

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as ‘aggregation-prone’ by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted ‘aggregation-prone’ parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization. PMID:24069181

Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

PubMed Central

Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

2006-01-01

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

In silico genome-wide identification and characterization of the glutathione S-transferase gene family in Vigna radiata.

PubMed

Vaish, Swati; Awasthi, Praveen; Tiwari, Siddharth; Tiwari, Shailesh Kumar; Gupta, Divya; Basantani, Mahesh Kumar

2018-05-01

Plant glutathione S-transferases (GSTs) are integral to normal plant metabolism and biotic and abiotic stress tolerance. The GST gene family has been characterized in diverse plant species using molecular biology and bioinformatics approaches. In the current study, in silico analysis identified 44 GSTs in Vigna radiata. Of the total 44 GSTs identified, chromosomal locations of 31 GSTs were confirmed. The pI value of GST proteins ranged from 5.10 to 9.40. The predicted molecular weights ranged from 13.12 to 50 kDa. Subcellular localization analysis revealed that all GSTs were predominantly localized in the cytoplasm. The active site amino acids were confirmed to be serine in tau, phi, theta, zeta, and TCHQD; cysteine in lambda, DHAR, and omega; and tyrosine in EF1G. The gene architecture conformed to the two-exon/one-intron and three-exon/two-intron organization in the case of tau and phi classes, respectively. MEME analysis identified 10 significantly conserved motifs with the width of 8-50 amino acids. The motifs identified were either specific to a specific GST class or were shared by multiple GST classes. The results of the current study will be of potential importance in the characterization of the GST gene family in V. radiata, an economically important leguminous crop.

Proanthocyanidin-rich Pinus radiata bark extract inhibits mast cell-mediated anaphylaxis-like reactions.

PubMed

Choi, Yun Ho; Song, Chang Ho; Mun, Sung Phil

2018-02-01

Mast cells play a critical role in the effector phase of immediate hypersensitivity and allergic reactions. Pinus radiata bark extract exerts multiple biological effects and exhibits immunomodulatory and antioxidant properties. However, its role in mast cell-mediated anaphylactic reactions has not been thoroughly investigated. In this study, we examined the effects of proanthocyanidin-rich water extract (PAWE) isolated from P. radiata bark on compound 48/80-induced or antidinitrophenyl (DNP) immunoglobulin E (IgE)-mediated anaphylaxis-like reactions in vivo. In addition, we evaluated the mechanism underlying the inhibitory effect of PAWE on mast cell activation, with a specific focus on histamine release, using rat peritoneal mast cells. PAWE attenuated compound 48/80-induced or anti-DNP IgE-mediated passive cutaneous anaphylaxis-like reactions in mice, and it inhibited histamine release triggered by compound 48/80, ionophore A23187, or anti-DNP IgE in rat peritoneal mast cells in vitro. Moreover, PAWE suppressed compound 48/80-elicited calcium uptake in a concentration-dependent manner and promoted a transient increase in intracellular cyclic adenosine-3',5'-monophosphate levels. Together, these results suggest that proanthocyanidin-rich P. radiata bark extract effectively inhibits anaphylaxis-like reactions. Copyright © 2017 John Wiley & Sons, Ltd.

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements.

PubMed

Florman, H M; First, N L

1988-08-01

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

Host Genotype and Nitrogen Form Shape the Root Microbiome of Pinus radiata.

PubMed

Gallart, Marta; Adair, Karen L; Love, Jonathan; Meason, Dean F; Clinton, Peter W; Xue, Jianming; Turnbull, Matthew H

2018-02-01

A central challenge in community ecology is understanding the role that phenotypic variation among genotypes plays in structuring host-associated communities. While recent studies have investigated the relationship between plant genotype and the composition of soil microbial communities, the effect of genotype-by-environment interactions on the plant microbiome remains unclear. In this study, we assessed the influence of tree genetics (G), nitrogen (N) form and genotype-by-environment interaction (G x N) on the composition of the root microbiome. Rhizosphere communities (bacteria and fungi) and root-associated fungi (including ectomycorrhizal and saprotrophic guilds) were characterised in two genotypes of Pinus radiata with contrasting physiological responses to exogenous organic or inorganic N supply. Genotype-specific responses to N form influenced the composition of the root microbiome. Specifically, (1) diversity and composition of rhizosphere bacterial and root-associated fungal communities differed between genotypes that had distinct responses to N form, (2) shifts in the relative abundance of individual taxa were driven by the main effects of N form or host genotype and (3) the root microbiome of the P. radiata genotype with the most divergent growth responses to organic and inorganic N was most sensitive to differences in N form. Our results show that intraspecific variation in tree response to N form has significant consequences for the root microbiome of P. radiata, demonstrating the importance of genotype-by-environment interactions in shaping host-associated communities.

Lethal and Sublethal Impacts of Acaricides on Tamarixia radiata (Hemiptera: Eulophidae), an Important Ectoparasitoid of Diaphorina citri (Hemiptera: Liviidae).

PubMed

Lira, A C S; Zanardi, O Z; Beloti, V H; Bordini, G P; Yamamoto, P T; Parra, J R P; Carvalho, G A

2015-10-01

The use of synthetic acaricides for management of pest mites may alter the efficacy of the ectoparasitoid Tamarixia radiata (Waterston) in biological control of Diaphorina citri Kuwayama, the vector of the bacteria associated with huanglongbing (HLB) in citrus orchards. We evaluated the toxicity of 16 acaricides that are recommended for the control of citrus-pest mites to T. radiata. Acrinathrin, bifenthrin, carbosulfan, and fenpropathrin caused high acute toxicity and were considered harmful (mortality >77%) to T. radiata. Abamectin, diflubenzuron, etoxazole, fenbutatin oxide, fenpyroximate, flufenoxuron, hexythiazox, propargite, spirodiclofen, and sulfur caused low acute toxicity and affected the parasitism rate and emergence rate of adults (F1 generation), and were considered slightly harmful to T. radiata. Dicofol and pyridaben did not affect the survival and action of the ectoparasitoid, and were considered harmless. In addition to its acute toxicity, carbosulfan caused mortality higher than 25% for >30 d after application, and was considered persistent. Acrinathrin, bifenthrin, fenpropathrin, fenpyroximate, propargite, and sulfur caused mortalities over 25% until 24 d after application and were considered moderately persistent; abamectin was slightly persistent, and fenbutatin oxide was short lived. Our results suggest that most acaricides used to control pest mites in citrus affect the density and efficacy of T. radiata in the biological control of D. citri. However, further evaluations are needed in order to determine the effect of these products on this ectoparasitoid under field conditions. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Biogenic synthesis and spatial distribution of silver nanoparticles in the legume mungbean plant (Vigna radiata L.).

PubMed

Kumari, Rima; Singh, Jay Shankar; Singh, Devendra Pratap

2017-01-01

The present investigation aimed to study the in vivo synthesis of silver nanoparticles (AgNPs) in the legume Vigna radiata. The level of plant metabolites such as total phenolics, lipid, terpenoids, alkaloids and amino acid increased by 65%, 133%, 19%, 67% and 35%, respectively, in AgNO 3 (100 mg L -1 ) treated plants compared to control. Whereas protein and sugar contents in the treated plants were reduced by 38% and 27%, respectively. FTIR analysis of AgNO 3 (20-100 mg L -1 ) treated plants exhibited changes in the IR regions between 3297 and 3363 cm -1 , 1635-1619 cm -1 , 1249-1266 cm -1 and that corresponded to alterations in OH groups of carbohydrates, OH and NH groups of amide I and II regions of protein, when compared with the control. Transmission electron micrographs showed the spatial distribution of AgNPs in the chloroplast, cytoplasmic spaces, vacuolar and nucleolar plant regions. Metal quantification in different tissues of plants exposed to 20-100 mg L -1 AgNO 3 showed about a 22 fold accumulation of Ag in roots as compared to shoots. The phytotoxic parameters such as percent seed germination and shoot elongation remained almost unaltered at low AgNO 3 doses (20-50 mg L -1 ). However, at higher levels of exposure (100 mg L -1 ), the percent seed germination as well as root and shoot elongation exhibited concentration dependent decline. In conclusion, synthesis of AgNPs in V. radiata particularly at lower doses of AgNO 3 , could be used as a sustainable and environmentally safe technology for large scale production of metal nanoparticles. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Western gall rust -- A threat to Pinus radiata in New Zealand

Treesearch

Tod D. Ramsfield; Darren J. Kriticos; Detlev R. Vogler; Brian W. Geils

2007-01-01

Western gall rust (Peridermium harknessii J. P. Moore (syn. Endocronartium harknessii (J. P. Moore) Y. Hiratsuka) is potentially a serious threat to exotic Pinus radiata D. Don plantations of New Zealand although the pathogen has not been recorded here. Mechanisms that may have prevented invasion of the pathogen include geographic...

Improvement of non-key traits in radiata pine breeding programme when long-term economic importance is uncertain.

PubMed

Li, Yongjun; Dungey, Heidi; Yanchuk, Alvin; Apiolaza, Luis A

2017-01-01

Diameter at breast height (DBH), wood density (DEN) and predicted modulus of elasticity (PME) are considered as 'key traits' (KT) in the improvement in radiata pine breeding programmes in New Zealand. Any other traits which are also of interest to radiata pine breeders and forest growers are called 'non-key traits' (NKTs). External resin bleeding (ERB), internal checking (IC), number of heartwood rings (NHR) are three such non-key traits which affect wood quality of radiata pine timber. Economic importance of the KTs and NKTs is hard to define in radiata pine breeding programmes due to long rotation period. Desired-gain index (DGIs) and robust selection were proposed to incorporate NKTs into radiata pine breeding programme in order to deal with the uncertainty of economic importance. Four desired-gain indices A-D were proposed in this study. The desired-gain index A (DGI-A) emphasized growth and led to small decrease in ERB and small increase in IC and NHR. The expected genetic gains of all traits in the desired-gain index B (DGI-B) were in the favourable directions (positive genetic gains in the key traits and negative genetic gains in the non-key traits). The desired-gain index C (DGI-C) placed emphasis on wood density, leading to favourable genetic gain in the NKTs but reduced genetic gains for DBH and PME. The desired-gain index D (DGI-D) exerted a bit more emphasis on the non-key traits, leading large favourable reduction in the non-key traits and lower increase in the key traits compared with the other DGIs. When selecting both the key traits and the non-key traits, the average EBVs of six traits were all in the same directions as the expected genetic gains except for DBH in the DGI-D. When the key traits were measured and selected, internal checking always had a negative (favourable) genetic gain but ERB and NHR had unfavourable genetic gain in the most of time. After removing some individuals with high sensitivity to the change of economic weights, robust

Susceptibility of Diaphorina citri (Hemiptera: Liviidae) and Its Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) to Entomopathogenic Fungi under Laboratory Conditions.

PubMed

Ibarra-Cortés, K H; Guzmán-Franco, A W; González-Hernández, H; Ortega-Arenas, L D; Villanueva-Jiménez, J A; Robles-Bermúdez, A

2018-02-01

Diaphorina citri (Kuwayama) is a global pest of citrus that transmits the bacteria associated with the disease, Huanglongbing. Entomopathogenic fungi and the parasitoid Tamarixia radiata (Waterston) are important biological control agents of this pest and likely to interact in D. citri populations. As a basis for interaction studies, we determined the susceptibility of nymphs and adults of D. citri and adults of the parasitoid T. radiata to six fungal isolates from the species Beauveria bassiana s.l. (Bals.-Criv.) Vuill. (isolates B1 and B3), Metarhizium anisopliae s.s. (Metsch.) (Ma129 and Ma65) and Isaria fumosorosea Wize (I2 and Pae). We conducted experiments evaluating infection levels in all three insect groups following inoculation with a series of conidial concentrations (1 × 10 4 -1 × 10 8 conidia mL -1 ). Results showed that D. citri nymphs and T. radiata were more susceptible to fungal isolates than D. citri adults. Overall, B. bassiana and M. anisopliae isolates caused the greatest infection compared with I. fumosorosea isolates in all three groups of insects. Isolates B1 (B. bassiana) and Ma129 (M. anisopliae) infected a greater proportion of adults and nymphs of D. citri, respectively. Both isolates of B. bassiana caused greater infection in T. radiata compared with isolates of the other fungal species. We propose that isolates B1 and Ma129 are the strongest candidates for control of D. citri. Our results represent the first report of entomopathogenic fungi infecting T. radiata, and the basis for future studies to design a biological control programme that uses both agents more efficiently against D. citri populations.

Transfer of bovine demi-embryos with and without the zona pellucida.

PubMed

Warfield, S J; Seidel, G E; Elsden, R P

1987-09-01

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.

Dimensions of Attention Associated with the Microstructure of Corona Radiata White Matter

PubMed Central

Stave, Elise A.; Hooper, Stephen R.; Woolley, Donald P.; Chang, Suk Ki; Chen, Steven D.

2016-01-01

Mirsky proposed a model of attention that included these dimensions: focus/execute, sustain, stabilize, encode, and shift. The neural correlates of these dimensions were investigated within corona radiate subregions in healthy youth. Diffusion tensor imaging and neuropsychological assessments were conducted in 79 healthy, right-handed youth aged 4–17 years. Diffusion tensor imaging maps were analyzed using standardized parcellation methods. Partial Pearson correlations between neuropsychological standardized scores, representing these attention dimensions, and diffusion tensor imaging measures of corona radiate subregions were calculated after adjusting for gender and IQ. Significant correlations were found between the focus/execute, sustain, stabilize and shift dimensions and imaging metrics in hypothesized corona radiate subregions. Results suggest that greater microstructural white matter integrity of the corona radiata is partly associated with attention across four attention dimensions. Findings suggest that white matter microstructure of the corona radiata is a neural correlate of several, but not all, attention dimensions. PMID:28090797

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

PubMed Central

Macdonald, Lucy J.; Adams, Paul D.; Petzold, Christopher J.; Strabala, Timothy J.; Wagner, Armin; Heazlewood, Joshua L.

2013-01-01

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557. PMID:24416096

Changes in fats and resins of Pinus radiata associated with heartwood formation

Treesearch

Richard W. Hemingway; W.E. Hillis

1971-01-01

In an analysis of Australian grown P. radiata marked changes were found in the relative proportions and compositions of the resin acids, fatty acids, and fatty acid esters associated with heartwood formation. While the proportion of resin acids increased substantially in the heartwood, there was little change in resin acid composition from outer...

Transcriptional and computational study of expansins differentially expressed in response to inclination in radiata pine.

PubMed

Mateluna, Patricio; Valenzuela-Riffo, Felipe; Morales-Quintana, Luis; Herrera, Raúl; Ramos, Patricio

2017-06-01

Plants have the ability to reorient their vertical growth when exposed to inclination. This response can be as quick as 2 h in inclined young pine (Pinus radiata D. Don) seedlings, with over accumulation of lignin observed after 9 days s. Several studies have identified expansins involved in cell expansion among other developmental processes in plants. Six putative expansin genes were identified in cDNA libraries isolated from inclined pine stems. A differential transcript abundance was observed by qPCR analysis over a time course of inclination. Five genes changed their transcript accumulation in both stem sides in a spatial and temporal manner compared with non-inclined stem. To compare these expansin genes, and to suggest a possible mechanism of action at molecular level, the structures of the predicted proteins were built by comparative modeling methodology. An open groove on the surface of the proteins composed of conserved zresidues was observed. Using a cellulose polymer as ligand the protein-ligand interaction was evaluated, with the results showing differences in the protein-ligand interaction mode. Differences in the binding energy interaction can be explained by changes in some residues that generate differences in electrostatic surface in the open groove region, supporting the participation of six members of multifamily proteins in this specific process. The data suggests participation of different expansin proteins in the dissembling and remodeling of the complex cell wall matrix during the reorientation response to inclination. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The Combined Intervention with Germinated Vigna radiata and Aerobic Interval Training Protocol Is an Effective Strategy for the Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD) and Other Alterations Related to the Metabolic Syndrome in Zucker Rats.

PubMed

Kapravelou, Garyfallia; Martínez, Rosario; Nebot, Elena; López-Jurado, María; Aranda, Pilar; Arrebola, Francisco; Cantarero, Samuel; Galisteo, Milagros; Porres, Jesus M

2017-07-19

Metabolic syndrome (MetS) is a group of related metabolic alterations that increase the risk of developing non-alcoholic fatty liver disease (NAFLD). Several lifestyle interventions based on dietary treatment with functional ingredients and physical activity are being studied as alternative or reinforcement treatments to the pharmacological ones actually in use. In the present experiment, the combined treatment with mung bean ( Vigna radiata ), a widely used legume with promising nutritional and health benefits that was included in the experimental diet as raw or 4 day-germinated seed flour, and aerobic interval training protocol (65-85% VO₂ max) has been tested in lean and obese Zucker rats following a 2 × 2 × 2 (2 phenotypes, 2 dietary interventions, 2 lifestyles) factorial ANOVA (Analysis of Variance) statistical analysis. Germination of V. radiata over a period of four days originated a significant protein hydrolysis leading to the appearance of low molecular weight peptides. The combination of 4 day-germinated V. radiata and aerobic interval training was more efficient compared to raw V. radiata at improving the aerobic capacity and physical performance, hepatic histology and functionality, and plasma lipid parameters as well as reverting the insulin resistance characteristic of the obese Zucker rat model. In conclusion, the joint intervention with legume sprouts and aerobic interval training protocol is an efficient treatment to improve the alterations of glucose and lipid metabolism as well as hepatic histology and functionality related to the development of NAFLD and the MetS.

Host range testing of Tamarixia radiata (Hymenoptera: Eulophidae) sourced from the Punjab of Pakistan for classical biological control of Diaphorina citri (Hemiptera: Liviidae: Euphyllurinae: Diaphorinini) in California.

PubMed

Hoddle, Mark S; Pandey, Raju

2014-02-01

ABSTRACT Tests evaluating the host range of Tamarixia radiata (Waterson) (Hymenoptera: Eulophidae), a parasitoid of the pestiferous Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), sourced from the Punjab of Pakistan, were conducted in quarantine at the University of California, Riverside, CA. Seven nontarget psyllid species (five native and two self-introduced species) representing five families were exposed to T radiata under the following three different exposure scenarios: 1) sequential no-choice tests, 2) static no-choice tests, and 3) choice tests. Nontarget species were selected for testing based on the following criteria: 1) taxonomic relatedness to the target, D. citri; 2) native psyllids inhabiting native host plants related to citrus that could release volatiles attractive to T. radiata; 3) native psyllids with a high probability of occurrence in native vegetation surrounding commercial citrus groves that could be encountered by T. radiata emigrating from D. citri-infested citrus orchards; 4) a common native pest psyllid species; and 5) a beneficial psyllid attacking a noxious weed. The results of host range testing were unambiguous; T radiata exhibited a narrow host range and high host specificity, with just one species of nontarget psyllid, the abundant native pest Bactericera cockerelli Sulc, being parasitized at low levels (< 5%). These results suggest that the likelihood of significant nontarget impacts is low, and the establishment of T. radiata in southern California for the classical biological control of D. citri poses negligible environmental risk.

Lack of genetic variation in tree ring delta13C suggests a uniform, stomatally-driven response to drought stress across Pinus radiata genotypes.

PubMed

Rowell, Douglas M; Ades, Peter K; Tausz, Michael; Arndt, Stefan K; Adams, Mark A

2009-02-01

We assessed the variation in delta(13)C signatures of Pinus radiata D. Don stemwood taken from three genetic trials in southern Australia. We sought to determine the potential of using delta(13)C signatures as selection criteria for drought tolerance. Increment cores were taken from P. radiata and were used to determine the basal area increment and the delta(13)C signature of extracted cellulose. Both growth increment and cellulose delta(13)C were affected by water availability. Growth increment and delta(13)C were negatively correlated suggesting that growth was water-limited. While there was significant genetic variation in growth, there was no significant genetic variation in cellulose delta(13)C of tree rings. This suggests that different genotypes of P. radiata display significant differences in growth and yet respond similarly to drought stress. The delta(13)C response to drought stress was more due to changes in stomatal conductance than to the variation in photosynthetic capacity, and this may explain the lack of genetic variation in delta(13)C. The lack of genetic variation in cellulose delta(13)C of tree rings precludes its use as a selection criterion for drought tolerance among P. radiata genotypes.

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Treesearch

Rakesh Minocha; Dale R. Smith; Cathie Reeves; Kevin D. Steele; Subhash C. Minocha

1999-01-01

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no...

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

PubMed

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny; Huszar, Gabor

2014-05-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

PubMed Central

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

Ocean acidification reverses the positive effects of seawater pH fluctuations on growth and photosynthesis of the habitat-forming kelp, Ecklonia radiata.

PubMed

Britton, Damon; Cornwall, Christopher E; Revill, Andrew T; Hurd, Catriona L; Johnson, Craig R

2016-05-27

Ocean acidification (OA) is the reduction in seawater pH due to the absorption of human-released CO2 by the world's oceans. The average surface oceanic pH is predicted to decline by 0.4 units by 2100. However, kelp metabolically modifies seawater pH via photosynthesis and respiration in some temperate coastal systems, resulting in daily pH fluctuations of up to ±0.45 units. It is unknown how these fluctuations in pH influence the growth and physiology of the kelp, or how this might change with OA. In laboratory experiments that mimicked the most extreme pH fluctuations measured within beds of the canopy-forming kelp Ecklonia radiata in Tasmania, the growth and photosynthetic rates of juvenile E. radiata were greater under fluctuating pH (8.4 in the day, 7.8 at night) than in static pH treatments (8.4, 8.1, 7.8). However, pH fluctuations had no effect on growth rates and a negative effect on photosynthesis when the mean pH of each treatment was reduced by 0.3 units. Currently, pH fluctuations have a positive effect on E. radiata but this effect could be reversed in the future under OA, which is likely to impact the future ecological dynamics and productivity of habitats dominated by E. radiata.

Characterization of seasonal reproductive and stress steroid hormones in wild Radiated Tortoises, Astrochelys radiata.

PubMed

Currylow, Andrea F T; Rafeliarisoa, Tsilavo H; Louis, Edward E; Stanford, Craig B; Randrianjafizanaka, Soary T; Chinn, Sarah M; Crocker, Daniel E

2017-11-01

The critically endangered Radiated Tortoise (Astrochelys radiata) is endemic to the southern coastlines of Madagascar. Once common, wild populations of this tortoise have undergone dramatic declines in recent years. Although there have been studies documenting reproductive activities, reproductive physiological parameters are unknown yet may be crucial in the recovery of the species. Over four research seasons in remote field locations native to A. radiata, we surveyed for, radio-tracked, and sampled wild, free ranging tortoises. We sampled and measured stress and reproductive parameters (corticosterone [CORT], testosterone [T], estradiol-17β [E2], and progesterone [P]) in 311 plasma samples from 203 wild A. radiata, capturing their active period. Generally, hormone concentrations were associated with body condition, temperature, and humidity. There was wide variation in CORT that varied monthly and by group. Juvenile tortoises maintained more than twice the mean basal CORT concentrations than either adult sex, with the most dramatic distinctions in the middle of the wet season. For adult sex hormones, the last months of the dry season and into the wet season when ground humidities are low and just begin to rise prior to temperature declines, male T concentrations gradually increased to a peak before returning to near undetectable values into the dry season. We had limited data for T concentrations in females, but found average T concentrations were much lower than in males and positively correlated with larger female home range sizes. For female hormone cycles, E2 also peaked in the early 1/3 of the wet season along with male T, and was followed by an uptick in P which correlates to the putative ovulatory cycle. Females tracked over four years showed variation in patterns of P, indicating that number and frequency of clutches vary. Our results suggest that 1) there is high species plasticity in response to stress; 2) A. radiata reproductive cycling is somewhat

Characterization of a novel Y2K-type dehydrin VrDhn1 from Vigna radiata.

PubMed

Lin, Chia-Hui; Peng, Po-Hsin; Ko, Chia-Yun; Markhart, Albert H; Lin, Tsai-Yun

2012-05-01

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to ∼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

Dimensional Changes of Tracheids during Drying of Radiata Pine (Pinus radiata D. Don) Compression Woods: A Study Using Variable-Pressure Scanning Electron Microscopy (VP-SEM)

PubMed Central

Zhang, Miao; Smith, Bronwen G.; McArdle, Brian H.; Chavan, Ramesh R.; James, Bryony J.

2018-01-01

Variable-pressure scanning electron microscopy was used to investigate the dimensional changes in longitudinal, tangential and radial directions, on wetting and drying, of tracheids of opposite wood (OW) and three grades of compression woods (CWs), including severe CW (SCW) and two grades of mild compression wood (MCW) (MCW1 and MCW2) in corewood of radiata pine (Pinus radiata) saplings. The CW was formed on the underside and OW on the upper side of slightly tilted stems. In the longitudinal direction, the shrinkage of SCW tracheids was ~300% greater than that of OW tracheids, with the shrinkage of the MCW1 and MCW2 tracheids being intermediate. Longitudinal swelling was also investigated and hysteresis was demonstrated for the tracheids of all corewood types, with the extent of hysteresis increasing with CW severity. A statistical association was found between longitudinal shrinkage and the content of lignin and galactosyl residues in the cell-wall matrix. The galactosyl residues are present mostly as (1→4)-β-galactans, which are known to have a high capacity for binding water and swell on hydration. The small proportions of (1→3)-β-glucans in the CWs have similar properties. These polysaccharides may play a functional role in the longitudinal shrinking and swelling of CW tracheids. Tangential shrinkage of tracheids was greater than radial shrinkage but both were greatest for OW and least for SCW, with the MCW1 and MCW2 being intermediate. PMID:29495536

Evaluation of Nano Structured Slow Release Fertilizer on the Soil Fertility, Yield and Nutritional Profile of Vigna radiata.

PubMed

Mala, Rajendran; Selvaraj, Ruby Celsia Arul; Sundaram, Vidhya Barathi; Rajan, Raja Blessina Siva Shanmuga; Gurusamy, Uma Maheswari

2017-01-01

The excessive use of fertilizers and pesticides has distorted soil composition, fertility and integrity with non-desirable environmental and ecological consequences. A strategy was designed to prepare a nano structured slow release fertilizer system that delivers nutrients and plant growth promoting rhizobacteria simultaneously. Slow release nano phosphate and potash fertilizer was prepared by blending the nano emulsion of fertilizer with neem cake and PGPR. Slow release nano phosphate and potash fertilizer was prepared by blending the nano emulsion of fertilizer with neem cake and PGPR. Few patents relevant to the topic have been reviewed and cited. The influence of nano structured slow release fertilizer on the biochemical characteristics, soil and yield attributes of Vigna radiata was studied in the field by randomized block design. The treatments used to evaluate the effect of nano SRF were a control (without any fertilizer), neem cake, chemical fertilizer, PGPR and nano SRF. Germination, specific activity of enzymes, carbohydrates, protein, photosynthetic pigments, root nodule number and microbial population were assessed by standard methods. The size of the nano urea slow release fertilizer ranged from 52.41 nm to 69.86 nm, and the size of the phosphate and potash fertilizer ranged from 81.85 nm to 87 nm. The weights of 1000 grains were 31.8 g, 33.28 g, 33.39 g, 36.65 g and 44.90 g in the control, neem cake, chemical fertilizer, PGPR and nano SRF, respectively. The protein concentrations were 162 mg g-1 in the control, 231 mg g-1 in the neem cake, 192 mg g-1 in the chemical fertilizer, 285 mg g-1 in the PGPR and 336 mg g-1 in the nano SRF. Nano slow release fertilizer treatment has stimulated germination and biochemical characteristics in Vigna radiata that are positively reflected in the yield attributes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Acute Toxicity of Fresh and Aged Residues of Pesticides to the Parasitoid Tamarixia radiata and to the HLB-Bacteria Vector Diaphorina citri.

PubMed

Beloti, V H; Alves, G R; Moral, R A; Demétrio, C G B; Yamamoto, P T

2018-06-01

One method for controlling the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama, the vector of the putative causal agent of Huanglongbing, uses the parasitoid Tamarixia radiata (Waterston). However, the general intensive use of insecticides has reduced the numbers of this parasitoid. This study evaluated the effect of the residual action of 24 insecticides on T. radiata and also determined the differential toxicity of insecticides to D. citri and T. radiata, using three bioassays. In the first, when adults of the parasitoid were exposed to residues of the 24 insecticides, ten were considered short-life (class 1), six slightly persistent (class 2), five moderately persistent (class 3), and three insecticides were considered persistent (class 4), under the IOBC/WPRS classification system. The second bioassay evaluated the sublethal concentrations of the persistent insecticides (formetanate, dimethoate, spinosad). Increasing the concentrations of the insecticides increased the number that were classified as persistent. In the third bioassay, evaluation of the differential toxicity of eight insecticides to the ACP and the parasitoid showed that chlorpyrifos and bifenthrin were more harmful to T. radiata. Therefore, these two insecticides are not recommended for application at the time of parasitoid release. Cypermethrin, imidacloprid, and dimethoate caused higher mortality of D. citri and are most often recommended in IPM programs. The choice of an insecticide for the control of citrus pests must be made with care, aiming to preserve the natural enemies in the ecosystem, and thereby contribute to the success of biological control.

Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards.

PubMed

Guerin-Laguette, Alexis; Cummings, Nicholas; Butler, Ruth Catherine; Willows, Anna; Hesom-Williams, Nina; Li, Shuhong; Wang, Yun

2014-10-01

The cultivation of Lactarius deliciosus (saffron milk cap) in New Zealand began in 2002 when fruiting bodies were produced in an Otago plantation of Pinus radiata seedlings artificially mycorrhized by L. deliciosus. In 2007, 42 P. radiata seedlings mycorrhized by L. deliciosus under controlled conditions were planted in a grass field at Plant and Food Research (Lincoln, Canterbury). The effects of pine bark mulch application and initial degree of mycorrhization of seedlings were examined to determine their influence on tree growth, development of mycorrhizae (i.e. their multiplication on the root system and their degree of branching) and fruiting body production. Mulch application increased tree growth significantly over 4 years. High initial mycorrhization slightly stimulated tree growth over 2 years. The initial degree of mycorrhization was positively, but not strongly, related to the persistence and development of L. deliciosus mycorrhizae and rhizomorphs based on root sample analyses 2 years after planting. However, mulching strongly reduced the proportion of highly branched L. deliciosus mycorrhizae compared with poorly ramified ones. A positive correlation was observed between the fruiting of L. deliciosus and the development of mycorrhizae. Mulching delayed the onset of fruiting body production. In 2010, fruiting bodies were produced only from non-mulched trees with eight of these (38 %) producing a total of 12 fruiting bodies. In 2011, 19 non-mulched trees (90 %) and 9 mulched trees (45 %) produced 143 and 47 fruiting bodies, respectively, totalling 190 fruiting bodies. By 2012, 19 non-mulched trees (90 %) and 13 mulched trees (65 %) produced 333 and 236 fruiting bodies, respectively, totalling 569 fruiting bodies (c. 30 kg). This study presents new information on factors influencing the onset of fruiting and the development of yields in a plantation of P. radiata mycorrhized by L. deliciosus. Projected yields as high as c. 300 kg/ha from the

Spatial and temporal dynamics of the colonization of Pinus radiata by Fusarium circinatum, of conidiophora development in the pith and of traumatic resin duct formation.

PubMed

Martín-Rodrigues, Noemí; Espinel, Santiago; Sanchez-Zabala, Joseba; Ortíz, Amaia; González-Murua, Carmen; Duñabeitia, Miren K

2013-06-01

· Fusarium circinatum causes pitch canker disease in a wide range of pine trees, including Pinus radiata, with devastating economic consequences. · To assess the spatial and temporal dynamics of growth of this pathogen in radiata pine, we examined the process of infection using both real-time PCR to quantify fungal biomass inside the plant host, and confocal microscopy using a green fluorescent protein (GFP)-tagged strain of F. circinatum. · Pathogen growth exhibited three distinct phases: an initial exponential increase in fungal biomass, concomitant with pathogen colonization of the cortex and phloem; a slowdown in fungal growth coincident with sporulating hyphae deep within the host; and stabilization of the fungal biomass when the first wilting symptoms appeared. The number of resin ducts in the xylem was found to increase in response to infection and the fungus grew inside both constitutive and traumatic resin ducts. · These results indicate that conidiation may contribute to the spatial or temporal dissemination of the pathogen. Moreover, the present findings raise the intriguing possibility that the generation of traumatic resin ducts may be of more benefit to the fungus than to the plant. © 2013 The Authors New Phytologist © 2013 New Phytologist Trust.

Interdependence of plant water status with photosynthetic performance and root defense responses in Vigna radiata (L.) Wilczek under progressive drought stress and recovery.

PubMed

Sengupta, Debashree; Guha, Anirban; Reddy, Attipalli Ramachandra

2013-10-05

The present study investigates the interdependence of plant water status with foliar and root responses in Vigna radiata L.Wilczek under progressive drought. Vegetatively-mature V. radiata plants were subjected to water withdrawal for 3 and 6days (D3 and D6, respectively) and then re-watered subsequently for 6days (6R) for stress-recovery. Changes in plant water status were expressed in terms of leaf and root moisture contents (LMC and RMC, respectively) and leaf relative water content (LRWC). Progressive drought caused apparent decrease in LRWC, LMC and RMC depicting significant level of dehydration of leaf and root tissues. Stomatal limitation alone could not account for the observed decrease in net CO2 assimilation rates (Pn) due to comparatively less decrease in sub-stomatal CO2 (Ci) concentrations with respect to other gas exchange parameters indicating possible involvement of non-stomatal limitations. Analysis of polyphasic chl a fluorescence kinetics during progressive drought showed decreased energy connectivity among PSII units as defined by a positive L-band with highest amplitude during D6. Efficiency of electron flux from OEC towards PSII acceptor side was not significantly affected during drought conditions as evidenced by the absence of a positive K-band. Increasing root-level water-limitation enforced a gradual oxidative stress through H2O2 accumulation and membrane lipid peroxidation in V. radiata roots exhibiting drastic enhancement of proline content and a significant but gradual increase in ascorbic acid content as well as guaiacol peroxidase activity under progressive drought. Expression analysis of Δ(1) pyrroline-5-carboxylate synthetase (P5CS) through real time PCR and enzyme activity studies showed a strong positive correlation between VrP5CS gene expression, enzyme activity and proline accumulation in the roots of V. radiata under progressive drought and recovery. Drought-induced changes in root moisture content (RMC) showed positive linear

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

PubMed Central

Hung, Pei-hsuan; Miller, Marion G.; Meyers, Stuart A.; VandeVoort, Catherine A.

2008-01-01

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 μM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque. PMID:18480469

Resin in bisulfite pulp from Pinus radiata wood and its relationship to pitch troubles

Treesearch

P.J. Nelson; Richard W. Hemingway

1971-01-01

The resin content of bisulfite pulp from Pinus radiata D. Don was determined at various stages in its manufacture and the changes in the composition of the resin studied. About 50% of the resin present in the wood was removed during cooking, an additional 11% by blowpit washing, and a further 8% by screening. Resin acids and fatty acids were...

Targeting the zona pellucida for immunocontraception: a minireview.

PubMed

Tesarik, J

1995-12-01

This minireview summarizes the main data relevant to the development of contraceptive vaccines based on zona pellucida (ZP) antigens, as well as the pros and the cons of this immunocontraceptive strategy. Even though the antifertility efficacy of anti-ZP antibodies in humans is not corroborated by a clear relationship between spontaneous autoimmunization against the ZP and infertility, passive and active immunization studies in laboratory animals have provided convincing results. The contraceptive action of anti-ZP antibodies, targeting events situated upstream of gamete fusion, is devoid of potential ethical concerns related to the destruction of early embryos. The high protein content of the mammalian ZP, knowledge of the complete amino acid sequence of the major ZP proteins, and the high degree of sequence homology between individual species all favour the rapid advancement of anti-ZP vaccine projects. However, certain sequences of ZP proteins, when incorporated into the vaccine construct, activate CD4+ T cells of the recipient organism to direct a cellular immune attack (autoimmune oophoritis) to other functionally relevant ovarian components (primordial follicles, steroidogenic cells). The search for the optimal combination of B cell and T cell epitopes in the vaccine construct will hopefully overcome this problem.

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

DOE PAGES

Que, Emily L.; Duncan, Francesca E.; Bayer, Amanda R.; ...

2017-01-19

During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy,more » transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. Finally, this adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.« less

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

PubMed

Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

2013-07-01

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Mechanics of sperm-egg interaction at the zona pellucida.

PubMed Central

Baltz, J M; Katz, D F; Cone, R A

1988-01-01

Mammalian sperm traverse several layers of egg vestments before fertilization can occur. The innermost vestment, the zona pellucida, is a glycoprotein shell, which captures and tethers the sperm before they penetrate it. We report here direct measurements of the force required to tether a motile human sperm as well as independent calculations of this force using flagellar beat parameters observed for sperm of several species on their homologous zonae. We have compared these sperm-generated forces with the calculated tensile strength of sperm-zona bonds, and found that a motile sperm can be tethered, at least temporarily, by a single bond. Therefore, sperm can be captured by the first bond formed and tethered permanently by a few. The sperm cannot subsequently penetrate the zona unless the bonds are first eliminated. However, premature elimination would simply allow the sperm to escape. Therefore, not only must the bonds be eliminated, but the timing of this must be regulated so that the sperm is already oriented toward the egg and beginning to penetrate as the bonds are broken. Images FIGURE 6 PMID:3224150

Detection of possible Phytophthora pinifolia infection in pinus radiata green sawn timber produced in Chile

Treesearch

R. Ahumada; C. Díaz; M. Peredo; C. Barría; P. González; G. Cuevas

2010-01-01

A new needle blight disease was observed on Pinus radiata in Chile during 2004. The disease, known in Chile as Daño Foliar del Pino (DFP), stretches southward from the Arauco to Valdivia Provinces, and was present over an area of about 60 000 ha in 2006, with different levels of intensity. The disease is typified by needle infections and...

The anatomy of the caudal zona incerta in rodents and primates

PubMed Central

Watson, Charles; Lind, Christopher R P; Thomas, Meghan G

2014-01-01

The caudal zona incerta is the target of a recent modification of established procedures for deep brain stimulation (DBS) for Parkinson's disease and tremor. The caudal zona incerta contains a number of neuronal populations that are distinct in terms of their cytoarchitecture, connections, and pattern of immunomarkers and is located at a position where a number of major tracts converge before turning toward their final destination in the forebrain. However, it is not clear which of the anatomical features of the region are related to its value as a target for DBS. This paper has tried to identify features that distinguish the caudal zona incerta of rodents (mouse and rat) and primates (marmoset, rhesus monkey, and human) from the remainder of the zona incerta. We studied cytoarchitecture, anatomical relationships, the pattern of immunomarkers, and gene expression in both of these areas. We found that the caudal zona incerta has a number of histological and gene expression characteristics that distinguish it from the other subdivisions of the zona incerta. Of particular note are the sparse population of GABA neurons and the small but distinctive population of calbindin neurons. We hope that a clearer appreciation of the anatomy of the region will in the end assist the interpretation of cases in which DBS is used in human patients. PMID:24138151

Lymantria monacha (nun moth) and L. dispar (gypsy moth) survival and development on improved Pinus radiata

Treesearch

T.M. Withers; M.A. Keena

2001-01-01

The lymantriid forest defoliators, Lymantria monacha L. (nun moth) and Lymantria dispar L. (gypsy moth) are particularly severe pests in other countries in the world, but the ability of these moths to utilise and complete development on Pinus radiata D. Don had never been established. In laboratory trials, colonies of central European L. monacha and Russian far east (...

Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.

PubMed

Körschgen, Hagen; Kuske, Michael; Karmilin, Konstantin; Yiallouros, Irene; Balbach, Melanie; Floehr, Julia; Wachten, Dagmar; Jahnen-Dechent, Willi; Stöcker, Walter

2017-09-01

How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like

Family-site interaction in Pinus radiata: implications for progeny testing strategy and regionalised breeding in New Zealand.

Treesearch

G.R. Johnson; R.D. Brudon

1990-01-01

A progeny test of 170 open-pollinated families from second-generation plus trees of Pinus radiata was established on four sites in New Zealand in 1981. Two test sites were on volcanic purnice soils in the Central North Island region and two were on phosphate-retentive clay soils in the Northland region.Assessments of volume growth, stem straightness, mal-...

Transcription of lignocellulose-decomposition associated genes, enzyme activities and production of ethanol upon bioconversion of waste substrate by Phlebia radiata.

PubMed

Mäkinen, Mari A; Risulainen, Netta; Mattila, Hans; Lundell, Taina K

2018-05-04

Previously identified twelve plant cell wall degradation-associated genes of the white rot fungus Phlebia radiata were studied by RT-qPCR in semi-aerobic solid-state cultures on lignocellulose waste material, and on glucose-containing reference medium. Wood-decay-involved enzyme activities and ethanol production were followed to elucidate both the degradative and fermentative processes. On the waste lignocellulose substrate, P. radiata carbohydrate-active enzyme (CAZy) genes encoding cellulolytic and hemicellulolytic activities were significantly upregulated whereas genes involved in lignin modification displayed a more complex response. Two lignin peroxidase genes were differentially expressed on waste lignocellulose compared to glucose medium, whereas three manganese peroxidase-encoding genes were less affected. On the contrary, highly significant difference was noticed for three cellulolytic genes (cbhI_1, eg1, bgl1) with higher expression levels on the lignocellulose substrate than on glucose. This indicates expression of the wood-attacking degradative enzyme system by the fungus also on the recycled, waste core board material. During the second week of cultivation, ethanol production increased on the core board to 0.24 g/L, and extracellular activities against cellulose, xylan, and lignin were detected. Sugar release from the solid lignocellulose resulted with concomitant accumulation of ethanol as fermentation product. Our findings confirm that the fungus activates its white rot decay system also on industrially processed lignocellulose adopted as growth substrate, and under semi-aerobic cultivation conditions. Thus, P. radiata is a good candidate for lignocellulose-based renewable biotechnology to make biofuels and biocompounds from materials with less value for recycling or manufacturing.

Leaf area index, biomass carbon and growth rate of radiata pine genetic types and relationships with LiDAR

Treesearch

Peter N. Beets; Stephen Reutebuch; Mark O. Kimberley; Graeme R. Oliver; Stephen H. Pearce; Robert J. McGaughey

2011-01-01

Relationships between discrete-return light detection and ranging (LiDAR) data and radiata pine leaf area index (LAI), stem volume, above ground carbon, and carbon sequestration were developed using 10 plots with directly measured biomass and leaf area data, and 36 plots with modelled carbon data. The plots included a range of genetic types established on north- and...

Bionomics of Asian Citrus Psyllid (Hemiptera: Liviidae) Associated with Orange Jasmine Hedges in Southeast Central Florida, with Special Reference to Biological Control by Tamarixia radiata.

PubMed

Hall, David G; Rohrig, Eric

2015-06-01

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in Florida because it transmits bacteria responsible for citrus huanglongbing disease. In addition to infesting citrus, orange jasmine (Murraya exotica L.) is one of Asian citrus psyllid's preferred host plants and is widely grown as an ornamental hedge. We report on Asian citrus psyllid bionomics over three years at five urban plantings of orange jasmine and on biological control of Asian citrus psyllid by a parasitoid Tamarixia radiata (Waterston). T. radiata had been released in Florida shortly after Asian citrus psyllid was first found, and the parasitoid was known to be established at each planting. Additionally, three new T. radiata haplotypes were released every 3 wk at three plantings during the first study year (one haplotype per planting, over all releases an average of 17 parasitoids per linear meter of hedge); all three haplotypes were released at a fourth planting beginning midway through the study (over all releases, an average combined total of 202 parasitoids per linear meter of hedge). Asian citrus psyllid populations were present year-round at each planting, often at large levels. Such plantings may pose risk to commercial citrus as Asian citrus psyllid reservoirs. Releases of the new haplotypes did not cause any measurable reduction in Asian citrus psyllid population levels during the study, and ironically percentage parasitism was generally highest at a planting where no releases were made. Higher release rates might have been more effective. The probability is discussed that repetitive pruning of orange jasmine reduced the full potential of T. radiata against Asian citrus psyllid in this study. Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C.

PubMed

Liao, Ming-Ling; Zhang, Shu; Zhang, Guang-Ya; Chu, Yun-Meng; Somero, George N; Dong, Yun-Wei

2017-06-01

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, K M NADH ) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners ( E. malaccana and E. radiata ) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata , in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation. © 2017. Published by The Company of Biologists Ltd.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata).

PubMed

Rao, A J; Ramachandra, S G; Ramesh, V; Krishnamurthy, H N; Jayaraman, S; Gopalakrishnan, K; Juneja, H S

1998-01-01

Administration of Tamoxifen via the Alzet pump at a rate of 50 micrograms hr-1 for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Biological control of Asian citrus psyllid (Diaphorina citri) in Florida by the parasitoid Tamarixia radiata in urban plantings of orange jasmine

USDA-ARS?s Scientific Manuscript database

The Asian citrus psyllid is an important invasive citrus pest in the United States because it vectors a bacterium responsible for a devastating disease of citrus known as huanglongbing. A parasitoid of the psyllid, Tamarixia radiata (Hymenoptera: Eulophidae), was imported from Southeast Asia and re...

Zona-free oocyte fertilized with intracytoplasmic sperm injection and underwent further division: case report and literature review.

PubMed

Hsieh, Y Y; Chang, C C; Tsai, H D

2001-09-01

The zona pellucida (ZP) plays a protective role during fertilization and early embryonic development. It is related to sperm binding, the acrosome reaction, prevention of polyspermic fertilization, and holding blastomeres together before the morular stage. Zona-free oocytes are accidentally encountered. If these oocytes are healthy, they can be fertilized normally by intracytoplasmic sperm injection (ICSI). We reported on a couple with male infertility undergoing oocyte retrieval after ovarian hyperstimulation. Before the ICSI procedure, cumulus cells surrounding the oocytes were removed, which resulted in one oocyte escaping from its ZP. The zona-free oocyte was fertilized normally with ICSI and developed to the 8-cell stage. We observed that the zona-free zygote had the ability to further divide, despite its loose contact. The zona-free embryo was transferred with other zona-intact embryos, but the implantation failed. We conclude that zona-free oocytes can be rescued, fertilized with ICSI, and cultured for further transfer or cryopreservation.

A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin.

PubMed

Suckling, Ian D; Jack, Michael W; Lloyd, John A; Murton, Karl D; Newman, Roger H; Stuthridge, Trevor R; Torr, Kirk M; Vaidya, Alankar A

2017-01-01

Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route. We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine ( Pinus radiata ) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3-72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as

Genome‐wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata

PubMed Central

Guo, Yanan; Sim, Andre D.; Kabir, M. Shahjahan; Chettri, Pranav; Ozturk, Ibrahim K.; Hunziker, Lukas; Ganley, Rebecca J.; Cox, Murray P.

2015-01-01

Summary We present genome‐wide gene expression patterns as a time series through the infection cycle of the fungal pine needle blight pathogen, Dothistroma septosporum, as it invades its gymnosperm host, Pinus radiata. We determined the molecular changes at three stages of the disease cycle: epiphytic/biotrophic (early), initial necrosis (mid) and mature sporulating lesion (late). Over 1.7 billion combined plant and fungal reads were sequenced to obtain 3.2 million fungal‐specific reads, which comprised as little as 0.1% of the sample reads early in infection. This enriched dataset shows that the initial biotrophic stage is characterized by the up‐regulation of genes encoding fungal cell wall‐modifying enzymes and signalling proteins. Later necrotrophic stages show the up‐regulation of genes for secondary metabolism, putative effectors, oxidoreductases, transporters and starch degradation. This in‐depth through‐time transcriptomic study provides our first snapshot of the gene expression dynamics that characterize infection by this fungal pathogen in its gymnosperm host. PMID:25919703

28-Homobrassinolide mitigates boron induced toxicity through enhanced antioxidant system in Vigna radiata plants.

PubMed

Yusuf, Mohammad; Fariduddin, Qazi; Ahmad, Aqil

2011-11-01

The objective of this study was to establish relationship between boron induced oxidative stress and antioxidant system in Vigna radiata plants and also to investigate whether brassinosteroids will enhance the level of antioxidant system that could confer tolerance to the plants from the boron induced oxidative stress. The mung bean (V. radiata cv. T-44) plants were administered with 0.50, 1.0 and 2.0 mM boron at 6 d stage for 7 d along with nutrient solution. At 13 d stage, the seedlings were sprayed with deionized water (control) or 10(-8) M of 28-homobrassinolide and plants were harvested at 21 d stage to assess growth, leaf gas-exchange traits and biochemical parameters. The boron treatments diminished growth, water relations and photosynthetic attributes along with nitrate reductase and carbonic anhydrase activity in the concentration dependent manner whereas, it enhanced lipid peroxidation, electrolyte leakage, accumulation of H(2)O(2) as well as proline, and various antioxidant enzymes in the leaves of mung bean which were more pronounced at higher concentrations of boron. However, the follow-up application of 28-homobrassinolide to the boron stressed plants improved growth, water relations and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and content of proline. The elevated level of antioxidant enzymes as well as proline could have conferred tolerance to the B-stressed plants resulting in improved growth, water relations and photosynthetic attributes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata.

PubMed

Fraga, Mario F; Rodríguez, Roberto; Cañal, Maria Jesús

2002-08-01

In animals, DNA methylation is related to gene silencing during ontogenic development. Little is known about DNA methylation in plants, although occasional changes in the DNA methylation state of specific gene promoters have been reported in angiosperms during some developmental processes. We found large differences in the extent of DNA methylation between meristematic areas of juvenile and mature Pinus radiata D. Don. trees, whereas differences in the extent of DNA methylation between differentiated tissues of juvenile and mature trees were small. In meristematic areas, there was a gradual decrease in extent of DNA methylation as the degree of reinvigoration increased. The observed changes in extent of DNA methylation during aging and reinvigoration indicate that reinvigoration could be a consequence of epigenetic modifications opposite in direction to those that occur during aging.

Formation of post-fire water-repellent layers in Monterrey pine (Pinus radiata D. Don) plantations in south-central Chile

Treesearch

P. Garcia-Chevesich; R. Pizarro; C. L. Stropki; P. Ramirez de Arellano; P. F. Ffolliott; L. F. DeBano; Dan Neary; D. C. Slack

2010-01-01

A wildfire burned about 15,000 ha of Monterrey Pine (Pinus radiata D. Don) plantations near Yungay, Chile, in January of 2007. Post-fire water repellency (hydrophobicity) was measured using the water-drop-penetration-time (WDPT) method at depths of 0, 5, and 10 mm from the soil surface. These measurements were collected on burned sites of both young (4-years old) and...

Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding.

PubMed

Hou, S T; Ma, A; Jones, R; Hall, L

1996-10-01

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

PubMed Central

Jin, Mayuko; Fujiwara, Eiji; Kakiuchi, Yasutaka; Okabe, Masaru; Satouh, Yuhkoh; Baba, Shoji A.; Chiba, Kazuyoshi; Hirohashi, Noritaka

2011-01-01

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization. PMID:21383182

Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

PubMed

Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

2014-11-10

Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown. Copyright © 2014 Elsevier B.V. All rights reserved.

Nontarget effects of chemical pesticides and biological pesticide on rhizospheric microbial community structure and function in Vigna radiata.

PubMed

Singh, Sunil; Gupta, Rashi; Kumari, Madhu; Sharma, Shilpi

2015-08-01

Intensive agriculture has resulted in an indiscriminate use of pesticides, which demands in-depth analysis of their impact on indigenous rhizospheric microbial community structure and function. Hence, the objective of the present work was to study the impact of two chemical pesticides (chlorpyrifos and cypermethrin) and one biological pesticide (azadirachtin) at two dosages on the microbial community structure using cultivation-dependent approach and on rhizospheric bacterial communities involved in nitrogen cycle in Vigna radiata rhizosphere through cultivation-independent technique of real-time PCR. Cultivation-dependent study highlighted the adverse effects of both chemical pesticide and biopesticide on rhizospheric bacterial and fungal communities at different plant growth stages. Also, an adverse effect on number of genes and transcripts of nifH (nitrogen fixation); amoA (nitrification); and narG, nirK, and nirS (denitrification) was observed. The results from the present study highlighted two points, firstly that nontarget effects of pesticides are significantly detrimental to soil microflora, and despite being of biological origin, azadirachtin exerted negative impact on rhizospheric microbial community of V. radiata behaving similar to chemical pesticides. Hence, such nontarget effects of chemical pesticide and biopesticide in plants' rhizosphere, which bring out the larger picture in terms of their ecotoxicological effect, demand a proper risk assessment before application of pesticides as agricultural amendments.

Lethal and Sublethal Effects of Insecticides Used on Citrus, on the Ectoparasitoid Tamarixia radiata

PubMed Central

Beloti, Vitor Hugo; Alves, Gustavo Rodrigues; Araújo, Diogo Feliciano Dias; Picoli, Mateus Manara; Moral, Rafael de Andrade; Demétrio, Clarice Garcia Borges; Yamamoto, Pedro Takao

2015-01-01

Huanglongbing (HLB) is a disease associated with the bacteria “Candidatus Liberibacter spp.” and has been devastating citrus orchards around the world. Its management involves control of the insect vector, the Asian citrus psyllid Diaphorina citri Kuwayama. However, the indiscriminate use of chemicals has caused pest outbreaks and eliminated the natural enemies of the vector, such as the parasitoid Tamarixia radiata (Waterston), the main agent for biological control of D. citri. This study assessed the lethal and sublethal effects of insecticides recommended for integrated production of citrus on the parasitoid T. radiata. When adult parasitoids were exposed to residues of 25 insecticides, 20% of them, i.e., gamma-cyhalothrin, etofenprox, azadirachtin, tebufenozide and pyriproxyfen, were considered as harmless (Class 1), 12% as slightly harmful (Class 2), 12% as moderately harmful (Class 3) and 56% as harmful (Class 4), according to the classification proposed by the IOBC/WPRS. Afterward, 14 insecticides (5 harmless and 9 harmful) were sprayed on the parasitoid pupae. Of the 14 insecticides tested, only the organophosphates dimethoate and chlorpyrifos affected the parasitoid emergence. The effects of insecticides on the parasitism capacity of adults exposed to residues of azadirachtin, etofenprox, gamma-cyhalothrin, pyriproxyfen and tebufenozide (harmless) were also evaluated. Tebufenozide and gamma-cyhalothrin affected the parasitism of the F0 generation, but did not affect the emergence of the F1 and F2 generations. Therefore, for an effective IPM program, selective insecticides or harmful pesticides to adult parasitoids could be used in the field, provided that the adults do not occur naturally and the chemical applications do not coincide with parasitoid releases. PMID:26132327

Thermal effects in laser-assisted pre-embryo zona drilling

NASA Astrophysics Data System (ADS)

Douglas-Hamilton, Diarmaid H.; Conia, Jerome D.

2001-04-01

Diode lasers ((lambda) equals 1480 nm) are used with in vitro fertilization to dissect the zone pellucida (shell) of pre- embryos. A focused laser beam is applied in vitro to form a channel or trench in the zona pellucida. The procedure is used to facilitate biopsy or as a promoter of embryo hatching. We present examples and measurements of zona pellucida ablation using animal models. In using the laser it is vital not to damage pre-embryo cells, e.g., by overheating. In order to define safe regimes we have derived some thermal side effects of zona pellucida removal. The temperature profile in the beam and vicinity is predicted as function of laser pulse duration and power. In a crossed- beam experiment a HeNe laser probe is used to detect the temperature-induced change in the refractive index of an aqueous solution, and estimate local thermal gradient. We find that the diode laser beam produces superheated water approaching 200 degree(s)C on the beam axis. Thermal histories during and following the laser pulse are given for regions in the neighborhood of the beam. We conclude that an optimum regime exists with pulse duration

Suppression of 4-Coumarate-CoA Ligase in the Coniferous Gymnosperm Pinus radiata1[W

PubMed Central

Wagner, Armin; Donaldson, Lloyd; Kim, Hoon; Phillips, Lorelle; Flint, Heather; Steward, Diane; Torr, Kirk; Koch, Gerald; Schmitt, Uwe; Ralph, John

2009-01-01

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a “bonsai tree-like” appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated β-aryl ether (β-O-4 unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata. PMID:18971431

Analysis of disconnected diallel mating designs II: results from a third generation progeny test of the New Zealand radiata pine improvement programme.

Treesearch

J.N. King; M.J. Carson; G.R. Johnson

1998-01-01

Genetic parameters from a second generation (F2) disconnected diallel progeny test of the New Zealand radiata pine improvement programme are presented. Heritability estimates of growth and yield traits of 0.2 are similar to progeny test results of the previous generation (F1) generation tests. A trend of declining dominance...

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro.

PubMed

Buffone, Mariano G; Zhuang, Tiangang; Ord, Teri S; Hui, Ling; Moss, Stuart B; Gerton, George L

2008-05-02

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

PubMed Central

Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V.; Mueller, Thomas D.; Nickel, Joachim; Eichler, Jutta; Muller, Yves A.

2013-01-01

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3. PMID:23826237

Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse.

PubMed

Schimmel, Tim; Cohen, Jacques; Saunders, Helen; Alikani, Mina

2014-12-01

Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 μm and width of the thinned area was 35 μm. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P < 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona

Zona pellucida-binding protein 2 (ZPBP2) and several proteins containing BX7B motifs in human sperm may have hyaluronic acid binding or recognition properties.

PubMed

Torabi, F; Bogle, O A; Estanyol, J M; Oliva, R; Miller, D

2017-12-01

Are there novel hyaladherins in human sperm? Zona pellucida-binding protein 2 (ZPBP2), containing a Link-like hyaluronic acid (HA)-binding domain, and several other proteins containing BX7B motifs, such as ADAM32 and Midkine, may be novel hyaladherins with HA-binding properties. HA-binding proteins (hyaladherins), which can bind HA surrounding the cumulus-oophorus complex, are distinct from hyases such as PH 20 (SPAM1) and are expressed by mature spermatozoa. Although HABP1 and CD44 are reasonably well characterized hyaladherins and the former has been implicated in sperm-oocyte interactions, the overall significance of sperm hyaladherins for male fertility is still poorly understood. This was a laboratory-based investigation into human sperm hyaladherins undertaken as part of a three year PhD programme sponsored by the EU Marie Curie Training network, Reprotrain. Protein homogenates of sperm obtained from young men of unknown fertility (N = 4) were partitioned into HA-binding and non-binding fractions by a protein affinity 'panning' method; their subsequent characterization was by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and partitioning behaviour was confirmed by western blotting. Sequences of proteins from both fractions were submitted to PDBsum to look for orthologous entries (PDB codes) and all returned codes were queried against the matching protein using SAS (Sequences Annotated by Structure) looking for structural similarities between them. A systematic search for other common features of hyaladherins was also undertaken. The presence of BX7B sequence motifs found in several well-described hyaladherins including RHAMM was used to assess efficacy of potential hyaladherin partitioning by the HA substrate. The data showed that 50% (14/28) and 34.5% (28/81) of proteins in the bound and unbound fractions, respectively, contained these motifs (one-tailed Z-score = 1.45; P = 0.074), indicating weak discrimination by the substrate. Querying PDBsum

Oviposition behavior and survival of Tamarixia radiata (Hymenoptera: Eulophidae), an ectoparasitoid of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), on hosts exposed to an entomopathogenic fungus

USDA-ARS?s Scientific Manuscript database

Antagonistic interactions between the nymphal parasitoid, Tamarixia radiata Waterston (Hymenoptera: Eulophidae), and the ARSEF 3581 isolate of the entomopathogenic fungus, Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae) could disrupt biological control of the Asian citrus psyllid, Diaphorina ...

Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine.

PubMed

De Diego, N; Saiz-Fernández, I; Rodríguez, J L; Pérez-Alfocea, P; Sampedro, M C; Barrio, R J; Lacuesta, M; Moncaleán, P

2015-09-01

Studies of metabolic and physiological bases of plant tolerance and hardening against drought are essential to improve genetic breeding programs, especially in productive species such as Pinus radiata. The exposure to different drought cycles is a highly effective tool that improves plant conditioning, but limited information is available about the mechanisms that modulate this process. To clarify this issue, six P. radiata breeds with well-known differences in drought tolerance were analyzed after two consecutive drought cycles. Survival rate, concentration of several metabolites such as free soluble amino acids and polyamines, and main plant hormones varied between them after drought hardening, while relative growth ratio and water potential at both predawn and dawn did not. Hardening induced a strong increase in total soluble amino acids in all breeds, accumulating mainly those implicated in the glutamate metabolism (GM), especially L-proline, in the most tolerant breeds. Other amino acids from GM such as γ-aminobutyric acid (GABA) and L-arginine (Arg) were also strongly increased. GABA pathway could improve the response against drought, whereas Arg acts as precursor for the synthesis of spermidine. This polyamine showed a positive relationship with the survival capacity, probably due to its role as antioxidant under stress conditions. Finally, drought hardening also induced changes in phytohormone content, showing each breed a different profile. Although all of them accumulated indole-3-acetic acid and jasmonic acid and reduced zeatin content in needles, significant differences were observed regarding abscisic acid, salicylic acid and mainly zeatin riboside. These results confirm that hardening is not only species-dependent but also an intraspecific processes controlled through metabolite changes. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization.

PubMed

Cohen, Débora J; Maldera, Julieta A; Weigel Muñoz, Mariana; Ernesto, Juan I; Vasen, Gustavo; Cuasnicu, Patricia S

2011-01-01

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion.

PubMed

Romero, Damian G; Zhou, Ming Yi; Yanes, Licy L; Plonczynski, Maria W; Washington, Tanganika R; Gomez-Sanchez, Celso E; Gomez-Sanchez, Elise P

2007-08-01

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Phase-change related epigenetic and physiological changes in Pinus radiata D. Don.

PubMed

Fraga, Mario F; Cañal, Maria Jesús; Rodríguez, Roberto

2002-08-01

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.

Evaluation of tight junction protein 1 encoding zona occludens 1 as a candidate gene for albuminuria in a Mexican American population.

PubMed

Lehman, D M; Leach, R J; Johnson-Pais, T; Hamlington, J; Fowler, S; Almasy, L; Duggirala, R; Stern, M P; Abboud, H E

2006-09-01

Albuminuria, a hallmark of diabetic nephropathy, has been shown to be significantly heritable in multiple studies. Therefore, the identification of genes that affect susceptibility to albuminuria may lead to novel avenues of intervention. Current evidence suggests that the podocyte and slit diaphragm play a key role in controlling the selective sieve of the glomerular filtration barrier, and podocyte-specific genes have been identified that are necessary for maintaining its integrity. We therefore investigated the role of gene variants of tight junction protein (TJP1) which encodes another slit diaphragm-associated protein zona occludens 1 as risk factors for albuminuria in the San Antonio Family Diabetes/Gallbladder Study (SAFDGS), which consists of extended Mexican-American families with a high prevalence of type 2 diabetes. Albuminuria, defined as an albumin (mg/dl) to creatinine (mg/dl) ratio (ACR) of 0.03, which is approximately equivalent to a urinary albumin excretion (UAE) >30 mg/day, was present in a total of 14.9% of participants, and 31% had type 2 diabetes. The TJP1 exons, flanking intronic sequence, and putative proximal promoter regions were investigated in this population. Twentynine polymorphisms, including 7 nonsynonymous SNPs, were identified and genotyped in all subjects of this study for association analysis. Three sets of correlated SNPs, which include 3 exonic SNPs, were nominally associated with ACR (p value range 0.007-0.049); however, the association with the discrete trait albuminuria was not significant (p value range 0.094-0.338). We conclude that these variants in TJP1 do not appear to be major determinants for albuminuria in the SAFDGS; however, they may play a minor role in its severity in this Mexican-American population. Further examination of the TJP1 gene region in this and other cohorts will be useful to determine whether ZO-1 plays a significant role in glomerular permselectivity.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep

PubMed Central

Liu, Kai; Kim, Juhyun; Kim, Dong Won; Zhang, Yi Stephanie; Bao, Hechen; Denaxa, Myrto; Lim, Szu-Aun; Kim, Eileen; Liu, Chang; Wickersham, Ian R.; Pachnis, Vassilis; Hattar, Samer; Song, Juan; Brown, Solange P.; Blackshaw, Seth

2017-01-01

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified1. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep2,3. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep–wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep. PMID:28847002

Relationship between diffusion tensor fractional anisotropy and motor outcome in patients with hemiparesis after corona radiata infarct.

PubMed

Koyama, Tetsuo; Marumoto, Kohei; Miyake, Hiroji; Domen, Kazuhisa

2013-11-01

This study examined the relationship between fractional anisotropy (FA) values of magnetic resonance-diffusion tensor imaging (DTI) and motor outcome (1 month after onset) in 15 patients with hemiparesis after ischemic stroke of corona radiata lesions. DTI data were obtained on days 14-18. FA values within the cerebral peduncle were analyzed using a computer-automated method. Motor outcome of hemiparesis was evaluated according to Brunnstrom stage (BRS; 6-point scale: severe to normal) for separate shoulder/elbow/forearm, wrist/hand, and lower extremity functions. The ratio of FA values in the affected hemisphere to those in the unaffected hemisphere (rFA) was assessed in relation to the BRS data (Spearman rank correlation test, P<.05). rFA values ranged from .715 to 1.002 (median=.924). BRS ranged from 1 to 6 (median=4) for shoulder/elbow/forearm, from 1 to 6 (median=5) for wrist/hand, and from 2 to 6 (median=4) for the lower extremities. Analysis revealed statistically significant relationships between rFA and upper extremity functions (correlation coefficient=.679 for shoulder/elbow/forearm and .706 for wrist/hand). Although slightly less evident, the relationship between rFA and lower extremity function was also statistically significant (correlation coefficient=.641). FA values within the cerebral peduncle are moderately associated with the outcome of both upper and lower extremity functions, suggesting that DTI may be applicable for outcome prediction in stroke patients with corona radiata infarct. Copyright © 2013 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics

PubMed Central

2011-01-01

Background The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile Pinus radiata trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics. Results Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA. Conclusions Microarray expression profiles in Pinus radiata juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further

Identification of ZASP, a novel protein associated to Zona occludens-2.

PubMed

Lechuga, Susana; Alarcón, Lourdes; Solano, Jesús; Huerta, Miriam; Lopez-Bayghen, Esther; González-Mariscal, Lorenza

2010-11-15

With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds. Copyright © 2010 Elsevier Inc. All rights reserved.

Halopriming of seeds imparts tolerance to NaCl and PEG induced stress in Vigna radiata (L.) Wilczek varieties.

PubMed

Jisha, K C; Puthur, Jos T

2014-07-01

The investigation was carried out to study the effect of halopriming on NaCl and polyethylene glycol-6000 (PEG-6000) induced stress tolerance potential of three Vigna radiata (L.) Wilczek varieties, with varied abiotic stress tolerance potential. Halopriming is a seed priming technique in which the seeds were soaked in various salt solutions (in this study NaCl was used). The results of the study indicated that the application of stresses (both NaCl and PEG) induced retardation of growth attributes (measured in terms of shoot length, fresh weight, dry weight) and decrease in physiological attributes like total chlorophyll content, metabolites, photosynthetic and mitochondrial activity of the seedlings in all three V. radiata (L.) varieties. However, halopriming of the seeds could reduce the extent of decrease in these biological attributes. NaCl and PEG stress also caused increase in MDA content (a product of membrane lipid peroxidation) in all the varieties studied and this increase was significantly minimized under halopriming. From the present investigation it was evident that among the green gram varieties studied, Pusa Vishal, a NaCl tolerant variety showed enhanced tolerance to NaCl and PEG induced stress, when the seeds were subjected to halopriming followed by Pusa Ratna (stress sensitive variety). Pusa 9531 (drought tolerant variety) also showed positive halopriming effects but it was less significant when compared to other two varieties. It could be concluded that halopriming improved the drought and salinity stress tolerance potential of all varieties and it was significantly higher in the Pusa Vishal as compared to Pusa 9531 and Pusa Ratna.

Early detection of secondary damage in ipsilateral thalamus after acute infarction at unilateral corona radiata by diffusion tensor imaging and magnetic resonance spectroscopy

PubMed Central

2011-01-01

Background Traditional magnetic resonance (MR) imaging can identify abnormal changes in ipsilateral thalamus in patients with unilateral middle cerebral artery (MCA) infarcts. However, it is difficult to demonstrate these early changes quantitatively. Diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy (MRS) are potentially sensitive and quantitative methods of detection in examining changes of tissue microstructure and metabolism. In this study, We used both DTI and MRS to examine possible secondary damage of thalamus in patients with corona radiata infarction. Methods Twelve patients with unilateral corona radiata infarction underwent MR imaging including DTI and MRS at one week (W1), four weeks (W4), and twelve weeks (W12) after onset of stroke. Twelve age-matched controls were imaged. Mean diffusivity (MD), fractional anisotropy (FA), N-acetylaspartate (NAA), choline(Cho), and creatine(Cr) were measured in thalami. Results T1-weighted fluid attenuation inversion recovery (FLAIR), T2-weighted, and T2-FLAIR imaging showed an infarct at unilateral corona radiate but no other lesion in each patient brain. In patients, MD was significantly increased at W12, compared to W1 and W4 (all P< 0.05). NAA was significantly decreased at W4 compared to W1, and at W12 compared to W4 (all P< 0.05) in the ipsilateral thalamus. There was no significant change in FA, Cho, or Cr in the ipsilateral thalamus from W1 to W12. Spearman's rank correlation analysis revealed a significant negative correlation between MD and the peak area of NAA, Cho, and Cr at W1, W4, and W12 and a significant positive correlation of FA with NAA at W1. Conclusions These findings indicate that DTI and MRS can detect the early changes indicating secondary damage in the ipsilateral thalamus after unilateral corona radiata infarction. MRS may reveal the progressive course of damage in the ipsilateral thalamus over time. PMID:21542942

Integrated Physiological, Proteomic, and Metabolomic Analysis of Ultra Violet (UV) Stress Responses and Adaptation Mechanisms in Pinus radiata*

PubMed Central

Pascual, Jesús; Cañal, María Jesús; Escandón, Mónica; Meijón, Mónica; Weckwerth, Wolfram

2017-01-01

Globally expected changes in environmental conditions, especially the increase of UV irradiation, necessitate extending our knowledge of the mechanisms mediating tree species adaptation to this stress. This is crucial for designing new strategies to maintain future forest productivity. Studies focused on environmentally realistic dosages of UV irradiation in forest species are scarce. Pinus spp. are commercially relevant trees and not much is known about their adaptation to UV. In this work, UV treatment and recovery of Pinus radiata plants with dosages mimicking future scenarios, based on current models of UV radiation, were performed in a time-dependent manner. The combined metabolome and proteome analysis were complemented with measurements of + physiological parameters and gene expression. Sparse PLS analysis revealed complex molecular interaction networks of molecular and physiological data. Early responses prevented phototoxicity by reducing photosystem activity and the electron transfer chain together with the accumulation of photoprotectors and photorespiration. Apart from the reduction in photosynthesis as consequence of the direct UV damage on the photosystems, the primary metabolism was rearranged to deal with the oxidative stress while minimizing ROS production. New protein kinases and proteases related to signaling, coordination, and regulation of UV stress responses were revealed. All these processes demonstrate a complex molecular interaction network extending the current knowledge on UV-stress adaptation in pine. PMID:28096192

Gill tissue reactions in walleye Stizostedion vitreum vitreum and common carp Cyprinus carpio to glochidia of the freshwater mussel Lampsilis radiata siliquoidea

USGS Publications Warehouse

Waller, D.L.; Mitchell, L.G.

1989-01-01

The glochidia of many freshwater mussels, which are obligate parasites on the gills, fins, and other body parts of specific fishes, attach to a suitable host, become encapsulated, and develop to the free-living juvenile stage. Using light and electron microscopy we compared gill tissue reactions in a suitable host (walleye Stizostedion vitreum vitreum) and unsuitable host (common carp Cyprinus carpio) infected with Lampsilis radiata siliquoidea. Encapsulation of glochidia on walleye gills was completed by 6 h post-infection at 20 to 22°C. Capsular formation and compaction were accompanied by a general increase in epithelioid cells. Fibrotic material appeared in capsules at about 48 h and virtually filled capsular cells from about Day 5 to Day 11 post-infection. Liberation of juvenile mussels was accompanied by thinning of the capsule from about Day 11 to Day l7. Although glochidia attached to the gills of common carp, few became encapsulated. By 48 h post-infection, preliminary capsular growth was evident and necrotic cells and cellular debris appeared at the edges of the growth. However, all glochidia were sloughed from carp gills by 60 h. Host specificity of L. radiata siliquoidea apparently depended on a combination of the attachment response of glochidia, differences in the encapsulation process, and tissue reactions in the fish.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

PubMed

Kitchener, Anne L; Harman, Amanda; Kay, David J; McCartney, Carmen A; Mate, Karen E; Rodger, John C

2009-01-01

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Laboratory evaluations of the foraging success of Tamarixia radiata (Hymenoptera: Eulophidae) on flowers & extra-floral nectaries: Potential use of nectary plants for conservation biological control of Asian citrus psyllid

USDA-ARS?s Scientific Manuscript database

Tamarixia radiata is natural enemy of the Asian citrus psyllid, the insect that transmits citrus greening disease. It is currently being mass reared for releases in Florida, Texas, and California for psyllid control. However, biological control of the psyllid following mass releases of Tamarixia rad...

Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using internal transcribed spacer and cytochrome oxidase subunit l DNA sequences

USDA-ARS?s Scientific Manuscript database

The genetic diversity of Tamarixia radiata laboratory colonies derived from collections in China, northern Vietnam, Pakistan, and a mixed colony from Taiwan and southern Vietnam was evaluated using the internal transcribed spacer region 1 (ITS-1), internal transcribed spacer region 2 (ITS-2) and the...

Wood formation from the base to the crown in Pinus radiata: gradients of tracheid wall thickness, wood density, radial growth rate and gene expression

Treesearch

Sheree Cato; Lisa McMillan; Lloyd Donaldson; Thomas Richardson; Craig Echt; Richard Gardner

2006-01-01

Wood formation was investigated at five heights along the bole for two unrelated trees of Pinus radiataBoth trees showed clear gradients in wood properties from the base to the crown. Cambial cells at the base of the tree were dividing 3.3-fold slower than those at the crown, while the average thickness of cell walls in wood was highest at the base....

Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek).

PubMed

Shrivastava, Divya; Verma, Priyanka; Bhatia, Sabhyata

2014-09-01

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard's similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.

Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bleil, J.D.; Wassarman, P.M.

1990-07-01

During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm.more » These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.« less

The Effect of Estradiol-17(beta), Goitrogen (T3), and Flutamide on Gene Expression in Medaka, Oryzias latipes

DOE Office of Scientific and Technical Information (OSTI.GOV)

E.Haut, J

Concern has been generated over the discovery of endocrine disrupting chemicals in rivers near sewage outflows. The presence of endocrine disrupting chemicals such as estradiol-17{beta} has been associated with a reduction of reproductive success in fish and an increase in the female phenotype and gonadal intersex in fish downstream of sewage treatment facilities. Such effects are believed to result from a disruption in the normal estrogenic pathways since estrogen plays a vital role in reproduction, sexual differentiation, the developments of secondary sex characteristics, and ovulation. Most studies have focused on the effect of a single endocrine disruptor on a singlemore » gene which does not provide for the interaction between genes. Microarray technology has made it possible to put an entire genome on a single chip so that researchers can get a clearer picture of the interaction of genes expressed in a cell and changes of said interactions when those cells are exposed to various conditions. Medaka males were exposed to known endocrine disruptors, estradial-17{beta} and goitrogen, and medaka females were exposed to flutamide. All treatments were then compared to controls. Total RNA was extracted from the livers of both treated and untreated males and hybridized to a microarray chip designed to have EST sequences specific to medaka. ESTs were identified through two-channel microarray analysis and compared to GenBank using blastn searches to identify up regulated genes. Choriogenins H and L, zona radiata, and vitellogenin, previously shown to be estrogen-induced in male fish were identified. Heat shock proteins (hsp70, hsp90, and hsp8) were also induced by estradiol-17{beta}, as was choriogenin Hminor. Exposure to goitrogen (T3) resulted in the induced expression of glutathione S-transferase and a GABA receptor protein in male medaka. Treatment with flutamide, an antiandrogen, caused the up regulation of choriogenin L, choriogenin Hminor, and zona radiata-2 in

Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism.

PubMed

Uppangala, Shubhashree; D'Souza, Fiona; Pudakalakatti, Shivanand; Atreya, Hanudatta S; Raval, Keyur; Kalthur, Guruprasad; Adiga, Satish Kumar

2016-12-01

Laser assisted zona hatching (LAH) is a routinely used therapeutic intervention in assisted reproductive technology for patients with poor prognosis. However, results are not conclusive in demonstrating the benefits of zona hatching in improving the pregnancy rate. Recent observations on LAH induced genetic instability in animal embryos prompted us to look into the effects of laser assisted zona hatching on the human preimplantation embryo quality and metabolic uptake using high resolution nuclear magnetic resonance (NMR) technology. This experimental prospective study included fifty embryos from twenty-five patients undergoing intra cytoplasmic sperm injection. Embryo quality assessment followed by profiling of spent media for the non-invasive evaluation of metabolites was performed using NMR spectroscopy 24 hours after laser treatment and compared with that of non-treated sibling embryos. Both cell number and embryo quality on day 3 of development did not vary significantly between the two groups at 24 hours post laser treatment interval. Time lapse monitoring of the embryos for 24 hours did not reveal blastomere fragmentation adjacent to the point of laser treatment. Similarly, principal component analysis of metabolites did not demonstrate any variation across the groups. These results suggest that laser assisted zona hatching does not affect human preimplantation embryo morphology and metabolism at least until 24 hours post laser assisted zona hatching. However, studies are required to elucidate laser induced metabolic and developmental changes at extended time periods. AH: assisted hatching; ART: assisted reproductive technology; DNA: deoxy-ribo nucleic acid; LAH: laser assisted hatching; MHz: megahertz; NMR: nuclear magnetic resonance; PCA: principal component analysis; PGD: preimplantation genetic diagnosis; TLM: time lapse monitoring.

[Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility].

PubMed

Serres, Catherine; Auer, Jana; Petit, François; Patrat, Catherine; Jouannet, Pierre

2008-01-01

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

PubMed

Wong, Chi-Wai; Lam, Kevin K W; Lee, Cheuk-Lun; Yeung, William S B; Zhao, Wei E; Ho, Pak-Chung; Ou, Jian-Ping; Chiu, Philip C N

2017-04-01

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes).

PubMed

Maria, Alexandre N; Orfão, Laura H; Rizzo, Elizete; Ninhaus-Silveira, Alexandre; Viveiros, Ana T M

2015-06-01

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 μm in thickness with eight pore canals/μm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Histochemical Characterization of Oocytes in the Pink Cuskeel (Genypterus blacodes).

PubMed

Cohen, Stefanía; Petcoff, Gladys; Freijo, Roberto O; Portiansky, Enrique L; Barbeito, Claudio G; Macchi, Gustavo J; Díaz, Alcira O

2015-08-01

In the present study we histochemically and lectinhistochemically characterized the growing oocytes of the pink cuskeel (Genypterus blacodes). We used histochemical methods for the localization and characterization of glycoconjugates (GCs) and lectin histochemical techniques for the identification of specific sugar residues. We analyzed presence and distribution of GCs in the different structures of the growing follicles (cortical alveoli, globules, yolk granules and zona radiata). During the initial stage of vitellogenesis, the oocytes presented small yolk granules composed of GCs that gradually increased during exogenous vitellogenesis. These GCs contained moderate quantities of α-D-mannose, D-glucose, N-acetylglucosamine and N-acetyl-neuraminic acid. The cortical alveoli contained both neutral and carboxylated GCs, and lectin techniques detected N-acetylgalactosamine, galactose and L-fucose. The zona radiata showed a strong positive reaction to PAS and it reacted weakly with more specific techniques, such as KOH/PA*S and PA/Bh/KOH/PAS. This structure showed GCs with oxidizable vicinal diols, O-acyl sugars and sialic acid residues with different substitution types and presented N-acetylgalactosamine and L-fucose specific residues. The oocytes follicular envelope evidenced neutral and acidic non-sulfated GCs and high concentrations of α-D-mannose, D-glucose, galactose and N-acetylgalactosamine. The intergranular cytoplasmic GCs were mainly rich in α-D-mannose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine and N-acetyl-neuraminic acid. These results enhance the comprehension of the structure and functionality of the pink cuskeel ovarian follicles, and provide a useful tool for the study of this tissue in other teleost species.

The variations in the nuclear proteome reveal new transcription factors and mechanisms involved in UV stress response in Pinus radiata.

PubMed

Pascual, Jesús; Alegre, Sara; Nagler, Matthias; Escandón, Mónica; Annacondia, María Luz; Weckwerth, Wolfram; Valledor, Luis; Cañal, María Jesús

2016-06-30

The importance of UV stress and its side-effects over the loss of plant productivity in forest species demands a deeper understanding of how pine trees respond to UV irradiation. Although the response to UV stress has been characterized at system and cellular levels, the dynamics within the nuclear proteome triggered by UV is still unknown despite that they are essential for gene expression and regulation of plant physiology. To fill this gap this work aims to characterize the variations in the nuclear proteome as a response to UV irradiation by using state-of-the-art mass spectrometry-based methods combined with novel bioinformatics workflows. The combination of SEQUEST, de novo sequencing, and novel annotation pipelines allowed cover sensing and transduction pathways, endoplasmic reticulum-related mechanisms and the regulation of chromatin dynamism and gene expression by histones, histone-like NF-Ys, and other transcription factors previously unrelated to this stress source, as well as the role of alternative splicing and other mechanisms involved in RNA translation and protein synthesis. The determination of 33 transcription factors, including NF-YB13, Pp005698_3 (NF-YB) and Pr009668_2 (WD-40), which are correlated to stress responsive mechanisms like an increased accumulation of photoprotective pigments and reduced photosynthesis, pointing them as strong candidate biomarkers for breeding programs aimed to improve UV resistance of pine trees. The description of the nuclear proteome of Pinus radiata combining a classic approach based on the use of SEQUEST and the use of a mass accuracy precursor alignment (MAPA) allowed an unprecedented protein coverage. This workflow provided the methodological basis for characterizing the changes in the nuclear proteome triggered by UV irradiation, allowing the depiction of the nuclear events involved in stress response and adaption. The relevance of some of the discovered proteins will suppose a major advance in stress biology

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

PubMed Central

Aagaard, Jan E.; Vacquier, Victor D.; MacCoss, Michael J.; Swanson, Willie J.

2010-01-01

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm–egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization. PMID:19767347

Utilization of Diamine Oxidase Enzyme from Mung Bean Sprouts (Vigna radiata L) for Histamine biosensors

NASA Astrophysics Data System (ADS)

Karim, Abdul; Wahab, A. W.; Raya, I.; Natsir, H.; Arif, A. R.

2018-03-01

This research is aimed to utilize the diamine oxidase enzyme (DAO) which isolated from mung bean sprouts (Vigna radiata L) to develop histamine biosensors based on electode enzyme with the amperometric method (cyclic voltammetry).The DAO enzyme is trapped inside the membrane of chitin-cellulose acetate 2:1 and glutaraldehyde which super imposed on a Pt electrode. Histamine will be oxidized by DAO enzyme to produce aldehydes and H2O2 that acting as electron transfer mediators.The performance of biosensors will be measured at various concentrations of glutaraldehyde, temperature changes and different range of pH. Recently, it has been found that the optimal conditions obtained from the paramaters as follows; at 25% of glutaraldehyde, temperature of 37°C and pH of 7.4. Eventually, the results provided an expectation for applying histamine biosensors in determining the freshness and safety of fish specifically skombroidae families.

Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

PubMed Central

Walther, Cornelia

2015-01-01

The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

PubMed

Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

2017-05-01

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in

Body mass index is not associated with sperm-zona pellucida binding ability in subfertile males.

PubMed

Sermondade, Nathalie; Dupont, Charlotte; Faure, Céline; Boubaya, Marouane; Cédrin-Durnerin, Isabelle; Chavatte-Palmer, Pascale; Sifer, Christophe; Lévy, Rachel

2013-09-01

Lifestyle factors, such as weight and nutritional status may affect male fertility, including sperm fertilization ability. The objective of this retrospective study was to evaluate the association between body mass index (BMI) and sperm-zona pellucida binding ability assessed according to the zona binding (ZB) test, which has been described to be a relevant diagnostic tool for the prediction of in vitro fertilization (IVF) ability. Three hundred and six male patients from couples diagnosed with primary idiopathic or mild male factor infertility were included. Correlations between BMI and semen parameters according to ZB test indices were assessed, together with frequencies of positive and negative tests across the BMI categories. In this selected population, BMI was not related to conventional semen parameters or sperm quality assessed according to the ability of spermatozoa to bind to the zona pellucida. The previously described poor outcomes of IVF procedures in cases of male obesity could be due to other sperm defects, such as alterations of sperm capacitation or acrosome reaction. The link between male BMI and biological outcomes during IVF procedures, such as fertilization rates, should be further evaluated.

Epididymal protein CRISP1 plays different roles during the fertilization process.

PubMed

Cohen, Débora J; Maldera, Julieta A; Vasen, Gustavo; Ernesto, Juan I; Muñoz, Mariana Weigel; Battistone, María A; Cuasnicú, Patricia S

2011-01-01

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization.

PubMed

Ferrer, Marvin; Rodriguez, Hilma; Zara, Lindsay; Yu, Yang; Xu, Wei; Oko, Richard

2012-09-01

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.

Assessment of holocellulose for the production of bioethanol by conserving Pinus radiata cones as renewable feedstock.

PubMed

Victor, Amudhavalli; Pulidindi, Indra Neel; Gedanken, Aharon

2015-10-01

Renewable and green energy sources are much sought. Bioethanol is an environmentally friendly transportation fuel. Pine cones from Pinus radiata were shown to be a potential feedstock for the production of bioethanol. Alkaline (NaOH) pretreatment was carried out to delignify the lignocellulosic material and generate holocellulose (72 wt. % yield). The pretreated biomass was hydrolysed using HCl as catalyst under microwave irradiation and hydrothermal conditions. Microwave irradiation was found to be better than the hydrothermal process. Microwave irradiation accelerated the hydrolysis of biomass (42 wt. % conversion) with the reaction conditions being 3 M HCl and 5 min of irradiation time. Interestingly, even the xylose, which is the major component of the hydrolyzate was found to be metabolized to ethanol using Baker's yeast (Saccharomyces cerevisiae) under the experimental conditions. 5.7 g of ethanol could be produced from 100 g of raw pine cones. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

PubMed

Yao, Junlan; McHedlishvili, David; McIntire, William E; Guagliardo, Nick A; Erisir, Alev; Coburn, Craig A; Santarelli, Vincent P; Bayliss, Douglas A; Barrett, Paula Q

2017-08-01

Ca 2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca 2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K + channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K + channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells. © 2017 American Heart Association, Inc.

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction.

PubMed

Morales, P; Llanos, M; Yovich, J L; Cummins, J M; Vigil, P

1993-01-01

Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml-1 of pentoxifylline at 37 degrees C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 x 10(6) cells ml-1. One hundred microlitres of each sperm suspension was then deposited under oil and 30-40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.

No impact of transgenic nptII-leafy Pinus radiata (Pinales: Pinaceae) on Pseudocoremia suavis (Lepidoptera: Geometridae) or its endoparasitoid Meteorus pulchricornis (Hymenoptera: Braconidae).

PubMed

Burgess, E P J; Barraclough, E I; Kean, A M; Walter, C; Malone, L A

2011-10-01

To investigate the biosafety to insects of transgenic Pinus radiata D. Don containing the antibiotic resistance marker gene nptII and the reproductive control gene leafy, bioassays were conducted with an endemic lepidopteran pest of New Zealand plantation pine forests and a hymenopteran endoparasitoid. Larvae of the common forest looper, Pseudocoremia suavis (Butler), were fed from hatching on P. radiata needles from either one of two nptII-leafy transgenic clones, or an isogenic unmodified control line. For both unparasitized P. suavis and those parasitized by Meteorus pulchricornis (Wesmael), consuming transgenic versus control pine had no impact on larval growth rate or mass at any age, larval duration, survival, pupation or successful emergence as an adult. Total larval duration was 1 d (3%) longer in larvae fed nptII-2 than nptII-1, but this difference was considered trivial and neither differed from the control. In unparasitized P. suavis larvae, pine type consumed did not affect rate of pupation or adult emergence, pupal mass, or pupal duration. Pine type had no effect on the duration or survival of M. pulchricornis larval or pupal stages, mass of cocoons, stage at which they died, adult emergence, or fecundity. Parasitism by M. pulchricornis reduced P. suavis larval growth rate, increased the duration of the third larval stadium, and resulted in the death of all host larvae before pupation. The lack of impact of an exclusive diet of nptII-leafy transgenic pines on the life history of P. suavis and M. pulchricornis suggests that transgenic plantation pines expressing nptII are unlikely to affect insect populations in the field.

FLB1, a human protein of epididymal origin that is involved in the sperm-oocyte recognition process.

PubMed

Boué, F; Duquenne, C; Lassalle, B; Lefèvre, A; Finaz, C

1995-02-01

CA6 antibody was selected out of a monoclonal antibody library raised against human sperm proteins primarily for its ability to recognize an epididymal antigen and to modify sperm adhesion to zona-free hamster oocytes. In the present study, CA6 was shown to decrease sperm binding to zona-free hamster and human oocytes by 40-92% and 38-48%, respectively. The corresponding protein, which was referred to as FLB1, was found to be secreted by the epididymis and to bind specifically to a human, macaque, and rodent subacrosomal sperm region. Western blotting revealed a molecular mass of 94 kDa in human epididymal extracts and of 100 kDa in human, macaque, mouse, rat, and hamster sperm, suggesting further modifications after its binding to sperm. An equivalent protein was not observed in human liver, ovary, testis, plasma, or epidermis. Two-dimensional electrophoresis showed that FLB1 is formed of two subunits with the same 47-kDa molecular mass and slightly different pI (5.8, 5.9). Microsequencing of the protein revealed a partial homology with human cytokeratins 1 and 10. These results suggest that FLB1 is an epididymis-specific cytokeratin-like protein that is involved in the sperm-oocyte recognition process.

Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demura, T.; Driscoll, W.J.; Lee, Y.C.

1991-01-01

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

Viscous forces are predominant in the zona pellucida mechanical resistance

NASA Astrophysics Data System (ADS)

Papi, Massimiliano; Maiorana, Alessandro; Douet, Cécile; Maulucci, Giuseppe; Parasassi, Tiziana; Brunelli, Roberto; Goudet, Ghylène; De Spirito, Marco

2013-01-01

The zona pellucida (ZP) is a multilayer glycoprotein spherical shell surrounding mammalian eggs. The ZP's mechanical response plays a crucial role in mammalian fertilization and is a parameter commonly adopted in "in vitro fertilization" to characterize the oocytes quality. While it is assumed that ZP mechanical response is purely elastic, here we prove that dissipative forces cannot be neglected. Physiologically, this evidence implies that an increase in the spermatozoa motility can induce dramatic changes on the ZP reaction force turning ZP shell in an impenetrable barrier leading to fertility impairments.

SLXL1, a novel acrosomal protein, interacts with DKKL1 and is involved in fertilization in mice.

PubMed

Zhuang, Xin-jie; Hou, Xiao-jun; Liao, Shang-Ying; Wang, Xiu-Xia; Cooke, Howard J; Zhang, Ming; Han, Chunsheng

2011-01-01

Spermatogenesis is a complex cellular developmental process which involves diverse families of genes. The Xlr (X-linked, lymphocyte regulated) family includes multiple members, only a few of which have reported functions in meiosis, post-meiotic maturation, and fertilization of germ cells. Slx-like1 (Slxl1) is a member of the Xlr family, whose expression and function in spermatogenesis need to be elucidated. The mRNA and protein expression and localization of Slxl1 were investigated by RT-PCR, Western blotting and immunohistochemistry in different tissues and at different stages of spermatogenesis. The interacting partner of SLXL1 was examined by co-immunoprecipitation and co-localization. Assessment of the role of SLXL1 in capacitation, acrosome reaction, zona pellucida binding/penetration, and fertilization was carried out in vitro using blocking antisera. The results showed that Slxl1 mRNA and protein were specifically expressed in the testis. SLXL1 was exclusively located in the acrosome of post-meiotic germ cells and interacts with DKKL1 (Dickkopf-like1), which is an acrosome-associated protein and plays an important role in fertilization. The rates of zona pellucida binding/penetration and fertilization were significantly reduced by the anti-SLXL1 polyclonal antiserum. SLXL1 is the first identified member of the XLR family that is associated with acrosome and is involved in zona pellucid binding/penetration and subsequent fertilization. These results, together with previous studies, suggest that Xlr family members participate in diverse processes from meiosis to fertilization during spermatogenesis.

Fine structure and morphogenesis of spironolactone bodies in the zona glomerulosa of the human adrenal cortex

PubMed Central

Kovacs, K.; Horvath, E.; Singer, W.

1973-01-01

Numerous spironolactone bodies have been detected in the zona glomerulosa cells of the adrenal cortex of a 36-year-old spironolactone-treated woman whose non-tumorous right adrenal gland was removed surgically because of primary hyperaldosteronism. Electron microscopy revealed spherical laminated whorls which consisted of a central core composed of an amorphous electron-dense material surrounded by numerous smooth-walled concentric membranes. Continuous with and deriving from the endoplasmic reticulum, they were present in viable cells and were not associated with ultrastructural features indicating cellular injury. Cytoplasmic inclusions similar to spironolactone bodies can be detected in other organs after the administration of various compounds. Thus, they can be regarded as neither specific to spironolactone treatment nor exclusively inducible in the zona glomerulosa of the adrenal cortex. Images PMID:4131694

Cellular modelling of secondary radial growth in conifer trees: application to Pinus radiata (D. Don).

PubMed

Forest, Loïc; Demongeot, Jacques; Demongeota, Jacques

2006-05-01

The radial growth of conifer trees proceeds from the dynamics of a merismatic tissue called vascular cambium or cambium. Cambium is a thin layer of active proliferating cells. The purpose of this paper was to model the main characteristics of cambial activity and its consecutive radial growth. Cell growth is under the control of the auxin hormone indole-3-acetic. The model is composed of a discrete part, which accounts for cellular proliferation, and a continuous part involving the transport of auxin. Cambium is modeled in a two-dimensional cross-section by a cellular automaton that describes the set of all its constitutive cells. Proliferation is defined as growth and division of cambial cells under neighbouring constraints, which can eliminate some cells from the cambium. The cell-growth rate is determined from auxin concentration, calculated with the continuous model. We studied the integration of each elementary cambial cell activity into the global coherent movement of macroscopic morphogenesis. Cases of normal and abnormal growth of Pinus radiata (D. Don) are modelled. Abnormal growth includes deformed trees where gravity influences auxin transport, producing heterogeneous radial growth. Cross-sectional microscopic views are also provided to validate the model's hypothesis and results.

Injury of leg somatotopy of corticospinal tract at corona radiata by ventriculoperitoneal shunt

PubMed Central

Jang, Sung Ho; Kwon, Younghyeon

2018-01-01

Abstract Rationale: A 45-year-old right-handed female patient suffered head trauma after being hit by a truck that ran into a house. Patient concerns: The patient lost consciousness for 1 hour and experienced posttraumatic amnesia for 1 month after the accident. Diagnoses: She underwent conservative management for a subdural hematoma in the left frontotemporal lobes and intracerebral hematoma in the left frontal lobe. Interventions: The patient's Glasgow Coma Scale score was 11. She underwent a VP shunt operation, approached through the right posterior parietal area of the brain, at 4 months after onset. Approximately, 6 months after onset, she was admitted to the rehabilitation department of a university hospital. She presented with moderate weakness of the left leg: Medical Research Council scores: hip flexor; 3, knee extensor; 3+, ankle dorsiflexor; 3–. Brain magnetic resonance imaging revealed a leukomalactic lesion in the right posterior corona radiata along the shunt. Outcomes: On 6-month (2 months after the shunt operation) diffusion tensor tractography, the left CST showed partial injury in the posterior portion compared with the right CST. On 6-month transcranial magnetic stimulation study, the motor-evoked potential obtained at the left tibialis anterior muscle revealed lower amplitude than that on the right side. Lessons: Injury of leg somatotopy of a CST was demonstrated in a patient with leg weakness following a VP shunt operation. PMID:29517704

Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores.

PubMed

Moros-Nicolás, C; Leza, A; Chevret, P; Guillén-Martínez, A; González-Brusi, L; Boué, F; Lopez-Bejar, M; Ballesta, J; Avilés, M; Izquierdo-Rico, M J

2018-01-01

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Copyright 2009 Wiley-Liss, Inc.

Solute accumulation and elastic modulus changes in six radiata pine breeds exposed to drought.

PubMed

De Diego, N; Sampedro, M C; Barrio, R J; Saiz-Fernández, I; Moncaleán, P; Lacuesta, M

2013-01-01

Drought is one of the main abiotic factors that determine forest species growth, survival and productivity. For this reason, knowledge of plant drought response and the identification of physiological traits involved in stress tolerance will be of interest to breeding programs. In this work, several Pinus radiata D. Don breeds from different geographical origins were evaluated along a water stress period (4 weeks) and subsequent rewatering (1 week), showing different responses among them. Leaf water potential (Ψ(leaf)) and osmotic potential decreases were accompanied by a variation in the total relative water content (RWC, %). The most tolerant breeds presented the lowest leaf water potential and RWC at turgor loss point, and showed the lowest elastic modulus (ε) values. A high ε value was a characteristic of a less-drought-tolerant plant and was related to membrane alterations (high electrolyte leakage percentages) that could favor cell water loss. Of the group of solutes that contributed to osmotic adjustment, soluble carbohydrates were the most abundant, although stressed plants also increased their content of free amino acids [mainly proline (Pro) and glutamic acid (Glu), and γ-aminobutyric acid (GABA)] and free polyamines. In addition, the most sensitive breeds had a higher GABA/Glu ratio. After rewatering, Pro and GABA were higher in rehydrated plants than in controls.

Inhibition of endothelin- and phorbol ester-stimulated tyrosine kinase activity by corticotrophin in the rat adrenal zona glomerulosa.

PubMed Central

Kapas, S; Hinson, J P

1996-01-01

1. The experiments described in this study were carried out to investigate the role of tyrosine kinase in the acute adrenal response to peptide hormone stimulation, and to determine whether the activity of this kinase may be subject to regulation by other intracellular signalling mechanisms in the adrenal zona glomerulosa. 2. Previous studies from this laboratory have shown that angiotensin II stimulates tyrosine kinase activity in the rat adrenal cortex. This study has shown, for the first time, that endothelin-1 also stimulates tyrosine kinase activity in this tissue. 3. Using the specific inhibitor of protein kinase C (PKC) activity, Ro 31-8220, we have shown that stimulation of tyrosine kinase activity, in response to endothelin-1, angiotensin II or the phorbol ester phorbol 12-myristate 13-acetate, is at least partly dependent on increased PKC activity. 4. The data presented also provide further evidence of cross-talk between signalling systems in the adrenal cortex. Corticotrophin and its intracellular second messenger, cyclic AMP, significantly attenuate the increment in tyrosine kinase activity seen in response to each of the effectors used. 5. The results of this study provide important new evidence for the regulation of protein kinases by other intracellular second messenger systems. PMID:8611168

Effect of exogenous ATP on the postharvest properties and pectin degradation of mung bean sprouts (Vigna radiata).

PubMed

Chen, Lin; Zhou, Yige; He, Zhenyun; Liu, Qin; Lai, Shaojuan; Yang, Hongshun

2018-06-15

The effects of exogenous ATP on the postharvest quality, browning and softening of mung bean (Vigna radiata) sprouts were evaluated. ATP treatment significantly alleviated the quality loss and browning events during the storage of 3 days. It also reduced the oxidant damage by inducing high activities of peroxidase (9.3-13.9%) and superoxide dismutase (8.8-10.3%) which scavenged the reactive oxygen species (ROS) effectively. Transcriptional results indicated that ATP treatment decreased VrPL1, VrPME and VrPG1 gene expression levels more than 2 folds at some time points. Furthermore, the atomic force microscope (AFM) images revealed that the pectin degradation was notably slowed by ATP treatment and the width and height of pectin backbone were better maintained (47.1% and 45.6% higher than control without ATP treatment). The cooperative effects of ROS scavenging and decreased expressions of pectin-related genes might contribute to the deferred pectin deterioration and firmness loss by ATP treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptome profiling of radiata pine branches reveals new insights into reaction wood formation with implications in plant gravitropism.

PubMed

Li, Xinguo; Yang, Xiaohui; Wu, Harry X

2013-11-08

Formation of compression (CW) and opposite wood (OW) in branches and bent trunks is an adaptive feature of conifer trees in response to various displacement forces, such as gravity, wind, snow and artificial bending. Several previous studies have characterized tracheids, wood and gene transcription in artificially or naturally bent conifer trunks. These studies have provided molecular basis of reaction wood formation in response to bending forces and gravity stimulus. However, little is known about reaction wood formation and gene transcription in conifer branches under gravity stress. In this study SilviScan® technology was used to characterize tracheid and wood traits in radiate pine (Pinus radiata D. Don) branches and genes differentially transcribed in CW and OW were investigated using cDNA microarrays. CW drastically differed from OW in tracheids and wood traits with increased growth, thicker tracheid walls, larger microfibril angle (MFA), higher density and lower stiffness. However, CW and OW tracheids had similar diameters in either radial or tangential direction. Thus, gravity stress largely influenced wood growth, secondary wall deposition, cellulose microfibril orientation and wood properties, but had little impact on primary wall expansion. Microarray gene transcription revealed about 29% of the xylem transcriptomes were significantly altered in CW and OW sampled in both spring and autumn, providing molecular evidence for the drastic variation in tracheid and wood traits. Genes involved in cell division, cellulose biosynthesis, lignin deposition, and microtubules were mostly up-regulated in CW, conferring its greater growth, thicker tracheid walls, higher density, larger MFA and lower stiffness. However, genes with roles in cell expansion and primary wall formation were differentially transcribed in CW and OW, respectively, implicating their similar diameters of tracheid walls and different tracheid lengths. Interestingly, many genes related to hormone

Antidiabetic, antioxidant and anti inflammatory properties of water and n-butanol soluble extracts from Saharian Anvillea radiata in high-fat-diet fed mice.

PubMed

Kandouli, Chouaib; Cassien, Mathieu; Mercier, Anne; Delehedde, Caroline; Ricquebourg, Emilie; Stocker, Pierre; Mekaouche, Mourad; Leulmi, Zineb; Mechakra, Aicha; Thétiot-Laurent, Sophie; Culcasi, Marcel; Pietri, Sylvia

2017-07-31

According to Saharian traditional medicine, Anvillea radiata Coss. & Dur. (Asteraceae) has been valued for treating a variety of ailments such as gastro-intestinal, liver and pulmonary diseases, and has gained awareness for its beneficial effect on postprandial hyperglycemia. However, to best of our knowledge, no detailed study of the antidiabetic curative effects of this plant has been conducted yet. To determine the hypoglycemic and antidiabetic effect of dietary supplementation with Anvillea radiata extracts on high-fat-diet (HFD)-induced obesity and insulin resistance in C57BL/6J mice in relation with antioxidant, anti-inflammatory, pancreatic beta-cells and skeletal muscle protection, and digestive enzyme inhibiting properties. Six extracts (water soluble and organic) from aerial parts of the plant were analyzed phytochemically (total phenolic and flavonoid content) and screened for in vitro superoxide (by chemiluminescence) and hydroxyl radical (by electron paramagnetic resonance spin-trapping) scavenging, antioxidant (DPPH, TRAP and ORAC assays), xanthine oxidase, metal chelating, α-amylase and α-glucosidase inhibitory property, and protective effects on copper-induced lipoprotein oxidation. Then selected hydroalcoholic and aqueous extracts were assessed for toxicity in normal human lung fibroblasts and A549 cancer cells using FMCA and MTT assays. Two water-soluble extracts having the best overall properties were assessed for their (i) protective effect at 1-15µg/mL on metabolic activity of rat insulinoma-derived INS-1 cells exposed to hyperglycemic medium, and (ii) acute hypoglycemic effect on 16-weeks HFD-induced diabetic mice. Then diabetic mice were administered HFD supplemented by extracts (up to 150mg/kg/day) for 12 additional weeks using standard diet as control and the antidiabetic drug, metformin (150mg/kg), as positive control. Then the antidiabetic, anti-inflammatory and antioxidant activity of extracts were determined. Of the highly efficient

Protein Mobilization in Germinating Mung Bean Seeds Involves Vacuolar Sorting Receptors and Multivesicular Bodies1[W][OA

PubMed Central

Wang, Junqi; Li, Yubing; Lo, Sze Wan; Hillmer, Stefan; Sun, Samuel S.M.; Robinson, David G.; Jiang, Liwen

2007-01-01

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination. PMID:17322331

Connectivity of the habitat-forming kelp, Ecklonia radiata within and among estuaries and open coast.

PubMed

Coleman, Melinda A

2013-01-01

With marine protected areas being established worldwide there is a pressing need to understand how the physical setting in which these areas are placed influences patterns of dispersal and connectivity of important marine organisms. This is particularly critical for dynamic and complex nearshore marine environments where patterns of genetic structure of organisms are often chaotic and uncoupled from broad scale physical processes. This study determines the influence of habitat heterogeneity (presence of estuaries) on patterns of genetic structure and connectivity of the common kelp, Ecklonia radiata. There was no genetic differentiation of kelp between estuaries and the open coast and the presence of estuaries did not increase genetic differentiation among open coast populations. Similarly, there were no differences in level of inbreeding or genetic diversity between estuarine and open coast populations. The presence of large estuaries along rocky coastlines does not appear to influence genetic structure of this kelp and factors other than physical heterogeneity of habitat are likely more important determinants of regional connectivity. Marine reserves are currently lacking in this bioregion and may be designated in the future. Knowledge of the factors that influence important habitat forming organisms such as kelp contribute to informed and effective marine protected area design and conservation initiatives to maintain resilience of important marine habitats.

The AVRDC - The World Vegetable Center mungbean (Vigna radiata) core and mini core collections.

PubMed

Schafleitner, Roland; Nair, Ramakrishnan Madhavan; Rathore, Abhishek; Wang, Yen-wei; Lin, Chen-yu; Chu, Shu-hui; Lin, Pin-yun; Chang, Jian-Cheng; Ebert, Andreas W

2015-04-29

Large ex situ germplasm collections generally harbor a wide range of crop diversity. AVRDC--The World Vegetable Center is holding in trust the world's second largest mungbean (Vigna radiata) germplasm collection with more than 6,700 accessions. Screening large collections for traits of interest is laborious and expensive. To enhance the access of breeders to the diversity of the crop, mungbean core and mini core collections have been established. The core collection of 1,481 entries has been built by random selection of 20% of the accessions after geographical stratification and subsequent cluster analysis of eight phenotypic descriptors in the whole collection. Summary statistics, especially the low differences of means, equal variance of the traits in both the whole and core collection and the visual inspection of quantile-quantile plots comparing the variation of phenotypic traits present in both collections indicated that the core collection well represented the pattern of diversity of the whole collection. The core collection was genotyped with 20 simple sequence repeat markers and a mini core set of 289 accessions was selected, which depicted the allele and genotype diversity of the core collection. The mungbean core and mini core collections plus their phenotypic and genotypic data are available for distribution to breeders. It is expected that these collections will enhance the access to biodiverse mungbean germplasm for breeding.

Time-lapse monitoring of zona pellucida-free embryos obtained through in vitro fertilization: a retrospective case series.

PubMed

Bodri, Daniel; Kato, Ryutaro; Kondo, Masae; Hosomi, Naoko; Katsumata, Yoshinari; Kawachiya, Satoshi; Matsumoto, Tsunekazu

2015-05-01

To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators. Retrospective case series. Private infertility clinic. Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014. Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle. In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns. Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Response of green gram (Vigna radiata L.) to an application of Minjingu Mazao fertilizer grown on Olasiti soils from Minjingu-Manyara, Tanzania.

PubMed

Kisetu, Eliakira; Teveli, Christina Ngomzee Medutieki

2013-11-15

A screen-house pot experiment was conducted to assess the response of green gram (Vigna radiata L.) to the application of Minjingu Mazao fertilizer (31% P2O5) on Olasiti soil, Manyara Region-Tanzania. This study was prompted by very low or limited use of Minjingu Mazao fertilizer by smallholder farmers in the country while yields turnout of most crops, green gram inclusive, is not promising. The soil was clay with medium pH (pH 5.5-7.0) and neutral reaction (pH 6.6-7.3). The results showed that the number of pods and seeds increased from 3-6 and 7-9, respectively, at 40 to 160 mg per 4 kg soil of fertilizer applied. Similarly, the tissue N and P increased with treatment levels. The number of pods per plant and seeds per pod showed similar increase, signifying the role of these nutrients in protein synthesis in leguminous plants like green gram. Soil properties could be the spearhead to low responses obtained at low (< 80 mg per 4 kg soil) and high (> 320 mg per 4 kg soil) rates of Minjingu Mazao fertilizer applied. It was concluded that to optimize green gram production in Olasiti soil, Minjingu Mazao fertilizer containing 31% P2O5 should be applied at a rate of 160-320 kg ha(-1) while considering other necessary agronomic practices. However, field studies to confirm the findings of this study and verify the usefulness of this fertilizer brand to green gram in Olasiti soil under field conditions could practically be the viable option before its recommendation to the smallholder farmers.

Effect of gamma and e-beam radiation on the essential oils of Thymus vulgaris thymoliferum, Eucalyptus radiata, and Lavandula angustifolia.

PubMed

Haddad, Mohamed; Herent, Marie-France; Tilquin, Bernard; Quetin-Leclercq, Joëlle

2007-07-25

The microbiological contamination of raw plant materials is common and may be adequately reduced by radiation processing. This study evaluated the effects of gamma- and e-beam ionizing radiations (25 kGy) on three plants used as food or as medicinal products (Thymus vulgaris L., Eucalyptus radiata D.C., and Lavandula angustifolia Mill.) as well as their effects on extracted or commercial essential oils and pure standard samples. Comparison between irradiated and nonirradiated samples was performed by GC/FID and GC/MS. At the studied doses, gamma and e-beam ionizing radiation did not induce any detectable qualitative or quantitative significant changes in the contents and yields of essential oils immediately after ionizing radiation of plants or commercial essential oils and standards. As the maximum dose tested (25 kGy) is a sterilizing dose (much higher than doses used for decontamination of vegetable drugs), it is likely that even decontamination with lower doses will not modify yields or composition of essential oils of these three plants.

The mitochondrial genome of the legume Vigna radiata and the analysis of recombination across short mitochondrial repeats.

PubMed

Alverson, Andrew J; Zhuo, Shi; Rice, Danny W; Sloan, Daniel B; Palmer, Jeffrey D

2011-01-20

The mitochondrial genomes of seed plants are exceptionally fluid in size, structure, and sequence content, with the accumulation and activity of repetitive sequences underlying much of this variation. We report the first fully sequenced mitochondrial genome of a legume, Vigna radiata (mung bean), and show that despite its unexceptional size (401,262 nt), the genome is unusually depauperate in repetitive DNA and "promiscuous" sequences from the chloroplast and nuclear genomes. Although Vigna lacks the large, recombinationally active repeats typical of most other seed plants, a PCR survey of its modest repertoire of short (38-297 nt) repeats nevertheless revealed evidence for recombination across all of them. A set of novel control assays showed, however, that these results could instead reflect, in part or entirely, artifacts of PCR-mediated recombination. Consequently, we recommend that other methods, especially high-depth genome sequencing, be used instead of PCR to infer patterns of plant mitochondrial recombination. The average-sized but repeat- and feature-poor mitochondrial genome of Vigna makes it ever more difficult to generalize about the factors shaping the size and sequence content of plant mitochondrial genomes.

[Effects of infrasound on activities of 3beta hydroxysteroid dehydrogenase and acid phosphatase of polygonal cells in adrenal cortex zona fasciculate in mice].

PubMed

Dang, Wei-min; Wang, Sheng; Tian, Shi-xiu; Chen, Bing; Sun, Fei; Li, Wei; Jiao, Yan; He, Li-hua

2007-02-01

To explore the biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation in mice. The biological effects of infrasound on the activities of 3beta hydroxysteroid dehydrogenase (3-betaHSDH) and acid phosphatase(ACP) of the polygonal cells in adrenal cortex zona fasciculate were observed when exposure to 8 and 16 Hz infrasound at 80, 90, 100, 110, 120 and 130 dB for 1 day, 7 days and 14 days or 14 days after the exposure. When exposure to 8 Hz infrasound, the enzyme activities of 3-betaHSDH increase as the sound pressure levels increase. Only when the sound pressure levels reach 130 dB, the enzyme activities began to decrease exceptionally. When exposure to 16 Hz, 80 dB infrasound, no significant difference between the treatment and control group in the activities of 3-betaHSDH could be observed, but the injury of the polygonal cells had appeared. When exposure to 16 Hz, 100 dB infrasound, the activities of 3-betaHSDH started to increase. The cell injury still existed. When exposed to 16 Hz, 120 dB infrasound, the local tissue damage represented. Fourteen days after the mice exposure to 8 Hz, 90 dB and 130 dB infrasound for 14 days continuously, the local tissue injury of the adrenal cortex zona fasciculation began to recover at certain extent, but the higher the exposure sound pressure level, the poorer the tissue recovery. The biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation response to the frequency of the infrasound are found at certain action strength range, but this characteristic usually is covered by the severe tissue injury. When exposure to infrasound is stopped for a period of time, the local tissue injury of the adrenal cortex zona fasciculation could recovers at certain extent, but the higher the exposure sound pressure level, the more poorer the tissue recovery.

New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis

PubMed Central

Tingaud-Sequeira, Angèle; Chauvigné, François; Lozano, Juanjo; Agulleiro, María J; Asensio, Esther; Cerdà, Joan

2009-01-01

Background The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. Results Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

PubMed Central

Patrat, Catherine; Auer, Jana; Fauque, Patricia; Leandri, Roger L; Jouannet, Pierre; Serres, Catherine

2006-01-01

Background The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes. Results The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. Conclusion The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans. PMID:17147816

Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles.

PubMed

Shi, Wei; Xu, Bo; Wu, Li-Min; Jin, Ren-Tao; Luan, Hong-Bing; Luo, Li-Hua; Zhu, Qing; Johansson, Lars; Liu, Yu-Sheng; Tong, Xian-Hong

2014-01-01

The morphological assessment of oocytes is important for embryologists to identify and select MII oocytes in IVF/ICSI cycles. Dysmorphism of oocytes decreases viability and the developmental potential of oocytes as well as the clinical pregnancy rate. Several reports have suggested that oocytes with a dark zona pellucida (DZP) correlate with the outcome of IVF treatment. However, the effect of DZP on oocyte quality, fertilization, implantation, and pregnancy outcome were not investigated in detail. In this study, a retrospective analysis was performed in 268 infertile patients with fallopian tube obstruction and/or male factor infertility. In 204 of these patients, all oocytes were surrounded by a normal zona pellucida (NZP, control group), whereas 46 patients were found to have part of their retrieved oocytes enclosed by NZP and the other by DZP (Group A). In addition, all oocytes enclosed by DZP were retrieved from 18 patients (Group B). No differences were detected between the control and group A. Compared to the control group, the rates of fertilization, good quality embryos, implantation and clinical pregnancy were significantly decreased in group B. Furthermore, mitochondria in oocytes with a DZP in both of the two study groups (A and B) were severely damaged with several ultrastructural alterations, which were associated with an increased density of the zona pellucida and vacuolization. Briefly, oocytes with a DZP affected the clinical outcome in IVF/ICSI cycles and appeared to contain more ultrastructural alterations. Thus, DZP could be used as a potential selective marker for embryologists during daily laboratory work.

Surface alterations of the mouse zona pellucida and ovum following in vivo fertilization: correlation with the cell cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackowski, S.; Dumont, J.N.

1979-01-01

The zona pellucida and cell surface of in vivo fertilized mouse ova exhibit time dependent changes which can be detected with the scanning electron microscope. The periods of ovulation, fertilization and first cleavage in superovulated C3D2/F/sub 1/ hybrids were determined and times corresponding to G/sub 1/, S, G/sub 2/, and M were calculated. The zona of a mature unfertilized ovum has a rough texture with deep furrows; at fertilization and thereafter the zona develops a smoother, ropy and seemingly porous surface. The cell surface of the unfertilized ovum is characterized by uniform microvilli, small blebs and rounded, mound-like elevations. Aftermore » fertilization and development to G/sub 1/, the ovum loses its blebs but retains the mound-like elevations and microvilli which are now less uniform. As the ovum progresses toward S, it loses the mound-like elevations but retains microvilli in the same density as found in G/sub 1/. The ovum in G/sub 2/ exhibits smaller but more numerous microvilli which vary considerably in length. Some appear to bifurcate. The fertilized ovum developing through M and G/sub 1/ of the 2 cell stage exhibits a less dense population of relatively uniform microvilli, periodic blebs and, again, rounded elevations. The data are reminiscent of surface changes associated with the cell cycle in tissue culture cells and indicate a cyclic progression of the in vivo fertilized mouse ovum through the first cleavage division to the 2 cell stage.« less

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

PubMed

Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

2015-03-08

Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Cotton Stalk Pretreatment Using Daedalea flavida, Phlebia radiata, and Flavodon flavus: Lignin Degradation, Cellulose Recovery, and Enzymatic Saccharification.

PubMed

Meehnian, Harmanpreet; Jana, Asim K

2017-04-01

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.

Hydroxyl radical production by a heterogeneous Fenton reaction supported in insoluble tannin from bark of Pinus radiata.

PubMed

Romero, Romina; Contreras, David; Segura, Cristina; Schwederski, Brigitte; Kaim, Wolfgang

2017-03-01

Fenton reactions driven by dihydroxybenzenes (DHBs) have been used for pollutant removal via advanced oxidation processes (AOPs), but such systems have the disadvantage of DHB release into the aqueous phase. In this work, insoluble tannins from bark can be used to drive Fenton reactions and as a heterogeneous support. This avoids the release of DHBs into the aqueous phase and can be used for AOPs. The production of ·OH was investigated using a spin-trapping electron paramagnetic resonance technique (5-dimethyl-1-pyrroline-N-oxide/·OH) in the first minute of the reaction and a high-performance liquid chromatography-fluorescence technique (coumarin/7-hydroxycoumarin) for 20 min. The ·OH yield achieved using insoluble tannins from Pinus radiata bark was higher than that achieved using catechin to drive the Fenton reaction. The Fenton-like system driven by insoluble tannins achieved 92.6 ± 0.3 % degradation of atrazine in 30 min. The degradation kinetics of atrazine was linearly correlated with ·OH production. The increased reactivity in ·OH production and insolubility of the ligand are promising for the development of a new technique for degradation of pollutants in wastewater using heterogeneous Fenton systems.

Injury of leg somatotopy of corticospinal tract at corona radiata by ventriculoperitoneal shunt: A case report.

PubMed

Jang, Sung Ho; Kwon, Younghyeon

2018-03-01

A 45-year-old right-handed female patient suffered head trauma after being hit by a truck that ran into a house. The patient lost consciousness for 1 hour and experienced posttraumatic amnesia for 1 month after the accident. She underwent conservative management for a subdural hematoma in the left frontotemporal lobes and intracerebral hematoma in the left frontal lobe. The patient's Glasgow Coma Scale score was 11. She underwent a VP shunt operation, approached through the right posterior parietal area of the brain, at 4 months after onset. Approximately, 6 months after onset, she was admitted to the rehabilitation department of a university hospital. She presented with moderate weakness of the left leg: Medical Research Council scores: hip flexor; 3, knee extensor; 3+, ankle dorsiflexor; 3-. Brain magnetic resonance imaging revealed a leukomalactic lesion in the right posterior corona radiata along the shunt. On 6-month (2 months after the shunt operation) diffusion tensor tractography, the left CST showed partial injury in the posterior portion compared with the right CST. On 6-month transcranial magnetic stimulation study, the motor-evoked potential obtained at the left tibialis anterior muscle revealed lower amplitude than that on the right side. Injury of leg somatotopy of a CST was demonstrated in a patient with leg weakness following a VP shunt operation.

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

PubMed

Morales, P; Vantman, D; Barros, C; Vigil, P

1991-03-01

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

PubMed

Wong, Christopher Yee; Mills, James K

2017-03-01

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation.

PubMed

Gadella, B M

2012-01-01

Recent findings have refined our thinking on sperm interactions with the cumulus-oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona-cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm-ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer, Zona Pellucida, and Perivitelline Space

PubMed Central

Liu, Ying-Lei; Chen, Ying; Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Liang, Cheng-Guang

2014-01-01

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications. PMID:25144310

Seasonal Terpene Variation in Needles of Pinus radiata (Pinales: Pinaceae) Trees Attacked by Tomicus piniperda (Coleoptera: Scolytinae) and the Effect of Limonene on Beetle Aggregation

PubMed Central

Romón, Pedro; Aparicio, Domitila; Palacios, Francisco; Iturrondobeitia, Juan Carlos; Hance, Thierry

2017-01-01

Abstract Concentrations of four monoterpenes were determined in needles of Pinus radiata (D.Don) (Pinales: Pinaceae) trees that were attacked or nonattacked by Tomicus piniperda (L.) (Coleoptera: Scolytinae). Compounds were identified and quantified by gas chromatography–mass spectrometry. The mean ambient temperature was obtained using climate-recording data loggers. The effect of limonene on field aggregation was also evaluated at three limonene release rates using Lindgren attractant-baited traps and trap logs. Attacked trees produced less α-pinene in March, July, and November than nonattacked trees, less β-pinene in July and November, and less limonene from May to November. Limonene reduced the attraction of T. piniperda to attractant-baited traps and trap logs. Results were linked to better responses to high temperatures, with respect to terpene contents, by the nonattacked trees after the spring attack. PMID:29117373

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

PubMed

Cowan, A E; Myles, D G; Koppel, D E

1991-03-01

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Mobile phone radiation inhibits Vigna radiata (mung bean) root growth by inducing oxidative stress.

PubMed

Sharma, Ved Parkash; Singh, Harminder Pal; Kohli, Ravinder Kumar; Batish, Daizy Rani

2009-10-15

During the last couple of decades, there has been a tremendous increase in the use of cell phones. It has significantly added to the rapidly increasing EMF smog, an unprecedented type of pollution consisting of radiation in the environment, thereby prompting the scientists to study the effects on humans. However, not many studies have been conducted to explore the effects of cell phone EMFr on growth and biochemical changes in plants. We investigated whether EMFr from cell phones inhibit growth of Vigna radiata (mung bean) through induction of conventional stress responses. Effects of cell phone EMFr (power density: 8.55 microW cm(-2); 900 MHz band width; for 1/2, 1, 2, and 4 h) were determined by measuring the generation of reactive oxygen species (ROS) in terms of malondialdehyde and hydrogen peroxide (H(2)O(2)) content, root oxidizability and changes in levels of antioxidant enzymes. Our results showed that cell phone EMFr significantly inhibited the germination (at > or =2 h), and radicle and plumule growths (> or =1 h) in mung bean in a time-dependent manner. Further, cell phone EMFr enhanced MDA content (indicating lipid peroxidation), and increased H(2)O(2) accumulation and root oxidizability in mung bean roots, thereby inducing oxidative stress and cellular damage. In response to EMFr, there was a significant upregulation in the activities of scavenging enzymes, such as superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases, catalases and glutathione reductases, in mung bean roots. The study concluded that cell phone EMFr inhibit root growth of mung bean by inducing ROS-generated oxidative stress despite increased activities of antioxidant enzymes.

Effects of biochar on enhanced nutrient use efficiency of green bean, Vigna radiata L.

PubMed

Prapagdee, Songkrit; Tawinteung, Nukoon

2017-04-01

Biochar is the carbonized material produced from biomass and is used in several environmental applications. The biochar characteristics depend on the carbonization conditions and feedstock. The suitability of a given biochar for soil improvement depends on the biochar characteristics, soil properties, and target plants. Biochar has been applied at 1-20% (w/w) in the soil, but currently there is a lack of information on what type and concentration of biochar are most suitable for a specific plant and soil quality. Too much biochar will reduce plant growth because of the high alkalinity of biochar, which will cause long-term soil alkalinity. In contrast, too little biochar might be insufficient to enhance plant productivity. In this study, a suitable concentration of cassava stem (an abundant agricultural waste in Thailand) biochar produced at 350 °C was evaluated for green bean (Vigna radiata L.) growth from germination to seed production in pots over 8 weeks. The soil fertility was increased with increasing biochar concentration. At 5% (w/w) biochar, the soil fertility and plant growth were significantly enhanced, while 10% (w/w) biochar significantly enhanced bean growth and bean pod production. The increased biochar concentration in the soil significantly increased the soil total nitrogen and extractable potassium (K) levels but did not affect the amount of available phosphorous. Biochar at 10% (w/w) significantly induced the accumulation of K in the stems, leaves, nut shells, and roots but not in nut seeds. Moreover, biochar not only increased the K concentration in soil but also increased the plant nutrient use efficiency of K, which is important for plant growth. Graphical abstract ᅟ.

Incentivos para atraer y retener personal de salud de zonas rurales del Perú: un estudio cualitativo

PubMed Central

Huicho, Luis; Canseco, Francisco Díez; Lema, Claudia; Miranda, J. Jaime; Lescano, Andrés G.

2014-01-01

El objetivo fue identificar incentivos de atracción y retención en zonas rurales y distantes de Ayacucho, Perú. Fueron realizadas entrevistas en profundidad con 80 médicos, enfermeras, obstetras y técnicos (20 por grupo) de las zonas más pobres y con 11 funcionarios. No existen políticas sistemáticas de atracción y retención de personal de salud en Ayacucho. Los principales incentivos, en orden de importancia, fueron mejoras salariales, oportunidades de formación y capacitación, estabilidad laboral y nombramiento, mejoras en infraestructura y equipos, e incremento del personal. Se mencionaron también mejoras en la vivienda y alimentación, mayor cercanía con la familia y reconocimiento por el sistema de salud. Existen coincidencias y singularidades entre los distintos grupos sobre los incentivos clave para estimular el trabajo rural, que deben considerarse al diseñar políticas públicas. Las iniciativas del Estado deben comprender procesos rigurosos de monitoreo y evaluación, para asegurar que las mismas tengan el impacto deseado. PMID:22488318

Studies on the management of root-knot nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease complex of green gram, Vigna radiata cv ML-1108

PubMed Central

Haseeb, Akhtar; Sharma, Anita; Shukla, Prabhat Kuma

2005-01-01

Studies were conducted under pot conditions to determine the comparative efficacy of carbofuran at 1 mg a.i./kg soil, bavistin at 1 mg a.i./kg soil, neem (Azadirachta indica) seed powder at 50 mg/kg soil, green mould (Trichoderma harzianum) at 50.0 ml/kg soil, rhizobacteria (Pseudomonas fluorescens) at 50.0 ml/kg soil against root-knot nematode, Meloidogyne incognita–wilt fungus, Fusarium oxysporum disease complex on green gram, Vigna radiata cv ML-1108. All the treatments significantly improved the growth of the plants as compared to untreated inoculated plants. Analysis of data showed that carbofuran and A. indica seed powder increased plant growth and yield significantly more in comparison to bavistin and P. fluorescens. Carbofuran was highly effective against nematode, bavistin against fungus, A. indica seed powder against both the pathogens and both the bioagents were moderately effective against both the pathogens. PMID:16052706

Effect of soaking and fermentation on content of phenolic compounds of soybean (Glycine max cv. Merit) and mung beans (Vigna radiata [L] Wilczek).

PubMed

María Landete, José; Hernández, Teresa; Robredo, Sergio; Dueñas, Montserrat; de Las Rivas, Blanca; Estrella, Isabel; Muñoz, Rosario

2015-03-01

Mung beans (Vigna radiata [L] Wilczek) purchased from a Spanish company as "green soybeans", showed a different phenolic composition than yellow soybeans (Glycine max cv. Merit). Isoflavones were predominant in yellow soybeans, whereas they were completely absent in the green seeds on which flavanones were predominant. In order to enhance their health benefits, both types of bean were subjected to technological processes, such as soaking and fermentation. Soaking increased malonyl glucoside isoflavone extraction in yellow beans and produced an increase in apigenin derivatives in the green beans. Lactobacillus plantarum CECT 748 T fermentation produced an increase in the bioactivity of both beans since a conversion of glycosylated isoflavones into bioactive aglycones and an increase of the bioactive vitexin was observed in yellow and green beans, respectively. In spite of potential consumer confusion, since soybean and "green soybean" are different legumes, the health benefits of both beans were enhanced by lactic fermentation.

An assessment of interspecific competition between two introduced parasitoids of Diaphorina citri (Hemiptera: Liviidae) on caged citrus plants.

PubMed

Vankosky, Meghan A; Hoddle, Mark S

2017-06-07

Two parasitoids attacking nymphs of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) (Hymenoptera: Encyrtidae) are being released in California, USA in a classical biological control program. To evaluate the effect of multiple parasitoid species on D. citri mortality, we conducted mesocosm experiments under controlled conditions using a complete block design with 6 treatments (D. citri nymphs exposed to: no parasitoids; D. aligarhensis or T. radiata alone; D. aligarhensis or T. radiata released first (by 48 h); and both species released simultaneously). Parasitism of D. citri nymphs by T. radiata exceeded 60% and was unchanged when D. aligarhensis were present. Parasitism by D. aligarhensis was greatest when T. radiata was absent (∼28%) and was reduced in all treatments with T. radiata present (<3%). D. citri mortality and parasitoid-related mortality of D. citri was consistent across parasitoid treatments. Laboratory results suggest that competition between D. aligarhensis and T. radiata is asymmetric and favors T. radiata. It may be difficult for D. aligarhensis to contribute significantly to D. citri biological control where T. radiata is present. However, results reported here suggest that competition between T. radiata and D. aligarhensis is not likely to reduce parasitism by T. radiata or reduce parasitoid-induced mortality of D. citri. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Toxicological features of maleilated polyflavonoids from Pinus radiata (D. Don.) as potential functional additives for biomaterials design.

PubMed

García, Danny E; Medina, Paulina A; Zúñiga, Valentina I

2017-11-01

Polyflavonoids from Pinus radiata (D. Don.) are an abundant natural oligomers highly desirable as renewable chemicals. However, structural modification of polyflavonoids is a viable strategy in order to use such polyphenols as macrobuilding-blocks for biomaterial design. Polyflavonoids were esterified with three five-member cyclic anhydrides (maleic, itaconic, and citraconic) at 20 °C during 24 h in order to diversify physicochemical-, and biological-properties for agricultural, and food-packaging applications. In addition, the influence of the chemical modification, as well as the chemical structure of the grafting on toxicological features was evaluated. Structural features of derivatives were analyzed by spectroscopy (FT-IR and 1 H-NMR), and the degree of substitution was calculated. Toxicological profile was assessed by using three target species in a wide range of concentration (0.01-100 mgL - 1 ). Effect of polyflavonoids on the growth rate (Selenastrum capricornutum), mortality (Daphnia magna), and germination and radicle length (Lactuca sativa) was determined. Chemical modification affects the toxicological profile on the derivatives in a high extent. Results described remarkable differences in function of the target specie. The bioassays indicate differences of the polyflavonoids toxicological profile associated to the chemical structure of the grafting. Results allowed conclude that polyflavonoids from pine bark show slight toxic properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa.

PubMed

Caballero, I; Vazquez, J M; García, E M; Parrilla, I; Roca, J; Calvete, J J; Sanz, L; Martínez, E A

2008-11-01

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.

Potential toxicological hazard due to endocrine-disrupting chemicals on Mediterranean top predators: State of art, gender differences and methodological tools

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fossi, M.C.; Casini, S.; Marsili, L.

Man-made endocrine-disrupting chemicals (EDCs) range across all continents and oceans. Some geographic areas are potentially more threatened than others: one of these is the Mediterranean Sea. Levels of some xenobiotics are much higher here than in other seas and oceans. In this paper we review the final results of a project supported by the Italian Ministry of the Environment, in which the hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs was investigated. We illustrate the need to develop and apply sensitive methodological tools, such as biomarkers (Vitellogenin,more » Zona Radiata proteins and CYP1A activities) for evaluation of toxicological risk in large pelagic fish top predators (Swordfish (Xiphias gladius), Bluefin Tuna (Thunnus thynnus thynnus)) and nondestructive biomarkers (CYP1A activities and fibroblast cell culture in skin biopsy), for the hazard assessment of threatened marine mammals species (Striped Dolphin, (Stenella coeruleoalba), Bottlenose Dolphin (Tursiops truncatus), Common Dolphin (Delphinus delphis) and Fin Whale (Balaenoptera physalus))exposed to EDCs. Differential gender susceptibility to EDCs is also explored both in large pelagic fish and in cetaceans. In cetaceans, male specimens showed higher cytochrome P450 induction (BPMO in skyn biopsies, CYP2B in fibroblasts cell cultures) by xenobiotics with respect to females.« less

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

PubMed

Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

2005-08-01

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

Pitch Variability in Patients with Parkinson's Disease: Effects of Deep Brain Stimulation of Caudal Zona Incerta and Subthalamic Nucleus

ERIC Educational Resources Information Center

Karlsson, Fredrik; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; van Doorn, Jan

2013-01-01

Purpose: The purpose of the present study was to examine the effect of deep brain stimulation (DBS) of the subthalamic nucleus (STN) and the caudal zona incerta (cZi) pitch characteristics of connected speech in patients with Parkinson's disease (PD). Method: The authors evaluated 16 patients preoperatively and 12 months after DBS surgery. Eight…

Seasonal Terpene Variation in Needles of Pinus radiata (Pinales: Pinaceae) Trees Attacked by Tomicus piniperda (Coleoptera: Scolytinae) and the Effect of Limonene on Beetle Aggregation.

PubMed

Romón, Pedro; Aparicio, Domitila; Palacios, Francisco; Iturrondobeitia, Juan Carlos; Hance, Thierry; Goldarazena, Arturo

2017-09-01

Concentrations of four monoterpenes were determined in needles of Pinus radiata (D.Don) (Pinales: Pinaceae) trees that were attacked or nonattacked by Tomicus piniperda (L.) (Coleoptera: Scolytinae). Compounds were identified and quantified by gas chromatography-mass spectrometry. The mean ambient temperature was obtained using climate-recording data loggers. The effect of limonene on field aggregation was also evaluated at three limonene release rates using Lindgren attractant-baited traps and trap logs. Attacked trees produced less α-pinene in March, July, and November than nonattacked trees, less β-pinene in July and November, and less limonene from May to November. Limonene reduced the attraction of T. piniperda to attractant-baited traps and trap logs. Results were linked to better responses to high temperatures, with respect to terpene contents, by the nonattacked trees after the spring attack. © The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata).

PubMed

Ravindranath, N; Ramesh, V; Krishnamurthy, H N; Rao, A J; Moudgal, R N

1992-03-01

To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Proven fertile male monkeys exhibiting normal testicular function. Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50 micrograms buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata.

PubMed

Jiang, Yumei; Xia, Bing; Liang, Lijian; Li, Xiaodan; Xu, Sheng; Peng, Feng; Wang, Ren

2013-03-01

Phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid biosynthesis, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds. In this study, we cloned a 2,326 bp full-length PAL2 gene from Lycoris radiata by using degenerate oligonucleotide primer PCR (DOP-PCR) and the rapid amplification of cDNA ends method. The cDNA contains a 2,124 bp coding region encoding 707 amino acids. The LrPAL2 shares about 77.0 % nucleic acid identity and 83 % amino acid identity with LrPAL1. Furthermore, genome sequence analysis demonstrated that LrPAL2 gene contains one intron and two exons. The 5' flanking sequence of LrPAL2 was also cloned by self-formed adaptor PCR (SEFA-PCR), and a group of putative cis-acting elements such as TATA box, CAAT box, G box, TC-rich repeats, CGTCA motif and TCA-element were identified. The LrPAL2 was detected in all tissues examined, with high abundance in bulbs at leaf sprouting stage and in petals at blooming stage. Besides, LrPAL2 drastically responded to MJ, SNP and UV, moderately responded to GA and SA, and a little increased under wounding. Comparison of LrPAL2 expression and LrPAL1 expression demonstrated that LrPAL2 can be more significantly induced than LrPAL1 under the above treatments, and LrPAL2 transcripts accumulated prominently at blooming stage, especially in petals, while LrPAL1 transcripts did not accumulated significantly at blooming stage. All these results suggested that LrPAL2 might play distinct roles in different branches of the phenylpropanoid pathway.

Superoxide and its metabolism during germination and axis growth of Vigna radiata (L.) Wilczek seeds

PubMed Central

Singh, Khangembam Lenin; Chaudhuri, Abira; Kar, Rup Kumar

2014-01-01

Involvement of reactive oxygen species in regulation of plant growth and development is recently being demonstrated with various results depending on the experimental system and plant species. Role of superoxide and its metabolism in germination and axis growth was investigated in case of Vigna radiata seeds, a non-endospermous leguminous species having epigeal germination, by studying the effect of different reactive oxygen species (ROS) inhibitors, distribution of O2•ˉ and H2O2 and ROS enzyme profile in axes. Germination percentage and axis growth were determined under treatment with ROS inhibitors and scavengers. Localization of O2•ˉ and H2O2 was done using nitroblue tetrazolium (NBT) and 3,3′,5,5′-tetramethyl benzidine dihydrochloride hydrate (TMB), respectively. Apoplastic level of O2•ˉ was monitored by spectrophotometric analysis of bathing medium of axes. Profiles of NADPH oxidase and superoxide dismutase (SOD) were studied by in-gel assay. Germination was retarded by treatments affecting ROS level except H2O2 scavengers, while axis growth was retarded by all. Superoxide synthesis inhibitor and scavenger prevented H2O2 accumulation in axes in later phase as revealed from TMB staining. Activity of Cu/Zn SOD1 was initially high and declined thereafter. Superoxide being produced in apoplast possibly by NADPH oxidase activity is further metabolized to •OH via H2O2. Germination process depends possibly on •OH production in the axes. Post-germinative axis growth requires O2•ˉ while the differentiating zone of axis (radicle) requires H2O2 for cell wall stiffening. PMID:25763616

Chromosome numbers of populations of three varieties of Bidens pilosa in Taiwan.

PubMed

Huang, Ya-Lun; Kao, Wen-Yuan

2015-12-01

Hairy beggar-ticks (Bidens pilosa L.) is a common invasive plant in tropical and subtropical regions. The Flora of Taiwan listed three varieties of B. pilosa in Taiwan, var. minor, var. pilosa and var. radiata. Among the three varieties, var. radiata was the most recently, in 1970s, introduced into Taiwan. However, after its introduction into Taiwan, var. radiata has become dominant over the other two varieties and is considered a serious invasive plant in lowland of Taiwan. Our previous study showed that var. radiata is self-incompatible and the other two varieties are self-fertile. Could it be possible that different chromosome numbers contribute to the different breeding systems of these three varieties? In addition, the heterogeneities of traits of var. radiata were found higher than those of var. minor and var. pilosa. Is the phenomenon resulting from the hybridization between var. radiata with other varieties? We counted chromosome numbers of populations of these three varieties distributed in Taiwan and conducted hand pollination treatment between var. radiata (as pollen receiver) and var. minor or var. pilosa (as pollen donor) to provide answer for the aforementioned questions. No difference was found in chromosome numbers among populations of the same variety. Forty-eight chromosomes (2n = 48) were counted for var. radiata while 72 (2n = 72) chromosomes for var. minor and var. pilosa. Therefore, var. radiata is tetraploid and var. minor and var. pilosa are hexaploid. No successful hybridization was found between var. radiata and var. minor or between var. radiata and var. pilosa. This study provided the evidence that the invasive plant (B. pilosa var. radiata) has different chromosome numbers from the other two varieties and is unlikely to hybridize with the other two varieties.

Ecotoxicological assessment of pesticides and their combination on rhizospheric microbial community structure and function of Vigna radiata.

PubMed

Walvekar, Varsha Ashok; Bajaj, Swati; Singh, Dileep K; Sharma, Shilpi

2017-07-01

India is one of the leading countries in production and indiscriminate consumption of pesticides. Owing to their xenobiotic nature, pesticides affect soil microorganisms that serve as mediators in plant growth promotion. Our study aimed to deliver a comprehensive picture, by comparing the effects of synthetic pesticides (chlorpyriphos, cypermethrin, and a combination of both) with a biopesticide (azadirachtin) at their recommended field application level (L), and three times the recommended dosage (H) on structure and function of microbial community in rhizosphere of Vigna radiata. Effect on culturable fraction was assessed by enumeration on selective media, while PCR-denaturing gradient gel electrophoresis (DGGE) was employed to capture total bacterial community diversity. This was followed by a metabolic sketch using community-level physiological profiling (CLPP), to obtain a broader picture of the non-target effects on rhizospheric microbial community. Although plant parameters were not significantly affected by pesticide application, the microbial community structure experienced an undesirable impact as compared to control devoid of pesticide treatment. Examination of DGGE banding patterns through cluster analysis revealed that microbial community structure of pesticide-treated soils had only 70% resemblance to control rhizospheric soil even at 45 days post application. Drastic changes in the metabolic profiles of pesticide-treated soils were also detected in terms of substrate utilization, rhizospheric diversity, and evenness. It is noteworthy that the effects exacerbated by biopesticide were comparable to that of synthetic pesticides, thus emphasizing the significance of ecotoxicological assessments before tagging biopesticides as "safe alternatives."

Polyamines Confer Salt Tolerance in Mung Bean (Vigna radiata L.) by Reducing Sodium Uptake, Improving Nutrient Homeostasis, Antioxidant Defense, and Methylglyoxal Detoxification Systems

PubMed Central

Nahar, Kamrun; Hasanuzzaman, Mirza; Rahman, Anisur; Alam, Md. Mahabub; Mahmud, Jubayer-Al; Suzuki, Toshisada; Fujita, Masayuki

2016-01-01

The physiological roles of PAs (putrescine, spermidine, and spermine) were investigated for their ability to confer salt tolerance (200 mM NaCl, 48 h) in mung bean seedlings (Vigna radiata L. cv. BARI Mung-2). Salt stress resulted in Na toxicity, decreased K, Ca, Mg, and Zn contents in roots and shoots, and disrupted antioxidant defense system which caused oxidative damage as indicated by increased lipid peroxidation, H2O2 content, O2•- generation rate, and lipoxygenase activity. Salinity-induced methylglyoxal (MG) toxicity was also clearly evident. Salinity decreased leaf chlorophyll (chl) and relative water content (RWC). Supplementation of salt affected seedlings with exogenous PAs enhanced the contents of glutathione and ascorbate, increased activities of antioxidant enzymes (dehydroascorbate reductase, glutathione reductase, catalase, and glutathione peroxidase) and glyoxalase enzyme (glyoxalase II), which reduced salt-induced oxidative stress and MG toxicity, respectively. Exogenous PAs reduced cellular Na content and maintained nutrient homeostasis and modulated endogenous PAs levels in salt affected mung bean seedlings. The overall salt tolerance was reflected through improved tissue water and chl content, and better seedling growth. PMID:27516763

Microanatomical diversification of the zona pellucida in aplochelioid killifishes.

PubMed

Thompson, A W; Furness, A I; Stone, C; Rade, C M; Ortí, G

2017-07-01

This study investigates zona pellucida (ZP) ultrastructure in fertilized eggs of annual killifishes (suborder Aplocheiloidei), a group of highly specialized fishes that are able to survive desiccation for several weeks to months before they hatch. Little is known about ZP or chorionic ultrastructure sustaining these life-history modes, so scanning electron microscopy (SEM) was used to describe this trait in a large number of aplocheiloids with a focus on the family Rivulidae and the genus Hypsolebias. New images of ZP ultrastructure for 52 aplocheiloid species are provided, more than doubling the number characterized thus far. The evolution of chorionic structure within this group is studied using these new data. Characters were coded into a morphological matrix and optimized onto a consensus phylogeny to assess phylogenetic signal and reconstruct ancestral character states. Although ZP characters seem highly homoplastic and exhibit a large amount of structural convergence among lineages, aplocheiloid killifishes have evolved a number of unique structures associated with the chorion. Some annual species seem to have lost long filaments because eggs are deposited in the soil instead of being adhered to aquatic plants. © 2017 The Fisheries Society of the British Isles.

Elevated [CO2] does not ameliorate the negative effects of elevated temperature on drought-induced mortality in Eucalyptus radiata seedlings.

PubMed

Duan, Honglang; Duursma, Remko A; Huang, Guomin; Smith, Renee A; Choat, Brendan; O'Grady, Anthony P; Tissue, David T

2014-07-01

It has been reported that elevated temperature accelerates the time-to-mortality in plants exposed to prolonged drought, while elevated [CO(2)] acts as a mitigating factor because it can reduce stomatal conductance and thereby reduce water loss. We examined the interactive effects of elevated [CO(2)] and temperature on the inter-dependent carbon and hydraulic characteristics associated with drought-induced mortality in Eucalyptus radiata seedlings grown in two [CO(2)] (400 and 640 μL L(-1)) and two temperature (ambient and ambient +4 °C) treatments. Seedlings were exposed to two controlled drying and rewatering cycles, and then water was withheld until plants died. The extent of xylem cavitation was assessed as loss of stem hydraulic conductivity. Elevated temperature triggered more rapid mortality than ambient temperature through hydraulic failure, and was associated with larger water use, increased drought sensitivities of gas exchange traits and earlier occurrence of xylem cavitation. Elevated [CO(2)] had a negligible effect on seedling response to drought, and did not ameliorate the negative effects of elevated temperature on drought. Our findings suggest that elevated temperature and consequent higher vapour pressure deficit, but not elevated [CO(2)], may be the primary contributors to drought-induced seedling mortality under future climates. © 2013 John Wiley & Sons Ltd.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs

PubMed Central

Bernhardt, Miranda L.; Lowther, Katie M.; Padilla-Banks, Elizabeth; McDonough, Caitlin E.; Lee, Katherine N.; Evsikov, Alexei V.; Uliasz, Tracy F.; Chidiac, Peter; Williams, Carmen J.; Mehlmann, Lisa M.

2015-01-01

During oocyte maturation, capacity and sensitivity of Ca2+ signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca2+ release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca2+ oscillations that drive egg activation and initiate early embryo development. Premature Ca2+ release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca2+ signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca2+ release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca2+ release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca2+ increases that were sufficient to cause premature zona pellucida conversion. Rgs2−/− females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2−/− eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca2+ release in eggs that are poised on the brink of development. PMID:26160904

Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Lean, A.; Cantin, M.

1986-03-05

The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levelsmore » of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.« less

Efficacy of a Simple Formulation Composed of Nematode-Trapping Fungi and Bidens pilosa var. radiata Scherff Aqueous Extracts (BPE) for Controlling the Southern Root-Knot Nematode

PubMed Central

Ajitomi, Atsushi; Taba, Satoshi; Ajitomi, Yoshino; Kinjo, Misa; Sekine, Ken-taro

2018-01-01

We tested a formulation composed of a mixture of Bidens pilosa var. radiata extract (BPE) and nematode-trapping fungi for its effects on Meloidogyne incognita. In earlier evaluations of the effects of plant extracts on the hyphal growth of 5 species of nematode-trapping fungi with different capture organs (traps), the growth of all species was slightly inhibited. However, an investigation on the number of capture organs and nematode-trapping rates revealed that Arthrobotrys dactyloides formed significantly more rings and nematode traps than those of the control. An evaluation of simple mixed formulations prepared using sodium alginate showed that nematodes were captured with all formulations tested. The simple mixed formulation showed a particularly high capture rate. Furthermore, in a pot test, although the effects of a single formulation made from the fungus or plant extract were acceptable, the efficacy of the simple mixed formulation against M. incognita root-knot formation was particularly high. PMID:29311429

Oviposition Behavior and Survival of Tamarixia radiata (Hymenoptera: Eulophidae), an Ectoparasitoid of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), on Hosts Exposed to an Entomopathogenic Fungus, Isaria fumosorosea (Hypocreales: Cordycipitaceae), Under Laboratory Conditions.

PubMed

Chow, Andrew; Dunlap, Christopher A; Jackson, Mark A; Flores, Daniel; Patt, Joseph M; Sétamou, Mamoudou

2016-10-01

Antagonistic interactions between the nymphal parasitoid, Tamarixia radiata Waterston (Hymenoptera: Eulophidae), and the ARSEF 3581 strain of the entomopathogenic fungus, Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), could disrupt biological control of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Three interactions were evaluated under laboratory conditions at 25 °C: 1) parasitoid survival if parasitized hosts were exposed to ARSEF 3581 blastospores before or after host mummification; 2) parasitoid survival if mummies containing larva or pupa were exposed to ARSEF 3581 hyphae; 3) parasitoid oviposition on infected hosts with visible or without visible hyphae. Topical application of blastospore formulation onto the dorsal surfaces of live nymphs parasitized with second-instar wasp larva (3 d after parasitism) reduced host mummification by 50% and parasitoid emergence by 85%. However, parasitoid emergence was not affected by topical application of blastospore formulation onto mummies that contained fourth-instar wasp larva (6 d after parasitism). Parasitoid emergence was reduced by 80% if mummies containing fourth-instar wasp larva were covered with blastospore formulation colonized by fungal hyphae. In comparison, parasitoid emergence was not affected if mummies containing wasp pupa (9 d after parasitism) were covered with formulation colonized by fungal hyphae. Female parasitoids oviposited on infected hosts without visible hyphae but not on infected hosts with visible hyphae. Our findings suggest that I. fumosorosea could detrimentally affect T. radiata, if both natural enemies are simultaneously deployed for biological control of D. citri However, temporal separation of the fungus and parasitoid could reduce antagonism and enhance control of D. citri. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

PubMed

Moro, L N; Jarazo, J; Buemo, C; Hiriart, M I; Sestelo, A; Salamone, D F

2015-10-01

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. © 2015 Blackwell Verlag GmbH.

GABA release in the zona incerta of the sheep in response to the sight and ingestion of food and salt.

PubMed

Kendrick, K M; Hinton, M R; Baldwin, B A

1991-05-31

In order to establish which neurotransmitters may influence the activity of zona incerta neurones in the sheep which respond selectively to the sight or ingestion of food, we have measured the release of amino acid and monoamine neurotransmitters from this region using microdialysis sampling. Co-ordinates for the placement of microdialysis probes in regions of the zona incerta where cells respond to the sight or ingestion of food were first established by making single-unit extracellular recordings. When animals were food-deprived results showed that release of gamma-aminobutyric acid (GABA) was increased in response to the sight and ingestion of food but not of aspartate, glutamate, taurine, noradrenaline, dopamine or serotonin. This release of GABA was absent when the animals were shown non-food objects or saw or ingested salt solutions. When the same animals were physiologically sodium-depleted GABA release was evoked by the sight and ingestion of salt solutions and release following the sight and ingestion of food was significantly reduced. These results provide further evidence that GABA is an important neurotransmitter in neural circuits controlling the regulation of food intake.

Characterization of oocyte retrieval cycles with empty zona pellucida.

PubMed

Oride, Aki; Kanasaki, Haruhiko; Hara, Tomomi; Ohta, Hiroko; Kyo, Satoru

2018-01-01

To identify the factors that characterize cycles with empty zona pellucida (EZP). Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

Articulatory Closure Proficiency in Patients with Parkinson's Disease Following Deep Brain Stimulation of the Subthalamic Nucleus and Caudal Zona Incerta

ERIC Educational Resources Information Center

Karlsson, Fredrik; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; Nordh, Erik; van Doorn, Jan

2014-01-01

Purpose: The present study aimed at comparing the effects of deep brain stimulation (DBS) treatment of the subthalamic nucleus (STN) and the caudal zona incerta (cZi) on the proficiency in achieving oral closure and release during plosive production of people with Parkinson's disease. Method: Nineteen patients participated preoperatively and…

NEFM (Neurofilament Medium) Polypeptide, a Marker for Zona Glomerulosa Cells in Human Adrenal, Inhibits D1R (Dopamine D1 Receptor)-Mediated Secretion of Aldosterone.

PubMed

Maniero, Carmela; Garg, Sumedha; Zhao, Wanfeng; Johnson, Timothy Isaac; Zhou, Junhua; Gurnell, Mark; Brown, Morris J

2017-08-01

Heterogeneity among aldosterone-producing adenomas (APAs) has been highlighted by the discovery of somatic mutations. KCNJ5 mutations predominate in large zona fasciculata (ZF)-like APAs; mutations in CACNA1D , ATP1A1, ATP2B3 , and CTNNB1 are more likely to be found in small zona glomerulosa (ZG)-like APAs. Microarray comparison of KCNJ5 mutant versus wild-type APAs revealed significant differences in transcriptomes. NEFM , encoding a neurofilament subunit which is a D1R (dopamine D1 receptor)-interacting protein, was 4-fold upregulated in ZG-like versus ZF-like APAs and 14-fold more highly expressed in normal ZG versus ZF. Immunohistochemistry confirmed selective expression of NEFM (neurofilament medium) polypeptide in ZG and in ZG-like APAs. Silencing NEFM in adrenocortical H295R cells increased basal aldosterone secretion and cell proliferation; silencing also amplified aldosterone stimulation by the D1R agonist, fenoldopam, and inhibition by the D1R antagonist, SCH23390. NEFM coimmunoprecipitated with D1R, and its expression was stimulated by fenoldopam. Immunohistochemistry for D1R was mainly intracellular in ZG-like APAs but membranous in ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZF-like APAs was higher than in cells from ZG-like APAs. Transfection of mutant KCNJ5 caused a large reduction in NEFM expression in H295R cells. We conclude that NEFM is a negative regulator of aldosterone production and cell proliferation, in part by facilitating D1R internalization from the plasma membrane. Downregulation of NEFM in ZF-like APAs may contribute to a D1R/D2R imbalance underlying variable pharmacological responses to dopaminergic drugs among patients with APAs. Finally, taken together, our data point to the possibility that ZF-like APAs are in fact ZG in origin. © 2017 American Heart Association, Inc.

Ultrastructural observations of previtellogenic ovarian follicles of dove.

PubMed

Zarnescu, Otilia

2004-11-01

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.

Expression analysis of sox3 during testicular development, recrudescence, and after hCG induction in catfish, Clarias batrachus.

PubMed

Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian

2014-01-01

In teleosts, the expression of steroidogenic enzymes and related transcription factor genes occurs in a stage- and tissue-specific manner causing sexual development. The role of sox3, an evolutionary ancestor of SRY, has not been studied in detail. Therefore, the full-length cDNA of sox3 (1,197 kb) was cloned from catfish testis, and mRNA expression was analyzed during gonadal development, during the seasonal reproductive cycle, and after human chorionic gonadotropin (hCG) induction. Tissue distribution analysis showed that sox3 expression was higher in testis, ovary, and brain compared to other tissues analyzed. Developing and mature testis showed higher sox3 expression than ovary of corresponding stages, and more sox3 transcripts were found during the spawning phase of the seasonal reproductive cycle. Expression of sox3 was upregulated by hCG after in vivo and in vitro induction, suggesting that gonadotropins might stimulate it. In situ hybridization and immunohistochemistry showed the presence of sox3 mRNA and protein in somatic and interstitial cell layers of the testis. Sox3 could also be found in the zona radiata of developing and mature oocytes. Exposure of methyltestosterone (1 µg/l) and ethinylestradiol (1 µg/l) for 21 days during testicular development showed lower sox3 expression levels in the testis and brain, indicating a certain feedback intervention. These results suggest a possible role for Sox3 in the regulation of testicular development and function. © 2014 S. Karger AG, Basel.

Division of Labor in Hand Usage Is Associated with Higher Hand Performance in Free-Ranging Bonnet Macaques, Macaca radiata

PubMed Central

Mangalam, Madhur; Desai, Nisarg; Singh, Mewa

2015-01-01

A practical approach to understanding lateral asymmetries in body, brain, and cognition would be to examine the performance advantages/disadvantages associated with the corresponding functions and behavior. In the present study, we examined whether the division of labor in hand usage, marked by the preferential usage of the two hands across manual operations requiring maneuvering in three-dimensional space (e.g., reaching for food, grooming, and hitting an opponent) and those requiring physical strength (e.g., climbing), is associated with higher hand performance in free-ranging bonnet macaques, Macaca radiata. We determined the extent to which the macaques exhibit laterality in hand usage in an experimental unimanual and a bimanual food-reaching task, and the extent to which manual laterality is associated with hand performance in an experimental hand-performance-differentiation task. We observed negative relationships between (a) the latency in food extraction by the preferred hand in the hand-performance-differentiation task (wherein, lower latency implies higher performance), the preferred hand determined using the bimanual food-reaching task, and the normalized difference between the performance of the two hands, and (b) the normalized difference between the performance of the two hands and the absolute difference between the laterality in hand usage in the unimanual and the bimanual food-reaching tasks (wherein, lesser difference implies higher manual specialization). Collectively, these observations demonstrate that the division of labor between the two hands is associated with higher hand performance. PMID:25806511

Capital social de los padres de escolares de una zona vulnerable.

PubMed

Cornejo, Susana; Herrera, Ariel; Hilas, Elena; Gigena, Y Pablo

2018-01-01

El capital social supone importantes inversiones materiales, simbólicas y de esfuerzos. Conocer el capital social de una comunidad puede facilitar la comprensión del beneficio de las relaciones comunitarias para la promoción de salud. Con el objetivo de reconocer los componentes del capital social de padres de escolares atendiendo una escuela en una zona vulnerable de la ciudad de Córdoba, Argentina, indagamos a través de grupos focales acerca de elementos esenciales del capital social. La asintonía socio-cultural entre los pobladores originarios y los reubicados, la estigmatización policial y el clientelismo político han generado desconfianza del uno hacia el otro en diferentes aspectos convivenciales, siendo la familia la única red de apoyo. La escuela se reconoce como espacio adonde la gente espontáneamente se organiza para invertir en redes sociales, presentándose así con potencialidad para la promoción de conductas saludables, por el lugar simbólico que ocupa para los padres.

Use of Both Cumulus Cells’ Transcriptomic Markers and Zona Pellucida Birefringence to Select Developmentally Competent Oocytes in Human Assisted Reproductive Technologies

PubMed Central

2015-01-01

Background Selection of the best oocyte for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in human infertility treatment procedure. Oocyte selection using morphological criteria mainly Zona pellucida (ZP) has been the gold standard method in assisted reproductive technologies (ART) clinics, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs-based approaches have allowed the identification of key molecular markers that quantitatively and non-invasively predict the oocyte quality for higher pregnancy rates and efficient infertility treatment. These biomarkers are a valuable reinforcement of the morphological selection criteria widely used in in vitro fertilization (IVF) clinics. In this context, this study was designed to investigate the relationship between transcriptomic predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality mainly the ZP birefringence. Results Microarray data revealed that 48 and 27 differentially expressed candidate genes in cumulus cells (CCs) were respectively overexpressed and underexpressed in the ZGP (Zona Good Pregnant) versus ZBNP (Zona Bad Non Pregnant) groups. More than 70% of previously reported transcriptomic biomarkers of oocyte developmental competence were confirmed in this study. The analysis of possible association between ZP birefringence versus molecular markers approach showed an absence of correlation between them using the current set of markers. Conclusions This study suggested a new integrative approach that matches morphological and molecular approaches used to select developmentally competent oocytes able to lead to successful pregnancy and the delivery of healthy baby. For each ZP birefringence score, oocytes displayed a particular CCs' gene expression pattern. However, no correlations were found between the 7 gene biomarkers of oocyte developmental

Swallowing Quality of Life After Zona Incerta Deep Brain Stimulation.

PubMed

Sundstedt, Stina; Nordh, Erik; Linder, Jan; Hedström, Johanna; Finizia, Caterina; Olofsson, Katarina

2017-02-01

The management of Parkinson's disease (PD) has been improved, but management of signs like swallowing problems is still challenging. Deep brain stimulation (DBS) alleviates the cardinal motor symptoms and improves quality of life, but its effect on swallowing is not fully explored. The purpose of this study was to examine self-reported swallowing-specific quality of life before and after caudal zona incerta DBS (cZI DBS) in comparison with a control group. Nine PD patients (2 women and 7 men) completed the self-report Swallowing Quality of Life questionnaire (SWAL-QOL) before and 12 months after cZI DBS surgery. The postoperative data were compared to 9 controls. Median ages were 53 years (range, 40-70 years) for patients and 54 years (range, 42-72 years) for controls. No significant differences were found between the pre- or postoperative scores. The SWAL-QOL total scores did not differ significantly between PD patients and controls. The PD patients reported significantly lower scores in the burden subscale and the symptom scale. Patients with PD selected for cZI DBS showed good self-reported swallowing-specific quality of life, in many aspects equal to controls. The cZI DBS did not negatively affect swallowing-specific quality of life in this study.

Transduction of Nonhuman Primate Brain with Adeno-Associated Virus Serotype 1: Vector Trafficking and Immune Response

PubMed Central

Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L.; Bankiewicz, Krystof S.

2009-01-01

Abstract We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohisto-chemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm3 coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4+ lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2. PMID:19292604

Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction.

PubMed

Beek, J; Nauwynck, H; Appeltant, R; Maes, D; Van Soom, A

2015-11-01

Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF. Copyright © 2015 Elsevier Inc. All rights reserved.

The preclinical evaluation of immunocontraceptive vaccines based on canine zona pellucida 3 (cZP3) in a mouse model.

PubMed

Wang, Ying; Li, Yijie; Zhang, Beibei; Zhang, Fuchun

2018-05-11

Stray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis. To control the overpopulation of stray dogs, zona pellucida 3 (ZP3), a primary receptor for sperm, is a potential antigen for developing contraceptive vaccines. To enhance the immune responses and contraceptive effects of canine ZP3 (cZP3), dog gonadotropin-releasing hormone (GnRH) and a T cell epitope of chicken ovalbumin (OVA) were selected to construct two fusion proteins with cZP3, ovalbumin-GnRH-ZP3 (OGZ) and ovalbumin-ZP3 (OZ), and their contraceptive effects were evaluated in mice. The synthesized DNA sequences of OGZ and OZ were cloned into plasmid pET-28a respectively. The fusion proteins OGZ and OZ were identified by SDS-PAGE and Western blot. Mice were immunized with OGZ, OZ and cZP3, and the infertility rates were monitored. Mice immunized with mouse ZP3 (mZP3) or adjuvant alone were used as positive control and negative control, respectively. cZP3- and GnRH-specific antibodies (Abs) were detected by ELISA. The bindings of the Abs to oocytes were detected by indirect immunofluorescence assay. The paraffin sections of mice ovaries were observed under microscope for analyzing pathological characteristics. SDS-PAGE and Western blot analyses showed that the two fusion proteins OGZ and OZ were correctly expressed. ELISA results showed that OGZ vaccine induced both cZP3- and GnRH-specific Abs, and OZ vaccine induced cZP3-specific Ab, which lasted for up to 168 days. The levels of follicle stimulating hormone (FSH) and estradiol (E2) in sera were significantly decreased in OGZ immunized mice. Indirect immunofluorescence results showed that Abs induced by cZP3 and mZP3 could bind to the mouse ZP and dog ZP each other. Compared with the adjuvant group, all vaccine immunized groups significantly decreased the fertility rate and mean litter size. Interestingly, the fertility rate in OGZ-immunized group is the lowest, and only 1 mouse out of 10 mice is fertile

Fundamental studies of adrenal retinoid-X-receptor: Protein isoform, tissue expression, subcellular distribution, and ligand availability.

PubMed

Cheng, Behling; Al-Shammari, Fatema H; Ghader, Isra'a A; Sequeira, Fatima; Thakkar, Jitendra; Mathew, Thazhumpal C

2017-07-01

Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55kDa), RXRβ (47kDa) and RXR (56kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

A facile biomimetic preparation of highly stabilized silver nanoparticles derived from seed extract of Vigna radiata and evaluation of their antibacterial activity

NASA Astrophysics Data System (ADS)

Choudhary, Manoj Kumar; Kataria, Jyoti; Cameotra, Swaranjit Singh; Singh, Jagdish

2016-01-01

The significant antibacterial activity of silver nanoparticles draws the major attention toward the present nanobiotechnology. Also, the use of plant material for the synthesis of metal nanoparticles is considered as a green technology. In this context, a non-toxic, eco-friendly, and cost-effective method has been developed for the synthesis of silver nanoparticles using seed extract of mung beans ( Vigna radiata). The synthesized nanoparticles have been characterized by UV-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), atomic absorption spectroscopy (AAS), and X-ray diffraction (XRD). The UV-visible spectrum showed an absorption peak at around 440 nm. The different types of phytochemicals present in the seed extract synergistically reduce the Ag metal ions, as each phytochemical is unique in terms of its structure and antioxidant function. The colloidal silver nanoparticles were observed to be highly stable, even after 5 months. XRD analysis showed that the silver nanoparticles are crystalline in nature with face-centered cubic geometry and the TEM micrographs showed spherical particles with an average size of 18 nm. Further, the antibacterial activity of silver nanoparticles was evaluated by well-diffusion method and it was observed that the biogenic silver nanoparticles have an effective antibacterial activity against Escherichia coli and Staphylococcus aureus. The outcome of this study could be useful for nanotechnology-based biomedical applications.

[A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

PubMed

Li, Chen-Chen; Yu, Ji-Yun; Jiang, Min; Tu, Yi-Xian; Ma, Xiao-Lin; Zhang, Fu-Chun

2011-09-01

To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway. Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA. ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (P<0.01). Antifertility experiment showed that the experimental group reduced the average fertility significantly different compared with the control group (P<0.01). Restriction analysis, RT-PCR and Western blot showed that the recombinant plasmid constructed correctly and can be the expression of mRNA and protein levels.It resulted that the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

Remodeling of the plasma membrane in preparation for sperm–egg recognition: roles of acrosomal proteins

PubMed Central

Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

2015-01-01

The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step. PMID:25994642

Associations between Ectomycorrhizal Fungi and Bacterial Needle Endophytes in Pinus radiata: Implications for Biotic Selection of Microbial Communities

PubMed Central

Rúa, Megan A.; Wilson, Emily C.; Steele, Sarah; Munters, Arielle R.; Hoeksema, Jason D.; Frank, Anna C.

2016-01-01

Studies of the ecological and evolutionary relationships between plants and their associated microbes have long been focused on single microbes, or single microbial guilds, but in reality, plants associate with a diverse array of microbes from a varied set of guilds. As such, multitrophic interactions among plant-associated microbes from multiple guilds represent an area of developing research, and can reveal how complex microbial communities are structured around plants. Interactions between coniferous plants and their associated microbes provide a good model system for such studies, as conifers host a suite of microorganisms including mutualistic ectomycorrhizal (ECM) fungi and foliar bacterial endophytes. To investigate the potential role ECM fungi play in structuring foliar bacterial endophyte communities, we sampled three isolated, native populations of Monterey pine (Pinus radiata), and used constrained analysis of principal coordinates to relate the community matrices of the ECM fungi and bacterial endophytes. Our results suggest that ECM fungi may be important factors for explaining variation in bacterial endophyte communities but this effect is influenced by population and environmental characteristics, emphasizing the potential importance of other factors — biotic or abiotic — in determining the composition of bacterial communities. We also classified ECM fungi into categories based on known fungal traits associated with substrate exploration and nutrient mobilization strategies since variation in these traits allows the fungi to acquire nutrients across a wide range of abiotic conditions and may influence the outcome of multi-species interactions. Across populations and environmental factors, none of the traits associated with fungal foraging strategy types significantly structured bacterial assemblages, suggesting these ECM fungal traits are not important for understanding endophyte-ECM interactions. Overall, our results suggest that both biotic

Análisis de los determinantes socioeconómicos del gasto de bolsillo en medicamentos en seis zonas geográficas de Panamá.

PubMed

Herrera-Ballesteros, Victor H; Castro, Franz; Gómez, Beatriz

2018-04-27

Caracterizar el gasto de bolsillo privado en medicamentos en función de los determinantes sociodemográficos y socioeconómicos. MATERIALES Y MéTODOS: La fuente de datos es la Encuesta de Gasto de Bolsillo en Medicamentos de 2014. Se caracterizó el gasto de bolsillo privado mediante variables explicativas sociodemográficas (SOD) y socioeconómicas (SES). Se hizo análisis factorial por componentes principales, regresión logística y lineal simple. Los Odds Ratio demuestran que la educación y la zona geográfica son determinantes fundamentales que inciden en el gasto de bolsillo. Los medicamentos son productos necesarios, en adición a que el gasto de bolsillo aumenta a un promedio del 2% por cada año de vida cronológica adicional. Existe mayor vulnerabilidad en las zonas más pauperizadas respecto del acceso a medicamentos, en especial en las indígenas e implica un mayor riesgo de gasto catastrófico a menor ingreso ante la mayor prevalencia de enfermedades crónicas. Copyright © 2018. Published by Elsevier Inc.

Advanced MRI in Acute Military TBI

DTIC Science & Technology

2014-09-01

symptoms onmental health and service discharge outcomes. J Neurotrauma. 2013; 30(16):1391-1397. 16. GalarneauMR,Woodruff SI, Dye JL, Mohrle CR, Wade...related mild traumatic brain injury acute symptoms on mental health and service discharge outcomes. J. Neurotrauma 30, 1391–1397. 22. Casscells, S. (2007...radiation (include optic radiation) left 37 Anterior corona radiata right 102 Anterior corona radiata left 38 Superior corona radiata right 103 Superior

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation.

PubMed

Zhang, Xiaobing; van den Pol, Anthony N

2017-05-26

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons. Copyright © 2017, American Association for the Advancement of Science.

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction.

PubMed

Cohen, Débora J; Busso, Dolores; Da Ros, Vanina; Ellerman, Diego A; Maldera, Julieta A; Goldweic, Nadia; Cuasnicu, Patricia S

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2). Further experiments analyzing both the ability of other CRISP proteins to bind to the rat egg and the amino acid sequence of their S2 regions show that the amino acid sequence of the S2 is needed for CRISP1 to interact with the egg. CRISP1 appears to be involved in the first step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. The observation that sperm testicular CRISP2 is also able to bind to the egg surface suggests a role for this protein in gamete fusion. Subsequent experiments confirmed the participation of CRISP2 in this step of fertilization and revealed that CRISP1 and CRISP2 interact with common egg surface binding sites. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. These observations contribute to a better understanding of the molecular mechanisms underlying mammalian fertilization.

Heterogeneous levels of oxidative phosphorylation enzymes in rat adrenal glands.

PubMed

Ogawa, Koichi; Harada, Keita; Endo, Yutaka; Sagawa, Sueko; Inoue, Masumi

2011-01-01

Mitochondria are organelles that produce ATP and reactive oxygen species, which are thought to be responsible for a decline in physiological function with aging. In this study, we morphologically and biochemically examined mitochondria in the rat adrenal gland. Immunohistochemistry showed that the rank order for intensity of immunolabelling for complex IV was zona reticularis > zona fasciculata > adrenal medulla, whereas for complex V α and β subunits, it was zona fasciculata > zona reticularis and adrenal medulla. The immunolabelling for complex I was homogeneous in the adrenal gland. The difference in immunolabelling between complexes I and IV indicates that the ratio of levels of complex I to that of complex IV in the zona reticularis was smaller than that in the zona fasciculata and the adrenal medulla. Electron microscopy revealed that aging rats had zona reticularis cells with many lysosomes and irregular nuclei. The result suggests that the level of proteins involved in oxidative phosphorylation is coordinated within the complex, but differs between the complexes. This might be responsible for degeneration of zona reticularis cells with aging. Copyright © 2009 Elsevier GmbH. All rights reserved.

Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurachi, H.; Wakimoto, H.; Sakumoto, T.

1984-02-01

The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using /sup 125/I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodiesmore » in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women.« less

From the epididymis to the egg: participation of CRISP proteins in mammalian fertilization.

PubMed

Da Ros, Vanina G; Muñoz, Mariana Weigel; Battistone, Maria A; Brukman, Nicolás G; Carvajal, Guillermo; Curci, Ludmila; Gómez-ElIas, MatIas D; Cohen, D Bora J; Cuasnicu, Patricia S

2015-01-01

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.

Localization of Cell Wall Polysaccharides in Normal and Compression Wood of Radiata Pine: Relationships with Lignification and Microfibril Orientation1

PubMed Central

Donaldson, Lloyd A.; Knox, J. Paul

2012-01-01

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides. PMID:22147521

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

PubMed

Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins

PubMed Central

2008-01-01

Background Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. Results A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. Conclusion The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated

Effects of Bidens pilosa L. var. radiata SCHERFF treated with enzyme on histamine-induced contraction of guinea pig ileum and on histamine release from mast cells.

PubMed

Matsumoto, Takayuki; Horiuchi, Masako; Kamata, Katsuo; Seyama, Yoshiyuki

2009-06-01

The medical mechanism against type I allergies is to block the release or production of chemical mediators from mast cells or to block the H(1)-receptor signaling. We previously reported that the anti-allergic action of the dry powder from Bidens pilosa L. var. radiata SCHERFF treated with the enzyme cellulosine (eMMBP) was dependent on the inhibition of histamine release from mast cells. Here, we investigate that the effect of fractions in eMMBP on the histamine-induced contraction in guinea pig ileum and on the release of histamine in rat peritoneal mast cells. The histamine-induced contraction in guinea pig ileum is dose-dependently inhibited by ketotifen, an antagonist of H(1)-receptor. Fractions contained caffeic acid, caffeoylquinic acid and fractions contained flavonoids such as hyperin and isoquercitrin in eMMBP inhibit histamine release from mast cells, but only flavonoids such as hyperin, isoquercitrin and rutin suppress the histamine-induced contraction in guinea pig ileum. Moreover, the histamine-induced contraction was not affected by caffeic acid, however, such contraction was significantly inhibited by rutin. These results suggest that the primary antagonists of H(1)- receptor are different from the components in eMMBP that inhibit histamine release, and that these components participate in the anti-allergic activity of eMMBP.

Modulation of xenobiotic biotransformation system and hormonal responses in Atlantic salmon (Salmo salar) after exposure to tributyltin (TBT).

PubMed

Mortensen, Anne Skjetne; Arukwe, Augustine

2007-04-01

Multiple biological effects of tributyltin (TBT) on juvenile salmon have been investigated. Fish were exposed for 7 days to waterborne TBT at nominal concentrations of 50 and 250 microg/L dissolved in dimethyl sulfoxide (DMSO). Hepatic samples were analyzed for gene expression patterns in the hormonal and xenobiotic biotransformation pathways using validated real-time PCR method. Immunochemical and several cytochrome P450 (CYP)-mediated enzyme activity (ethoxyresorufin: EROD, benzyloxyresorufin: BROD, methoxyresorufin: MROD and pentoxyresorufin: PROD) assays were analyzed. Our data show that TBT produced concentration-specific decrease of estrogen receptor-alpha (ERalpha), vitellogenin (Vtg), zona radiata protein (Zr-protein) and increase of estrogen receptor-beta (ERbeta) and androgen receptor-beta (ARbeta) in the hormonal pathway. In the xenobiotic biotransformation pathway, TBT produced apparent increase and decrease at respective low and high concentration, on aryl hydrocarbon receptor-alpha (AhRalpha), AhR nuclear translocator (ARNT) and AhR repressor (AhRR) mRNA. The expression of CYP1A1 and GST showed a TBT concentration-dependent decrease. The AhRbeta, CYP3A and uridine diphosphoglucuronosyl transferase (UGT) mRNA expressions were significantly induced after exposure to TBT. Immunochemical analysis of CYP3A and CYP1A1 protein levels confirmed the TBT effects observed at the transcriptional levels. The effect of TBT on the biotransformation enzyme gene expressions partially co-related but did not directly parallel enzyme activity levels for EROD, BROD, MROD and PROD. In general, these findings confirm previous reports on the endocrine effects of TBT, in addition to effects on hepatic CYP1A isoenzyme at the transcriptional level that transcends to protein and enzymatic levels. The induced expression patterns of CYP3A and UGT mRNA after TBT exposure, suggest the involvement of CYP3A and UGT in TBT metabolism in fish. The effect of TBT on CYP3A is proposed to

LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

PubMed Central

Huang, Peng; Li, Wenshu; Yang, Zhifang; Zhang, Ning; Xu, Yixin; Bao, Jianying; Jiang, Deke; Dong, Xianping

2017-01-01

Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract. PMID:28182716

Effects of porcine zona pellucida immunocontraceptives in zoo felids.

PubMed

Harrenstien, Lisa A; Munson, Linda; Chassy, Lisa M; Liu, Irwin K M; Kirkpatrick, Jay F

2004-09-01

Methods of contraception are necessary for management of zoo felids; however, the most commonly used contraceptive (melengestrol acetate implant) is associated with serious adverse reactions with long-term use. Porcine zona pellucida (pZP) vaccines are promising as contraceptives, but their safety in zoo felids has not been tested. pZP vaccine was administered to 27 female felids representing 10 species, including African lion (Panthera leo), Asian leopard (P. pardus), jaguar (P. onca), tiger (P. tigris), snow leopard (P. uncia), cougar (Felis concolor), Siberian lynx (F. lynx), Canada lynx (F. canadensis), serval (F. serval), and bobcat (F. rufus), in 15 facilities. Over 6 wk, each animal received three i.m. injections of 65 microg pZP with Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, or carbopol as the adjuvant. Behavioral signs of estrus were seen in 14 of the vaccinated felids. An unacceptably high incidence of adverse reactions was seen including injection site swelling, lameness, limb swelling, or abscessation (or all) in five felids after injection with FCA as the initial adjuvant. Adverse behavioral signs, including increased irritability and aggression, were seen in four felids. Six of the felids were assayed for antibodies against pZP during the 12 mo after vaccination; all showed antibody production. Antibody levels appeared to peak 1-4 mo after vaccination began, although elevated antibody levels persisted in two animals for > 12 mo after the first injection. All vaccinated felids were ovariohysterectomized 3-13 mo after vaccination. Folliculogenesis was present in all treated animals, and there was no histopathologic evidence of inflammatory damage to ovaries. Contraceptive efficacy was not specifically evaluated in this study; however, two of the three felids housed with an intact male became pregnant during the study, one of which gave birth to healthy cubs.

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

PubMed

Johnson, Wesley; Dai, Changsheng; Liu, Jun; Wang, Xian; Luu, Devin K; Zhang, Zhuoran; Ru, Changhai; Zhou, Chao; Tan, Min; Pu, Huayan; Xie, Shaorong; Peng, Yan; Luo, Jun; Sun, Yu

2018-03-01

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Characterization and localization of cysteine-rich secretory protein 3 (CRISP-3) in the human male reproductive tract.

PubMed

Udby, Lene; Bjartell, Anders; Malm, Johan; Egesten, Arne; Lundwall, Ake; Cowland, Jack B; Borregaard, Niels; Kjeldsen, Lars

2005-01-01

Mammalian members of the cysteine-rich secretory protein (CRISP) family are expressed predominantly in the male reproductive tract and are implicated in the process of reproduction from spermiogenesis, posttesticular sperm maturation, and capacitation to oocyte-sperm fusion, and possibly also penetration of the zona pellucida. Rodents express only 2 CRISPs (CRISP-1 and CRISP-2) in their male reproductive system, whereas humans and horses express an additional third member named CRISP-3. We have previously demonstrated that this protein is present in human seminal plasma as well as in other exocrine secretions, in blood plasma, and in neutrophilic granulocytes. To characterize the protein in seminal plasma and localize the production of CRISP-3 in the human male reproductive tract, we performed immunoblotting and enzyme-linked immunosorbent assay measurements of seminal plasma and immunohistochemistry and in situ hybridization of tissue specimens. We were able to show that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes. Furthermore, CRISP-3 expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens. Examination of seminal plasma from vasectomized males indicates that organs downstream of the epididymis are probably the major sources of seminal plasma CRISP-3.

Remediation of arsenic in mung bean (Vigna radiata) with growth enhancement by unique arsenic-resistant bacterium Acinetobacter lwoffii.

PubMed

Das, Joyati; Sarkar, Priyabrata

2018-05-15

Arsenic, a carcinogenic and toxic contaminant of soil and water, affects human health adversely. During last few decades, it has been an important global environmental issue. Among several arsenic detoxification methods remediation using arsenic resistant microbes is proved to be environment-friendly and cost-effective. This study aimed to test the effects of arsenic utilizing bacterial strain Acinetobacter lwoffii (RJB-2) on arsenic uptake and growth of mung bean plants (Vigna radiata). RJB-2 exhibited tolerance up to 125mM of arsenic (V) and 50mM of arsenic (III). RJB-2 produced plant growth promoting substances e.g. indole acetic acid (IAA), siderophores, exopolysaccharide (EPS) and phosphate solubilization in the absence and in presence of arsenic. Pot experiments were used to scrutinize the role of RJB-2 on arsenic uptake and growth of mung bean plants grown in soil amended with 22.5mgkg -1 of sodium arsenate (Na 2 HAsO 4 ·7H 2 O). RJB-2 could arrest arsenic uptake in just 7days and increase plant growth, number of plants per pot, chlorophyll and carotenoid content of the mung bean plants. RJB-2 formed biofilm and its root-association helped to abate arsenic uptake in mung bean. Confocal and light microscopic studies also revealed the abatement of arsenic uptake and increase in chlorophyll content in mung bean plants in presence of RJB-2. RJB-2 was also responsible for less production of reactive oxygen species (ROS) in mung bean plants reducing the oxidative damage caused by arsenic. The lower percentage of electrolytic leakage (EL) in RJB-2 inoculated mung bean plants proved arsenic abatement. The study also reported the distribution of arsenic in various parts of mung bean plant. RJB-2 owing to its intrinsic abilities of plant growth promotion even in presence of high concentrations of arsenic could inhibit arsenic uptake completely and therefore it could be used in large-scale cultivation for phytostabilization of plants. Copyright © 2017 Elsevier B

Sapwood development in Pinus radiata trees grown for three years at ambient and elevated carbon dioxide partial pressures.

PubMed

Atwell, B J; Henery, M L; Whitehead, D

2003-01-01

Clonal trees of Pinus radiata D. Don were grown in open-top chambers at a field site in New Zealand for 3 years at ambient (37 Pa) or elevated (65 Pa) carbon dioxide (CO2) partial pressure. Nitrogen (N) was supplied to half of the trees in each CO2 treatment, at 15 g N m-2 in the first year and 60 g N m-2 in the subsequent 2 years (high-N treatment). Trees in the low-N treatment were not supplied with N but received the same amount of other nutrients as trees in the high-N treatment. In the first year, stem basal area increased more in trees growing at elevated CO2 partial pressure and high-N supply than in control trees, suggesting a positive interaction between these resources. However, the relative rate of growth became the same across trees in all treatments after 450 days, resulting in trees growing at elevated CO2 partial pressure and high-N supply having larger basal areas than trees in the other treatments. Sapwood N content per unit dry mass was consistently about 0.09% in all treatments, indicating that N status was not suppressed by elevated CO2 partial pressure. Thus, during the first year of growth, an elevated CO2 partial pressure enhanced carbon (C) and N storage in woody stems, but there was no further stimulus to C and N deposition after the first year. The chemical composition of sapwood was unaffected by elevated CO2 partial pressure, indicating that no additional C was sequestered through lignification. However, independent of the treatments, early wood was 13% richer in lignin than late wood. Elevated CO2 partial pressure decreased the proportion of sapwood occupied by the lumina of tracheids by up to 12%, indicating increased sapwood density in response to CO2 enrichment. This effect was probably a result of thicker tracheid walls rather than narrower lumina.

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

PubMed Central

Hong, Y. P.; Hipkins, V. D.; Strauss, S. H.

1993-01-01

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga. PMID:7905846

Relationship between carbohydrate concentration and root growth potential in coniferous seedlings from three climates during cold hardening and dehardening

Treesearch

R.W. Tinus; K.E. Burr; N. Atzmon; J. Riov

2000-01-01

Greenhouse-cultured, container-grown seedlings of Aleppo pine (Pinus halepensis Mill.), radiata pine (Pinus radiata D. Don), and interior Douglas fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco) were cold acclimated and deacclimated in growth chambers over 24 weeks....

Identification of differentially expressed reproductive and metabolic proteins in the female abalone (Haliotis laevigata) gonad following artificial induction of spawning.

PubMed

Mendoza-Porras, Omar; Botwright, Natasha A; Reverter, Antonio; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

2017-12-01

Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning; (2) post-spawning; and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Detection of angiotensin II binding to single adrenal zona glomerulosa cells by confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem

1999-04-01

We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.

Quantitative trait locus mapping under irrigated and drought treatments based on a novel genetic linkage map in mungbean (Vigna radiata L.).

PubMed

Liu, Changyou; Wu, Jing; Wang, Lanfen; Fan, Baojie; Cao, Zhimin; Su, Qiuzhu; Zhang, Zhixiao; Wang, Yan; Tian, Jing; Wang, Shumin

2017-11-01

A novel genetic linkage map was constructed using SSR markers and stable QTLs were identified for six drought tolerance related-traits using single-environment analysis under irrigation and drought treatments. Mungbean (Vigna radiata L.) is one of the most important leguminous food crops. However, mungbean production is seriously constrained by drought. Isolation of drought-responsive genetic elements and marker-assisted selection breeding will benefit from the detection of quantitative trait locus (QTLs) for traits related to drought tolerance. In this study, we developed a full-coverage genetic linkage map based on simple sequence repeat (SSR) markers using a recombinant inbred line (RIL) population derived from an intra-specific cross between two drought-resistant varieties. This novel map was anchored with 313 markers. The total map length was 1010.18 cM across 11 linkage groups, covering the entire genome of mungbean with a saturation of one marker every 3.23 cM. We subsequently detected 58 QTLs for plant height (PH), maximum leaf area (MLA), biomass (BM), relative water content, days to first flowering, and seed yield (Yield) and 5 for the drought tolerance index of 3 traits in irrigated and drought environments at 2 locations. Thirty-eight of these QTLs were consistently detected two or more times at similar linkage positions. Notably, qPH5A and qMLA2A were consistently identified in marker intervals from GMES5773 to MUS128 in LG05 and from Mchr11-34 to the HAAS_VR_1812 region in LG02 in four environments, contributing 6.40-20.06% and 6.97-7.94% of the observed phenotypic variation, respectively. None of these QTLs shared loci with previously identified drought-related loci from mungbean. The results of these analyses might facilitate the isolation of drought-related genes and help to clarify the mechanism of drought tolerance in mungbean.

Assessment of cadmium accumulation, toxicity, and tolerance in Brassicaceae and Fabaceae plants--implications for phytoremediation.

PubMed

Anjum, Naser A; Umar, Shahid; Iqbal, Muhammad

2014-09-01

This study, based on a greenhouse pot culture experiment conducted with 15-day-old rapeseed (Brassica campestris L. cv. Pusa Gold; family Brassicaceae) and moong bean (Vigna radiata L. Wilczek cv. Pusa Ratna; family Fabaceae) plants treated with cadmium (Cd) concentrations (0, 50, and 100 mg kg(-1) soil), investigates their potential for Cd accumulation and tolerance, and dissects the underlying basic physiological/biochemical mechanisms. In both species, plant dry mass decreased, while Cd concentration of both root and shoot increased with increase in soil Cd. Roots harbored a higher amount of Cd (vs. shoot) in B. campestris, while the reverse applied to V. radiata. By comparison, root Cd concentration was higher in B. campestris than in V. radiata. The high Cd concentrations in B. campestris roots and V. radiata shoots led to significant elevation in oxidative indices, as measured in terms of electrolyte leakage, H2O2 content, and lipid peroxidation. Both plants displayed differential adaptation strategies to counteract the Cd burden-caused anomalies in their roots and shoots. In B. campestris, increasing Cd burden led to a significantly decreased reduced glutathione (GSH) content but a significant increase in activities of GSH reductase (GR), GSH peroxidase (GPX), and GSH sulfotransferase (GST). However, in V. radiata, increasing Cd burden caused significant increase in GSH content and GR activity, but a significant decline in activities of GPX and GST. Cross talks on Cd burden of tissues and the adapted Cd tolerance strategies against Cd burden-accrued toxicity indicated that B. campestris and V. radiata are good Cd stabilizer and Cd extractor, respectively, wherein a fine tuning among the major components (GR, GPX, GST, GSH) of the GSH redox system helped the plants to counteract differentially the Cd load-induced anomalies in tissues. On the whole, the physiological/biochemical characterization of the B. campestris and V. radiata responses to varying Cd

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (

Orexin Gene Transfer into Zona Incerta Neurons Suppresses Muscle Paralysis in Narcoleptic Mice

PubMed Central

Liu, Meng; Konadhode, RodaRani; Begum, Suraiya; Pelluru, Dheeraj; Gerashchenko, Dmitry; Sakurai, Takeshi; Yanagisawa, Masashi; van den Pol, Anthony N.

2011-01-01

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy. PMID:21508228

Nucleotide variation in genes invloved in wood formation in two pine species

Treesearch

David Pot; Lisa McMillan; Craig Echt; Gregoire Le Provost; Pauline Garnier-Gere; Sheree Cato; Christophe Plomion

2005-01-01

Nucleotide diversity in eight genes related to wood formation was investigated in two pine species, Pinus pinaster and P. radiata. The nucleotide diversity patterns observed and their properties were compared between the two species according to the specific characteristics of the samples analysed. A lower diversity was observed in P. radiata...

Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins.

PubMed

Dhanda, Saurabh; Sandhir, Rajat

2018-05-01

The present study was designed to investigate the mechanisms involved in blood-brain barrier (BBB) permeability in bile duct ligation (BDL) model of chronic hepatic encephalopathy (HE). Four weeks after BDL surgery, a significant increase was observed in serum bilirubin levels. Masson trichrome staining revealed severe hepatic fibrosis in the BDL rats. 99m Tc-mebrofenin retention was increased in the liver of BDL rats suggesting impaired hepatobiliary transport. An increase in permeability to sodium fluorescein, Evans blue, and fluorescein isothiocyanate (FITC)-dextran along with increase in water and electrolyte content was observed in brain regions of BDL rats suggesting disrupted BBB. Increased brain water content can be attributed to increase in aquaporin-4 mRNA and protein expression in BDL rats. Matrix metalloproteinase-9 (MMP-9) mRNA and protein expression was increased in brain regions of BDL rats. Additionally, mRNA and protein expression of tissue inhibitor of matrix metalloproteinases (TIMPs) was also increased in different regions of brain. A significant decrease in mRNA expression and protein levels of tight junction proteins, viz., occludin, claudin-5, and zona occluden-1 (ZO-1) was observed in different brain regions of BDL rats. VCAM-1 mRNA and protein expression was also found to be significantly upregulated in different brain regions of BDL animals. The findings from the study suggest that increased BBB permeability in HE involves activation of MMP-9 and loss of tight junction proteins.

Sequestration of PDC-109 protein by specific antibodies and egg yolk cryoprotects bull spermatozoa.

PubMed

Srivastava, N; Srivastava, S K; Ghosh, S K; Jerome, A; Das, G K; Mehrotra, S

2013-10-01

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures. © 2013 Blackwell Verlag GmbH.

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction.

PubMed

Busso, D; Cohen, D J; Hayashi, M; Kasahara, M; Cuasnicú, P S

2005-04-01

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

Target and non-target toxicity of fern extracts against mosquito vectors and beneficial aquatic organisms.

PubMed

Kamaraj, Chinnaperumal; Deepak, Paramasivam; Balasubramani, Govindasamy; Karthi, Sengodan; Arul, Dhayalan; Aiswarya, Dilipkumar; Amutha, Vadivel; Vimalkumar, Elangovan; Mathivanan, Damodaran; Suseem, Sundaram Renjitham; Muthu-Pandian, Chanthini Kanagaraj; Senthil-Nathan, Sengottayan; Perumal, Pachiappan

2018-06-06

Dengue and malaria are significant mosquito-borne diseases that are rapidly spread worldwide, mainly in temperate countries. Pteridophytes were identified to be a significant source of novel mosquitocidal agents. The present research was to explore the eco-friendly larvicides from methanol extracts of ferns, viz., Actiniopteris radiata, Adiantum caudatum, Cheilanthes swartzii, Hemionitis arifolia and Lycopodium clavatum. The larvicidal potential of the extracts screened using larvae of dengue vector Aedes aegypti (III and IV instar) and malarial vector Anopheles stephensi (III and IV instar), showed 10-100% mortality rates. Biosafety assessment was made on embryos of Danio rerio and Artemia nauplii. The phyto-constituents of the methanol extract of A. radiata leaves were identified through gas chromatography-mass spectrometry (GC-MS). Methanolic leaf extracts of A. radiata, A. caudatum and C. swartzii exhibited larvicidal activity against III and IV instar larvae of Ae. aegypti (LC 50 : 37.47, 74.51 and 152.38 and 67.58, 95.89 and 271.46 ppm) and An. stephensi (LC 50 : 70.35, 112.12 and 301.05 and 113.83, 175.30 and 315.19 ppm), respectively. The GC-MS of the methanol extract of A. radiata leaves revealed the presence of 7 phyto-components among which, Carbamic acid, phenyl-, (2-Nitrophenyl) methyl ester (1), Benzoic acid, 3- methylbenzoate (2) and 4-(benzylimino)- 1,4-dihydro-1-(p-toluoylmethyl) pyridine (3) were dominant. Biosafety assessment of methanol extract of A. radiata leaves on embryos of Danio rerio (Zebra fish) and Artemia nauplii (micro crustacean) revealed that there were no destructive or teratogenic effects. To conclude, the larvicidal activity and insignificant toxicity to non-target aquatic organisms of A. radiata leaves makes it a potential and environment safe biocontrol agent against dengue and malarial vectors. Copyright © 2018. Published by Elsevier Inc.

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

USGS Publications Warehouse

Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

2002-01-01

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

Time-of-Flight Adjustment Procedure for Acoustic Measurements in Structural Timber

Treesearch

Danbiel F. Llana; Guillermo Iñiguez-Gonzalez; Francisco Arriaga; Xiping Wang

2016-01-01

The effect of timber length on time-of-flight acoustic longitudinal measurements was investigated on the structural timber of four Spanish species: radiata pine (Pinus radiata D. Don), Scots pine (Pinus sylvestris L.), laricio pine (Pinus nigra Arn.), and maritime pine (Pinus pinaster Ait.). Time-of-flight longitudinal measurements were conducted on 120 specimens of...

Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis.

PubMed

Sano, Kaori; Kawaguchi, Mari; Katano, Keita; Tomita, Kenji; Inokuchi, Mayu; Nagasawa, Tatsuki; Hiroi, Junya; Kaneko, Toyoji; Kitagawa, Takashi; Fujimoto, Takafumi; Arai, Katsutoshi; Tanaka, Masaru; Yasumasu, Shigeki

2017-05-01

Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin. © 2017 Wiley Periodicals, Inc.

Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

PubMed

Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

2007-01-01

Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos.

PubMed

Novo, Sergi; Penon, Oriol; Barrios, Leonardo; Nogués, Carme; Santaló, Josep; Durán, Sara; Gómez-Matínez, Rodrigo; Samitier, Josep; Plaza, José Antonio; Pérez-García, Luisa; Ibáñez, Elena

2013-06-01

Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading

Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues.

PubMed

Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G; Yeo, Giles S H; Azizan, Elena A B; Brown, Morris J

2016-12-01

Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)-mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

PubMed Central

Da Ros, Vanina G.; Maldera, Julieta A.; Willis, William D.; Cohen, Débora J.; Goulding, Eugenia H.; Gelman, Diego M.; Rubinstein, Marcelo; Eddy, Edward M.; Cuasnicu, Patricia S.

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1−/− animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1−/− mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1−/− sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1+/+ and Crisp1−/− sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1−/− sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family. PMID:18571638

Parkin-mediated Monoubiquitination of the PDZ Protein PICK1 Regulates the Activity of Acid-sensing Ion Channels

PubMed Central

Joch, Monica; Ase, Ariel R.; Chen, Carol X.-Q.; MacDonald, Penny A.; Kontogiannea, Maria; Corera, Amadou T.; Brice, Alexis

2007-01-01

Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding– or E3 ubiquitin-ligase–defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease. PMID:17553932

Mapping the potential beverage quality of coffee produced in the Zona da Mata, Minas Gerais, Brazil.

PubMed

Silva, Samuel de Assis; de Queiroz, Daniel Marçal; Ferreira, Williams Pinto Marques; Corrêa, Paulo Cesar; Rufino, José Luis Dos Santos

2016-07-01

Detailed knowledge of coffee production systems enables optimization of crop management, harvesting and post-harvest techniques. In this study, coffee quality is mapped as a function of coffee variety, altitude and terrain aspect attributes. The work was performed in the Zona da Mata, Minas Gerais, Brazil. A large range of coffee quality grades was observed for the Red Catuai variety. For the Yellow Catuai variety, no quality grades lower than 70 were observed. Regarding the terrain aspect, samples from the southeast-facing slope (SEFS) and the northwest-facing slope (NWFS) exhibited distinct behaviors. The SEFS samples had a greater range of quality grades than did the NWFS samples. The highest grade was obtained from an NWFS point. The lowest quality values and the largest range of grades were observed at lower altitudes. The extracts from the highest-altitude samples did not produce any low-quality coffee. The production site's position and altitude are the primary variables that influenced the coffee quality. The study area has micro-regions with grades ranging from 80 to 94. These areas have the potential for producing specialty coffees. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Involvement of abscisic acid in regulating antioxidative defense systems and IAA-oxidase activity and improving adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings under cadmium stress.

PubMed

Li, Shi-Weng; Leng, Yan; Feng, Lin; Zeng, Xiao-Ying

2014-01-01

In vitro experiments were conducted to investigate the effects of abscisic acid (ABA) and Cd on antioxidative defense systems and indole-3-acetic acid (IAA) oxidase during adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings. The exogenous ABA significantly enhanced the number and fresh weight of the adventitious roots. CdCl2 strongly inhibited adventitious rooting. Pretreatment with 10 μM ABA clearly alleviated the inhibitory effect of Cd on rooting. ABA significantly reduced superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) activities, as well as the levels of glutathione (GSH) and ascorbic acid (ASA) during adventitious rooting. ABA strongly increased IAA-oxidase activity during the induction (0-12 h) and expression (after 48 h) phases and increased the phenols levels. Cd treatment significantly reduced the activities of SOD, APX, POD, and IAA oxidase, as well as GSH level. Cd strongly increased ASA levels. ABA pretreatment counteracted Cd-induced alterations of certain antioxidants and antioxidative enzymes, e.g., remarkably rescued APX and POD activities, reduced the elevated SOD and CAT activities and ASA levels, and recovered the reduced GSH levels, caused by Cd stress. Thus, the physiological effects of the combination of ABA and Cd treatments were opposite of those obtained with Cd treatment alone, suggesting that ABA involved in the regulation of antioxidative defense systems and the alleviation of wounding- and Cd-induced oxidative stress.

Resveratrol reverses the adverse effects of a diet-induced obese murine model on oocyte quality and zona pellucida softening.

PubMed

Jia, Zhenzhen; Feng, Zeyang; Wang, Lining; Li, Hao; Wang, Hongyu; Xu, Dingqi; Zhao, Xin; Feng, Daofu; Feng, Xizeng

2018-05-23

Reproductive dysfunction associated with obesity is increasing among women of reproductive age, including infertility and increasing risk of miscarriage. In females, reproductive disorders are linked to declining quality of oocytes. Using a model of diet-induced obesity, we have investigated the possible effects of obesity on oocyte quality, including metabolism, lipid accumulation, ROS levels, meiosis and changes in spindle structure in Metaphase II. Our study showed that obesity induced by a high fat diet can impair oocyte meiosis, destroy spindle assembly, and promote oxidative stress and abnormal mitochondrial distribution. With the addition of resveratrol, the negative impact of diet-induced obesity on the quality of oocytes was alleviated to some extent. In addition, we found that obesity causes mouse oocytes to soften, and resveratrol can restore the zona pellucida of oocytes to the same state as the control group. In conclusion, resveratrol can reverse the adverse effects of obesity on oocytes, which is beneficial for subsequent embryonic development.

Population genetic structure of Attalea vitrivir Zona (Arecaceae) in fragmented areas of southeast Brazil.

PubMed

Santos, R R M; Cavallari, M M; Pimenta, M A S; Abreu, A G; Costa, M R; Guedes, M L

2015-06-11

Attalea vitrivir Zona (synonym Orbignya oleifera) is one of the six species of Arecaceae known as "babassu". This species is used to make cosmetics, food, and detergents due to the high concentration of oil in the seeds. It is found only in fragmented areas of southern Bahia State and northern Minas Gerais State, southeast Brazil, and this fragmentation has affected both its ecological and genetic characteristics. We evaluated the genetic diversity and population genetic structure of A. vitrivir in six areas of two different regions at the extremes of its geographical range, in order to gain a better understanding of the factors that affect the distribution and partitioning of its diversity. Nine inter simple sequence repeat (ISSR) markers amplified 74 polymorphic bands, resulting in large diversity values (Shannon diversity index, 0.37-0.47; intrapopulation genetic diversity, 0.25-0.34). Analysis of molecular variance (AMOVA) revealed considerable differentiation between sampling sites (30.03%) and regions (12.08%), although most of the diversity was observed within sampling sites (69%). Further differentiation between sampling sites was noted more in the northern region than in the southern region, highlighting the genetic connectivity between the sampling sites within Rio Pandeiros Environmental Protection Area (southern region). The identification of two distinct genetic clusters (K = 2) corresponded to the northern and southern regions, and corroborated the AMOVA results. We suggest that the northern area, outside Rio Pandeiros Environmental Protection Area, must be included in future management plans for this species.

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study.

PubMed

Hammami, I; Nahdi, A; Atig, F; El May, A; El May, M V

2016-12-01

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin-43, Zona Occluding-1 and N-cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood-testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed. © 2016 Blackwell Verlag GmbH.

Foaling rates in feral horses treated with the immunocontraceptive porcine zona pellucida

USGS Publications Warehouse

Ransom, J.I.; Roelle, J.E.; Cade, B.S.; Coates-Markle, L.; Kane, A.J.

2011-01-01

Locally abundant feral horses (Equus caballus) can rapidly deplete available resources. Fertility control agents present promising nonlethal tools for reducing their population growth rates. We tested the effect of 2 forms of the immunocontraceptive porcine zona pellucida (PZP) on foaling rates in 3 populations of feral horses in the western United States. A liquid form requiring annual boosters was administered at Little Book Cliffs Wild Horse Range, Mesa County (CO), and Pryor Mountain Wild Horse Range, Bighorn County (WY) and Carbon County (MT), and a time-release pellet form designed to produce 2 yr of infertility was administered at McCullough Peaks Herd Management Area, Park County (WY). Average foaling rates (foals born/mare-yr) from direct observation of untreated and treated female horses (mares), 2004-2008, were 60.1% (n = 153 mare-yr) versus 6.6% (n = 91 mare-yr) at Little Book Cliffs, and 62.8% (n = 129 mare-yr) versus 17.7% (n = 79 mare-yr) at Pryor Mountain, respectively. At McCullough Peaks, mean annual foaling rates from 2006 to 2008 were 75.0% (n = 48 mare-yr) for untreated mares and 31.7% (n = 101 mare-yr) for treated mares. Controlling for age of mares and pretreatment differences in fertility, PZP reduced foaling rates in all 3 herds. The pellets used at McCullough Peaks (produced by cold evaporation) were less effective than pellets used in a previous trial and produced by heat extrusion. Immunocontraception with PZP may be a useful tool in reducing fertility rates in some western United States feral horse herds, but population growth reduction will depend on timely access to mares for inoculation and the proportion of mares that can be successfully treated. ?? 2011 The Wildlife Society.

Estimating Young's modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum

PubMed Central

Khalilian, Morteza; Navidbakhsh, Mahdi; Valojerdi, Mojtaba Rezazadeh; Chizari, Mahmoud; Yazdi, Poopak Eftekhari

2010-01-01

The zona pellucida (ZP) is the spherical layer that surrounds the mammalian oocyte. The physical hardness of this layer plays a crucial role in fertilization and is largely unknown because of the lack of appropriate measuring and modelling methods. The aim of this study is to measure the biomechanical properties of the ZP of human/mouse ovum and to test the hypothesis that Young's modulus of the ZP varies with fertilization. Young's moduli of ZP are determined before and after fertilization by using the micropipette aspiration technique, coupled with theoretical models of the oocyte as an elastic incompressible half-space (half-space model), an elastic compressible bilayer (layered model) or an elastic compressible shell (shell model). Comparison of the models shows that incorporation of the layered geometry of the ovum and the compressibility of the ZP in the layered and shell models may provide a means of more accurately characterizing ZP elasticity. Evaluation of results shows that although the results of the models are different, all confirm that the hardening of ZP will increase following fertilization. As can be seen, different choices of models and experimental parameters can affect the interpretation of experimental data and lead to differing mechanical properties. PMID:19828504

Environmental Influences on Kelp Performance across the Reproductive Period: An Ecological Trade-Off between Gametophyte Survival and Growth?

PubMed Central

Mohring, Margaret B.; Kendrick, Gary A.; Wernberg, Thomas; Rule, Michael J.; Vanderklift, Mathew A.

2013-01-01

Most kelps (order Laminariales) exhibit distinct temporal patterns in zoospore production, gametogenesis and gametophyte reproduction. Natural fluctuations in ambient environmental conditions influence the intrinsic characteristics of gametes, which define their ability to tolerate varied conditions. The aim of this work was to document seasonal patterns in reproduction and gametophyte growth and survival of Ecklonia radiata (C. Agardh) J. Agardh in south-western Australia. These results were related to patterns in local environmental conditions in an attempt to ascertain which factors explain variation throughout the season. E. radiata was fertile (produced zoospores) for three and a half months over summer and autumn. Every two weeks during this time, gametophytes were grown in a range of temperatures (16–22°C) in the laboratory. Zoospore densities were highly variable among sample periods; however, zoospores released early in the season produced gametophytes which had greater rates of growth and survival, and these rates declined towards the end of the reproductive season. Growth rates of gametophytes were positively related to day length, with the fastest growing recruits released when the days were longest. Gametophytes consistently survived best in the lowest temperature (16°C), yet exhibited optimum growth in higher culture temperatures (20–22°C). These results suggest that E. radiata releases gametes when conditions are favourable for growth, and E. radiata gametophytes are tolerant of the range of temperatures observed at this location. E. radiata releases the healthiest gametophytes when day length and temperature conditions are optimal for better germination, growth, and sporophyte production, perhaps as a mechanism to help compete against other species for space and other resources. PMID:23755217

Establishment of the onset of host specificity in four phyllobothriid tapeworm species (Cestoda: Tetraphyllidea) using a molecular approach.

PubMed

Randhawa, H S; Saunders, G W; Burt, M D B

2007-08-01

A parasitological survey in the Bay of Fundy, Canada, resulted in the recovery of mature specimens from 5 species of phyllobothriid tapeworms (Cestoda: Tetraphyllidea) from 4 rajid skates: Echeneibothrium canadensis and E. dubium abyssorum specimens from Amblyraja radiata; E. vernetae and Pseudanthobothrium n.sp. from Leucoraja erinacea and L. ocellata; and P. hanseni from A. radiata and Malacoraja senta. Partial sequence data of a variable region (D2) from the large subunit ribosomal DNA (LSU) were used here to determine the host distribution of immature specimens for 4 of these 5 species (E. d. abyssorum was not included in the analyses). Immature specimens from both Pseudanthobothrium spp. were identified in the same hosts as recorded previously for mature specimens, thus suggesting that there are mechanisms that prevent the attachment of the parasite in an 'unsuitable' host species. Immature E. canadensis specimens were recovered exclusively from A. radiata, whereas immature E. vernetae specimens were recovered from L. erinacea and A. radiata, despite the latter host species not harbouring mature E. vernetae specimens. Their presence in the latter host species may be explained by host restriction or resistance, which allows the attachment of the parasites in the 'wrong' host species, but not establishment or development.

Hepatitis B Virus S Protein Enhances Sperm Apoptosis and Reduces Sperm Fertilizing Capacity In Vitro

PubMed Central

Huang, JiHua; Zhong, Ying; Fang, XiaoWu; Xie, QingDong; Kang, XiangJin; Wu, RiRan; Li, FangZheng; Xu, XiaoQin; Lu, Hui; Xu, Lan; Huang, TianHua

2013-01-01

Objective Studying the impact of Hepatitis B virus S protein (HBs) on early apoptotic events in human spermatozoa and sperm fertilizing capacity. Methodology/Principal Findings Spermatozoa were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h, and then fluo-4 AM calcium assay, Calcein/Co2+ assay, protein extraction and ELISA, ADP/ATP ratio assay, sperm motility and hyperactivation and sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction (ZPIAR) tests were performed. The results showed that in the spermatozoa, with increasing concentration of HBs, (1) average cytosolic free Ca2+ concentration ([Ca2+]i) rose; (2) fluorescence intensity of Cal-AM declined; (3) average levels of cytochrome c decreased in mitochondrial fraction and increased in cytosolic fraction; (4) ADP/ATP ratios rose; (5) average rates of total motility and mean hyperactivation declined; (6) average rate of ZPIAR declined. In the above groups the effects of HBs exhibited dose dependency. However, there was no significant difference in the number of sperms bound to ZP between the control and all test groups. Conclusion HBs could induce early events in the apoptotic cascade in human spermatozoa, such as elevation of [Ca2+]i, opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c (cyt c) and increase of ADP/ATP ratio, but exerted a negative impact on sperm fertilizing capacity. PMID:23874723

White matter changes in preclinical Alzheimer's disease: a magnetic resonance imaging-diffusion tensor imaging study on cognitively normal older people with positive amyloid β protein 42 levels.

PubMed

Molinuevo, José Luis; Ripolles, Pablo; Simó, Marta; Lladó, Albert; Olives, Jaume; Balasa, Mircea; Antonell, Anna; Rodriguez-Fornells, Antoni; Rami, Lorena

2014-12-01

The aim of this study was to use diffusion tensor imaging measures to determine the existence of white matter microstructural differences in the preclinical phases of Alzheimer's disease, assessing cognitively normal older individuals with positive amyloid β protein (Aβ42) levels (CN_Aβ42+) on the basis of normal cognition and cerebrospinal fluid Aβ42 levels below 500 pg/mL. Nineteen CN_Aβ42+ and 19 subjects with Aβ42 levels above 500 pg/mL (CN_Aβ42-) were included. We encountered increases in axial diffusivity (AxD) in CN_Aβ42+ relative to CN_Aβ42- in the corpus callosum, corona radiata, internal capsule, and superior longitudinal fasciculus bilaterally, and also in the left fornix, left uncinate fasciculus, and left inferior fronto-occipital fasciculus. However, no differences were found in other diffusion tensor imaging indexes. Cognitive reserve scores were positively associated with AxD exclusively in the CN_Aβ42+ group. The finding of AxD alteration together with preserved fractional anisotropy, mean diffusivity, and radial diffusivity indexes in the CN_Aβ42+ group may indicate that, subtle axonal changes may be happening in the preclinical phases of Alzheimer's disease, whereas white matter integrity is still widely preserved. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein kinase Cβ as a therapeutic target stabilizing blood–brain barrier disruption in experimental autoimmune encephalomyelitis

PubMed Central

Lanz, Tobias V.; Becker, Simon; Osswald, Matthias; Bittner, Stefan; Schuhmann, Michael K.; Opitz, Christiane A.; Gaikwad, Sadanand; Wiestler, Benedikt; Litzenburger, Ulrike M.; Sahm, Felix; Ott, Martina; Iwantscheff, Simeon; Grabitz, Carl; Mittelbronn, Michel; von Deimling, Andreas; Winkler, Frank; Meuth, Sven G.; Wick, Wolfgang; Platten, Michael

2013-01-01

Disruption of the blood–brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cβ, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cβ in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS. PMID:23959874

Effects of acrolein on aldosterone release from zona glomerulosa cells in male rats.

PubMed

Wang, Kai-Lee; Huang, Wen-Ching; Chou, Jou-Chun; Weng, Ting-Chun; Hu, Sindy; Lieu, Fu-Kong; Lai, Wei-Ho; Idova, Galina; Wang, Paulus S; Wang, Shyi-Wu

2016-07-01

A positive correlation between smoking and hypertension has been well established. Acrolein is a major toxic volatile compound found in cigarette smoke. Human exposure to low levels of acrolein is unavoidable due to its production in daily activities, such as smoke from industrial, hot oil cooking vapors, and exhaust fumes from vehicles. The toxicity and the action mechanism of acrolein to induce apoptosis have been extensively studied, but the effects of acrolein on hypertension are still unknown. The present study aimed to examine the effects of acrolein on aldosterone release both in vivo and in vitro. Male rats were divided into three groups, and intraperitoneally injected with normal saline, or acrolein (2mg/kg) for 1 (group A-1) or 3 (group A-3) days, respectively. After sacrificing, rat blood samples were obtained to measure plasma aldosterone and angiotensin II (Ang II) levels. Zona glomerulosa (ZG) cells were prepared from rat adrenal cortex, and were incubated with or without stimulants. We found that the serum aldosterone was increased by 1.2-fold (p<0.05) in A-3 group as compared to control group. Basal aldosterone release from ZG cells in A-3 group was also increased significantly. Moreover, acrolein enhanced the stimulatory effects of Ang II and 8-bromo-cyclic AMP on aldosterone secretion from ZG cells prepared in both A-1 and A-3 groups. Furthermore, the enzyme activity of P450scc, the rate-limiting step of aldosterone synthesis, was elevated after acrolein injection. Plasma level of Ang II was increased in both A-1 and A-3 groups. These results suggested that acrolein exposure increased aldosterone production, at least in part, through elevating the level of plasma Ang II and stimulating steroidogenesis pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Two new gonad-infecting species of Philometra Costa, 1845 (Nematoda: Philometridae) from Trachinus spp. (Osteichthyes: Trachinidae) in the Gulf of Hammamet, Tunisia.

PubMed

Ghanmi, Nessrine; González-Solís, David; Gargouri, Lamia

2018-03-01

Based on light and scanning electron microscopical studies, two new gonad-infecting species of Philometra Costa, 1845, P. draco n. sp. and P. radiata n. sp. (Nematoda: Philometridae), are described from the marine perciform fishes Trachinus draco (Linnaeus) and T. radiatus (Linnaeus) (both Trachinidae), respectively, in the Gulf of Hammamet, off the northeastern coast of Tunisia. Philometra draco n. sp. and P. radiata n. sp. can be separated from other gonad-infecting species of this genus by the structures associated to the gubernaculum (e.g. dorsal protuberance, smooth field separating the dorsolateral longitudinal parts), as well as by the length of the body, spicules and gubernaculum. Philometra radiata n. sp. can be distinguished from P. draco n. sp. in having the dorsal side of the gubernaculum distal end provided with a median longitudinal smooth field demarcated by two dorsolateral lamellate parts. These two new species are the first philometrid species described from fishes of the family Trachinidae.

Mung bean (Vigna radiata (L.)) coat extract modulates macrophage functions to enhance antigen presentation: A proteomic study.

PubMed

Hashiguchi, Akiko; Hitachi, Keisuke; Zhu, Wei; Tian, Jingkui; Tsuchida, Kunihiro; Komatsu, Setsuko

2017-05-24

The immunomodulatory effect of mung bean is mainly attributed to antioxidant properties of flavonoids; however, the precise machinery for biological effect on animal cells remains uncertain. To understand the physiological change produced by mung bean consumption, proteomic and metabolomic techniques were used. In vitro assay confirmed the importance of synergistic interaction among multiple flavonoids by IL-6 expression. Proteomic analysis detected that the abundance of 190 proteins was changed in lipopolysaccharide-stimulated RAW264.7 cells by treatment with coat extract. Pathway mapping revealed that a range of proteins were regulated including an interferon-responsive antiviral enzyme (2'-5'-oligoadenylate synthetase), antigen processing factors (immunoglobulin heavy chain-binding protein and protein disulfide-isomerase), and proteins related to proteasomal degradation. Major histocompatibility complex pathway was activated. These results suggest that mung bean consumption enhances immune response toward a Th2-promoting polarization. This study highlighted the immunomodulation of RAW264.7 cells in response to treatment with mung bean seed coat extract, using gel-free proteomic technique. The mechanism of immunomodulation by mung bean has not been described until today except for a report which identified HMGB1 suppression as a pathway underlying the protective effect against sepsis. This study suggested that the mung bean is involved in the regulation of antigen processing and presentation, and thus shifts immune response from acute febrile illness to specific/systemic and long-lasting immunity to protect the host. Copyright © 2017 Elsevier B.V. All rights reserved.

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles.

PubMed

Celik-Ozenci, Ciler; Akkoyunlu, Gokhan; Kayisli, Umit Ali; Arici, Aydin; Demir, Ramazan

2003-11-01

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus-oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an

Two subunits of the 55 K porcine zona pellucida glycoprotein family are immunologically distinct

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subramanian, M.G.; Yurewicz, E.C.; Sacco, A.G.

1986-03-01

The 55K glycoprotein family (ZP3) of the porcine zona pellucida is comprised of two subunits of 46 K and 45 K which can be resolved by endo-..beta..-galactosidase digestion of ZP3 followed by reversed phase HPLC on Vydac C4 resin. Gel electrophoresis revealed that the 46 K component (EBDG..cap alpha..) is approx. 95% pure and the 45 K component (EBGD..beta..) is 100% pure. In the present study, these two subunits were evaluated immunologically by RIA. Under similar reaction protocols (chloramine-T iodination procedure) comparable specific activities were obtained for EBGD..cap alpha.. (33.06 +/- 7.5 ..mu..ci/..mu..gm), EBGD..beta.. (30.45 +/- 1.6) and ZP3 (26.3more » +/- 1.3). Antibody (Ab) titration studies revealed that EBGD..cap alpha.. and ..beta.. are potent immunogens and /sup 125/I-EBGD..cap alpha.. showed minimal cross reactivity to EBGD..beta..-Ab (8% bound at 1:500 dilution), whereas, /sup 125/I-EBGD..beta.. showed a greater degree of cross reactivity to EBGD..cap alpha..-Ab (23% bound at 1:500 dilution). Maximum binding for the two labeled antigens against homologous Abs (1:500) was > 60%. Dose response studies revealed that in the /sup 125/I-EBGD..cap alpha.. vs EBGD..cap alpha.. -Ab system, the 50% intercept was 3.25 +/- 0.32 ng for EBGD..cap alpha.. and 472.43 +/- 30.26 ng for EBGD..beta.. (p < 0.01), whereas, in the /sup 125/I-EBGD..beta.. vs EBGD..beta..-Ab system the 50% intercept was 3.51 +/- 0.58 for EBGD..beta.. and 166.77 +/- 49.20 for EBGD..cap alpha.. (p < 0.01). No significant differences were observed in the slopes of the dose response curves. It is concluded that the two subunits of ZP3 possess distinct immunologic characteristics as evaluated by RIA.« less

Expression of the GM2 activator protein in mouse testis.

PubMed

Li, Yu-Teh; Li, Su-Chen; Chen, I-Li

2017-12-01

The GM2-activator protein (GM2-AP), revealed by Li et al. in 1973 in human liver, was initially identified as a protein cofactor that stimulated β-hexosaminidase A to hydrolyze N-acetylgalactosamine from GM2 ganglioside. This cofactor was found to be missing in human variant AB Tay-Sachs disease. Over the years, the GM2-AP has also been shown to be involved in kidney vesicular transport, lipid presentation by CD1 molecule to T-cells, and interaction of human sperm with zona pellucida. Since the expression of the GM2-AP via mRNA detection in mouse tissues was found to be the highest in testis, we became interested in the localization of the GM2-AP at cellular level in mouse testis during spermatogenesis. Using immunohistochemical analysis and electron microscopy, we found that the GM2-AP was predominantly localized in the basal cytoplasm and the attenuated processes of Sertoli cells. The stained structure appeared to be lysosomes. The most interesting finding was the association of the GM2-AP with the acrosomal apparatus in early spermatids. A modest to intense staining was observed in some acrosomal granules and acrosomal caps. The GM2-AP seemed to disappear from acrosomal caps in the later stage of spermatids, in which the nucleus became elongated and condensed. These results suggest that the GM2-AP may be involved in the normal functions of Sertoli cells and play important roles during the development of acrosomal caps in the early spermatids. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

The transcriptome of corona radiata cells from individual MІІ oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women.

PubMed

Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David; Nguyen, Kho do; Belling, Kirstine; Høst, Thomas; Mikkelsen, Anne Lis

2014-11-29

Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs showed no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR < 0.05). Five of the genes contributing to the up-regulated cell cycle pathways in the PCOS CRCs were selected for qRT-PCR validation in ten PCOS and ten control CRC samples. qRT-PCR confirmed significant up-regulation in PCOS CRCs of cell cycle progression genes HIST1H4C (FC = 2.7), UBE2C (FC = 2.6) and cell cycle related transcription factor E2F4 (FC = 2.5). The overexpression of cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed to clarify whether the up-regulated cell cycle pathways in PCOS CRCs have any clinical implications.

Outcomes from stimulation of the caudal zona incerta and pedunculopontine nucleus in patients with Parkinson's disease.

PubMed

Khan, Sadaquate; Mooney, Lucy; Plaha, Puneet; Javed, Shazia; White, Paul; Whone, Alan L; Gill, Steven S

2011-04-01

Axial symptoms including postural instability, falls and failure of gait initiation are some of the most disabling motor symptoms of Parkinson's disease (PD). We performed bilateral deep brain stimulation (DBS) of the pedunculopontine nucleus (PPN) in combination with the caudal zona incerta (cZi) in order to determine their efficacy in alleviating these symptoms. Seven patients with predominant axial symptoms in both the 'on' and 'off' medication states underwent bilateral cZi and PPN DBS. Motor outcomes were assessed using the motor component of the Unified Parkinson's Disease Rating Scale (UPDRS 3) and a composite axial subscore was derived from items 27, 28, 29 and 30 (arising from chair, posture, gait and postural stability). Quality of life was measured using the PDQ39. Comparisons were made between scores obtained at baseline and those at a mean follow-up of 12 months. In both the off and on medication states, a statistically significant improvement in the UPDRS part 3 score was achieved by stimulation of the PPN, cZi and both in combination. In the off medication state, our composite axial subscore of the UPDRS part 3 improved with stimulation of the PPN, cZi and both in combination. The composite axial subscore, in the 'on' medication state, however, only showed a statistically significant improvement when a combination of cZi and PPN stimulation was used. This study provides evidence that a combination of PPN and cZi stimulation can achieve a significant improvement in the hitherto untreatable 'on' medication axial symptoms of PD.

Reusing ethyl acetate and aqueous exhausted fractions of dry olive mill residue by saprobe fungi.

PubMed

Aranda, E; García-Romera, I; Ocampo, J A; Carbone, V; Malorni, A; Sannino, F; De Martino, A; Capasso, R

2007-01-01

Some saprobe fungi (Phlebia radiata, Trametes versicolor, Coriolopsis rigida, Pycnoporus cinnabarinus, Fomes sclerodermus or Pleurotus pulmonarius) were able to bioconvert the ethyl acetate fraction (DEAF) and the corresponding aqueous exhausted fraction (EAF) of dry olive mill residue (DOR), reducing their phytotoxicity on Lepidium sativum seeds. Large amount of hydroxytyrosol together with other eight monomeric phenols were found in the native DEAF fraction, which represents a good source of antioxidants. P. radiata, T. versicolor and F. sclerodermus caused an effective phytotoxicity reduction of EAF in the concentration range of 25-3 gl(-1). In particular, in the range between 12.5 and 3 gl(-1), the EAF samples inoculated with P. radiata and F. sclerodermus surprisingly stimulated the germinability of L. sativum, suggesting their use as a potential biofertilizer. This is the first report which showed the bioconversion of the above fractions in shorter time with respect to the previous findings concerning DOR. The possible implications of laccase in the decrease of DEAF and EAF phytotoxicity was also discussed.

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts.

PubMed

Cervera, R P; Garcia-Ximénez, F

2003-10-01

The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

Enhanced contraception of canine zona pellucida 3 DNA vaccine via targeting DEC-205 in mice.

PubMed

Wang, Ying; Zhang, Beibei; Li, Jinyao; Aipire, Adila; Li, Yijie; Zhang, Fuchun

2018-06-01

Zona pellucida 3 (ZP3) is a potential antigen for the development of contraceptive vaccines to control animal population. In this study, we designed a canine ZP3 (CZP3) DNA vaccine through targeting DEC-205 (named as pcD-scFv-CZP3c) and investigated its contraceptive effect in mice. Female BALB/c mice were intramuscularly immunized 3 times at 2 weeks intervals. After immunization, humoral and cellular immune responses were detected by ELISA and flow cytometry. The results showed that pcD-CZP3 and pcD-scFv-CZP3c induced CZP3-specific antibody (Ab) responses both in serum and vaginal secretions compared to pcDNA3.1. Additionally, compared to pcD-CZP3, pcD-scFv-CZP3c increased the levels of CZP3-specific Abs after a third immunization. Abs induced by these two DNA vaccines could bind with mice and dogs oocytes. Moreover, pcD-scFv-CZP3c enhanced the activation of CD4 + T cells characterized by the increased frequencies of CD4 + CD44 + T cells. Finally, the contraceptive effect was evaluated in the immunized mice. These two DNA vaccines significantly decreased a mean litter size of mice compared to pcDNA3.1, but pcD-scFv-CZP3c group showed the smallest mean litter size. The mean litter size of pcD-scFv-CZP3 were 3.2 ± 0.742 and 4.6 ± 1.118 in two mating tests, which were significantly lower than pcDNA3.1(P < 0.001 and P < 0.05). Our results suggest that the CZP3 DNA vaccine targeted with DEC-205 may be a potential strategy for developing a contraceptive DNA vaccine. Copyright © 2018 Elsevier Inc. All rights reserved.

Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa.

PubMed

Hall, Sally E; Aitken, R John; Nixon, Brett; Smith, Nathan D; Gibb, Zamira

2017-01-01

Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece; however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species. © The Authors 2016. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction.

PubMed

Illmensee, K; Kaskar, K; Zavos, P M

2006-08-01

This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

PubMed Central

2010-01-01

Background The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations. PMID:20849572

Caudal Zona Incerta/VOP Radiofrequency Lesioning Guided by Combined Stereotactic MRI and Microelectrode Recording for Posttraumatic Midbrain Resting-Kinetic Tremor.

PubMed

Contreras Lopez, William Omar; Azevedo, Angelo R; Cury, Rubens G; Alencar, Francisco; Neville, Iuri S; Reis, Paul R; Navarro, Jessie; Monaco, Bernardo; da Silva, Fabio E Fernandes; Teixeira, Manoel J; Fonoff, Erich T

2016-02-01

Reporting the outcome of two patients who underwent unilateral ablative stereotactic surgery to treat pharmacologic resistant posttraumatic tremor (PTT). We present two patients (31 and 47 years old) with refractory PTT severely affecting their quality of life. Under stereotactic guidance, refined by T2-weighted magnetic resonance imaging and double-channel multiunit microelectrode recording (MER), three sequential radiofrequency lesions were performed in the caudal zona incerta (cZi) up to the base of thalamus (VOP). Effects of cZi/VOP lesion were prospectively rated with a tremor rating scale. Both patients demonstrated intraoperative tremor suppression with sustained results up to 18 months follow-up, with improvement of 92% and 84%, respectively, on the tremor rating scale. Tremor improvement was associated with enhancement functionality and quality of life for the patients. The patients returned to their work after the procedure. No adverse effects were observed up to the last follow-up. Radiofrequency lesion of the cZi/VOP target was effective for posttraumatic tremor in both cases. The use of T2-weighted images and MER was found helpful in increasing the precision and safety of the procedure, because it leads the RF probe by relying on neighbor structures based on thalamus and subthalamic nucleus. Copyright © 2016 Elsevier Inc. All rights reserved.

Voice Tremor Outcomes of Subthalamic Nucleus and Zona Incerta Deep Brain Stimulation in Patients With Parkinson Disease.

PubMed

Karlsson, Fredrik; Malinova, Elin; Olofsson, Katarina; Blomstedt, Patric; Linder, Jan; Nordh, Erik

2018-01-17

We aimed to study the effect of deep brain stimulation (DBS) in the subthalamic nucleus (STN) and caudal zona incerta (cZi) on level of perceived voice tremor in patients with Parkinson disease (PD). This is a prospective nonrandomized design with consecutive patients. Perceived voice tremor was assessed in patients with PD having received either STN-DBS (8 patients, 5 bilateral and 3 unilateral, aged 43.1-73.6 years; median = 61.2 years) or cZi-DBS (14 bilateral patients, aged 39.0-71.9 years; median = 56.6 years) 12 months before the assessment. Sustained vowels that were produced OFF and ON stimulation (with simultaneous l-DOPA medication) were assessed perceptually in terms of voice tremor by two raters on a four-point rating scale. The assessments were repeated five times per sample and rated in a blinded and randomized procedure. Three out of the 22 patients (13%) were concluded to have voice tremor OFF stimulation. Patients with PD with STN-DBS showed mild levels of perceived voice tremor OFF stimulation and a group level improvement. Patients with moderate/severe perceived voice tremor and cZi-DBS showed marked improvements, but there was no overall group effect. Six patients with cZi-DBS showed small increases in perceived voice tremor severity. STN-DBS decreased perceived voice tremor on a group level. cZi-DBS decreased perceived voice tremor in patients with PD with moderate to severe preoperative levels of the symptom. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems

PubMed Central

Vicens, Alberto; Borziak, Kirill; Karr, Timothy L.; Roldan, Eduardo R.S.

2017-01-01

Abstract Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete. PMID:28333336

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa.

PubMed

Bromfield, Elizabeth G; Aitken, R John; Gibb, Zamira; Lambourne, Sarah R; Nixon, Brett

2014-02-01

While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

PubMed

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Forage intake and weight gain of ewes is affected by roughage mixes during winter in northeastern China.

PubMed

Yang, Zhiming; Wang, Yunbo; Yuan, Xia; Wang, Ling; Wang, Deli

2017-08-01

We studied the effect of dietary roughage species and their combinations on forage intake and growth rate of ewes during winter in a pastoral-farming area of northeast China. Twenty-five Northeast crossbred ewes (fine-wool sheep × Small-tailed Han sheep) were randomly selected and divided into five groups (G1, G2, G3, G4 and G5). During a 30 day feeding trial, each group of ewes were offered the same basal diet (composed of 0.36 kg chopped maize stalk (10 mm), 0.14 kg corn meal, 0.05 kg soybean meal and 1.2 g NaCl) and one of the five supplementary roughage mixes, namely 100% Leymus chinensis hay (G1), 100% Vigna radiata stalk (G2), L. chinensis hay plus Suaeda glauca (G3), V. radiata stalk plus S. glauca (G4) and L. chinensis hay plus V. radiata stalk and S. glauca (G5). The results showed that roughage mixes had significant influences on daily roughage intake and daily weight gain of ewes. Ewes had greater daily roughage intake when supplemented with three species of roughage compared to the roughage with one species; however, there was no significant difference between G1 and G2, G3 and G4, or between G4 and G5. The average daily gain of ewes was also greater when they were supplemented with the roughage combination of L. chinensis, V. radiata stalk and S. glauca. No difference in average daily weight gain was observed between the G4 and G5 treatments (P > 0.05). The lowest average daily weight gain was observed when the ewes were supplemented with V. radiata stalk alone (G2) (P < 0.05). The results indicated that supplying ewes with various roughages simultaneously in winter could improve their forage intake and average daily weight gain compared to offering the ewes only one type of dietary roughage. Further, feeding roughage supplements containing a diverse mix of roughage species represents one method for increasing roughage utilization in livestock production during winter in the pastoral-farming areas of northeastern China. © 2016 Japanese

Acute Ischemic Stroke Infarct Topology: Association with Lesion Volume and Severity of Symptoms at Admission and Discharge.

PubMed

Payabvash, S; Taleb, S; Benson, J C; McKinney, A M

2017-01-01

Acute stroke presentation and outcome depend on both ischemic infarct volume and location. We aimed to determine the association between acute ischemic infarct topology and lesion volume and stroke severity at presentation and discharge. Patients with acute ischemic stroke who underwent MR imaging within 24 hours of symptom onset or last seen well were included. Infarcts were segmented and coregistered on the Montreal Neurological Institute-152 brain map. Voxel-based analyses were performed to determine the distribution of infarct lesions associated with larger volumes, higher NIHSS scores at admission and discharge, and greater NIHSS/volume ratios. A total of 238 patients were included. Ischemic infarcts involving the bilateral lentiform nuclei, insular ribbons, middle corona radiata, and right precentral gyrus were associated with larger infarct volumes (average, 76.7 ± 125.6 mL versus 16.4 ± 24.0 mL, P < .001) and higher admission NIHSS scores. Meanwhile, brain stem and thalami infarctions were associated with higher admission NIHSS/volume ratios. The discharge NIHSS scores were available in 218 patients, in whom voxel-based analysis demonstrated that ischemic infarcts of the bilateral posterior insular ribbons, middle corona radiata, and right precentral gyrus were associated with more severe symptoms at discharge, whereas ischemic lesions of the brain stem, bilateral thalami, and, to a lesser extent, the middle corona radiata were associated with higher ratios of discharge NIHSS score/infarct volume. Acute ischemic infarcts of the insulae, lentiform nuclei, and middle corona radiata tend to have larger volumes, more severe presentations, and worse outcomes, whereas brain stem and thalamic infarcts have greater symptom severity relative to smaller lesion volumes. © 2017 by American Journal of Neuroradiology.

[Sexuality and STD/AIDS prevention: social representations by rural men in a county in the Zona da Mata region in Pernambuco, Brazil].

PubMed

Alves, Maria de Fátima Paz

2003-01-01

This study analyzes the concepts displayed by rural men in the Zona da Mata region in the State of Pernambuco, Brazil, concerning their sexual practices and STD/AIDS prevention. The study adopts a qualitative methodology, having interviewed 22 men According to the interviews, their first sexual intercourse is characterized as a learning experience and is sometimes marked by violence. They make a distinction between the "woman at home" and "street women"; they acknowledge women's sexual desire and value reciprocity in sexual relations, differentiating between the kinds of sex they have with different categories of women. Seven men report homoerotic experiences during adolescence, which they ascribe to immaturity, not affecting their heterosexual identity. Condom use, perceived in a negative light, is inconstant and irregular, inversely proportional to knowing the female partner. STDs in general inspire little fear, while AIDS is associated with death; the interviewees do not see themselves at risk of acquiring HIV. Ambiguities in the men's discourse, together with a basically ineffective approach by health services and preventive campaigns, reveal a high level of exposure to the risk of contracting STDs/AIDS among the interviewees and their female or male partners.

Protein docking prediction using predicted protein-protein interface.

PubMed

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability.

PubMed

Da Ros, Vanina G; Munuce, María J; Cohen, Débora J; Marín-Briggiler, Clara I; Busso, Dolores; Visconti, Pablo E; Cuasnicú, Patricia S

2004-05-01

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Actinobacteria Associated With Arbuscular Mycorrhizal Funneliformis mosseae Spores, Taxonomic Characterization and Their Beneficial Traits to Plants: Evidence Obtained From Mung Bean (Vigna radiata) and Thai Jasmine Rice (Oryza sativa)

PubMed Central

Lasudee, Krisana; Tokuyama, Shinji; Lumyong, Saisamorn; Pathom-aree, Wasu

2018-01-01

In this study, we report on the isolation of actinobacteria obtained from spores of Funneliformis mosseae and provide evidence for their potential in agricultural uses as plant growth promoters in vitro and in vivo. Actinobacteria were isolated from spores of F. mosseae using the dilution plate technique and media designed for the selective isolation of members of specific actinobacterial taxa. Six strains namely 48, S1, S3, S4, S4-1 and SP, were isolated and identified based on16S rRNA gene sequences. Phylogenetic analysis showed that isolate SP belonged to the genus Pseudonocardia with P. nantongensis KLBMP 1282T as its closest neighbor. The remaining isolates belonged to the genus Streptomyces. Two isolates, 48 and S3 were most closely related to S. thermocarboxydus DSM 44293T. Isolates S4 and S4-1 shared the highest 16S RNA gene similarity with S. pilosus NBRC 127772T. Isolate S1 showed its closest relationship with the type strain of S. spinoverrucosus NBRC14228T. The ability of these isolates to produce indole-3-acetic acid (IAA), siderophores and the ability to solubilize phosphate in vitro were examined. All isolates produced siderophores, four isolates produced IAA and two isolates solubilized inorganic phosphate at varying levels. S. thermocarboxydus isolate S3 showed the highest IAA production with high activities of phosphate solubilization and siderophore production. The inoculation of mung beans (Vigna radiata) with this strain resulted in a significant increase in fresh weight, root length and total length as an effect of IAA production. In an experiment with rice (Oryza sativa), S. thermocarboxydus isolate S3 promoted the growth of rice plants grown in low nutritional soil under induced drought stress. This report supports the view that the inoculation of rice with plant growth promoting actinobacteria mitigates some adverse effects of low nutrient and drought stress on rice. PMID:29942292

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

77 FR 51819 - Endangered Species; Receipt of Applications for Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-27

...) Radiated tortoise (Astrochelys radiata) Nile crocodile (Crocodiles niloticus) Salmon-crested cockatoo..., and viable eggs of Masked bobwhite quail (Colinus virginianus ridgwayi), as part of the identified...

Uncontacted Waorani in the Yasuní Biosphere Reserve: Geographical Validation of the Zona Intangible Tagaeri Taromenane (ZITT).

PubMed

Pappalardo, Salvatore Eugenio; De Marchi, Massimo; Ferrarese, Francesco

2013-01-01

The Tagaeri Taromenane People are two indigenous groups belonging to the Waorani first nation living in voluntary isolation within the Napo region of the western Amazon rainforest. To protect their territory the Ecuadorean State has declared and geographically defined, by Decrees, the Zona Intangible Tagaeri Taromenane (ZITT). This zone is located within the UNESCO Yasuní Biosphere Reserve (1989), one of the most biodiverse areas in the world. Due to several hydrocarbon reserve exploitation projects running in the area and the advancing of a large-scale deforestation front, the survival of these groups is presently at risk. The general aim was to validate the ZITT boundary using the geographical references included in the Decree 2187 (2007) by analyzing the geomorphological characteristics of the area. Remote sensing data such as Digital Elevation Models (DEM), Landsat imagery, topographic cartography of IGM-Ecuador, and fieldwork geographical data have been integrated and processed by Geographical Information System (GIS). The ZITT presents two levels of geographic inconsistencies. The first dimension is about the serious cartographical weaknesses in the perimeter delimitation related to the impossibility of linking two rivers belonging to different basins while the second deals with the perimeter line not respecting the hydrographic network. The GIS analysis results clearly show that ZITT boundary is cartographically nonsense due to the impossibility of mapping out the perimeter. Furthermore, GIS analysis of anthropological data shows presence of Tagaeri Taromenane clans outside the ZITT perimeter, within oil production areas and in nearby farmer settlements, reflecting the limits of protection policies for non-contacted indigenous territory. The delimitation of the ZITT followed a traditional pattern of geometric boundary not taking into account the nomadic characteristic of Tagaeri Taromenane: it is necessary to adopt geographical approaches to recognize the

Uncontacted Waorani in the Yasuní Biosphere Reserve: Geographical Validation of the Zona Intangible Tagaeri Taromenane (ZITT)

PubMed Central

Pappalardo, Salvatore Eugenio; De Marchi, Massimo; Ferrarese, Francesco

2013-01-01

The Tagaeri Taromenane People are two indigenous groups belonging to the Waorani first nation living in voluntary isolation within the Napo region of the western Amazon rainforest. To protect their territory the Ecuadorean State has declared and geographically defined, by Decrees, the Zona Intangible Tagaeri Taromenane (ZITT). This zone is located within the UNESCO Yasuní Biosphere Reserve (1989), one of the most biodiverse areas in the world. Due to several hydrocarbon reserve exploitation projects running in the area and the advancing of a large-scale deforestation front, the survival of these groups is presently at risk. The general aim was to validate the ZITT boundary using the geographical references included in the Decree 2187 (2007) by analyzing the geomorphological characteristics of the area. Remote sensing data such as Digital Elevation Models (DEM), Landsat imagery, topographic cartography of IGM-Ecuador, and fieldwork geographical data have been integrated and processed by Geographical Information System (GIS). The ZITT presents two levels of geographic inconsistencies. The first dimension is about the serious cartographical weaknesses in the perimeter delimitation related to the impossibility of linking two rivers belonging to different basins while the second deals with the perimeter line not respecting the hydrographic network. The GIS analysis results clearly show that ZITT boundary is cartographically nonsense due to the impossibility of mapping out the perimeter. Furthermore, GIS analysis of anthropological data shows presence of Tagaeri Taromenane clans outside the ZITT perimeter, within oil production areas and in nearby farmer settlements, reflecting the limits of protection policies for non-contacted indigenous territory. The delimitation of the ZITT followed a traditional pattern of geometric boundary not taking into account the nomadic characteristic of Tagaeri Taromenane: it is necessary to adopt geographical approaches to recognize the

Analysis of metal Bioleaching from thermal power plant fly ash by Aspergillus niger 34770 culture supernatant and reduction of phytotoxicity during the process.

PubMed

Jadhav, Umesh U; Hocheng, Hong

2015-01-01

Aspergillus niger culture supernatant is used for bioleaching process. Before starting bioleaching process, fly ash was washed with distilled water. This removed 100 % sodium, 47 % (±0.45) boron, 38.07 % (±0.12) calcium, 29.89 % (±0.78) magnesium, and 11.8 % (±0.05) potassium. The pH was reduced from 10.5 to 8.5 after water washing. During bioleaching process, around 100 % metal removal was achieved in 4 h for all metals except chromium 93 % (±1.18), nickel 83 % (±0.32), arsenic 78 % (±0.52), and lead 70 % (±0.20). The process parameters including temperature, shaking speed, and solid/liquid ratio were optimized for bioleaching process. Experiments were conducted to evaluate effect of fly ash on growth of mung bean (Vigna radiata). At 20 g/100 ml fly ash concentration no germination of V. radiata seeds was observed. With an increasing concentration of untreated fly ash, a gradual decrease in root/shoot length was observed. After bioleaching process 78 % (±0.19) germination of V. radiata was observed with 20 g/100 ml fly ash. This study will help to develop an efficient process to remove the toxic metals from fly ash.

Syringyl lignin production in conifers: Proof of concept in a Pine tracheary element system.

PubMed

Wagner, Armin; Tobimatsu, Yuki; Phillips, Lorelle; Flint, Heather; Geddes, Barbara; Lu, Fachuang; Ralph, John

2015-05-12

Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.

Syringyl lignin production in conifers: Proof of concept in a Pine tracheary element system

PubMed Central

Wagner, Armin; Tobimatsu, Yuki; Phillips, Lorelle; Flint, Heather; Geddes, Barbara; Lu, Fachuang; Ralph, John

2015-01-01

Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing. PMID:25902506

Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs.

PubMed

Tanihara, Fuminori; Nakai, Michiko; Men, Nguyen Thi; Kato, Noriko; Kaneko, Hiroyuki; Noguchi, Junko; Otoi, Takeshige; Kikuchi, Kazuhiro

2014-04-01

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization. © 2014 Japanese Society of Animal Science.

Reproductive biomarkers responses induced by xenoestrogens in the characid fish Astyanax fasciatus inhabiting a South American reservoir: An integrated field and laboratory approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prado, Paula S.; Pinheiro, Ana Paula B.; Bazzoli, Nilo

2014-05-01

Field studies evaluating the effects of endocrine disruption chemicals (EDCs) on the fish reproduction are scarce worldwide. The goal of this study was to assess hepatic levels of vitellogenin (Vtg), zona radiata proteins (Zrp) and insulin-like growth factors (IGF-I and IGF-II), and relating them to reproductive endpoints in a wild fish population habiting a reservoir that receive domestic sewage, agricultural and industrial residues. Adult fish Astyanax fasciatus were sampled during the reproductive season in five sites from the Furnas Reservoir, Grande River, and Paraguay–Paraná basin. As a control to field data, fish were experimentally exposed via dietary intake, to oestradiolmore » benzoate (OB) for 7 days. Fish from site with little anthropogenic interference showed hepatic levels of Vtg, Zrp and IGF-I and IGF-II similar to those from the non-treated experimental group. In sites located immediately downstream from the municipal wastewater discharges, the water total oestrogen was >120 ng/l, and male fish displayed increased Vtg and Zrp and decreased IGF-I levels similar to OB treated fish. In females, levels of Vtg, Zrp, IGF-I and IGF-II suggest an impairment of final oocyte maturation and spawning, as also detected by frequency of over-ripening, follicular atresia and fecundity. At the sites that receive agricultural and industrial residues, the water total oestrogen was <50 ng/l and females showed decreased Zrp and increased IGF-II levels associated to reduced diameter of vitellogenic follicles, indicating an inhibition of oocyte growth. Overall, the current study reports oestrogenic contamination impairing the reproduction of a wild fish from a hydroeletric reservoir and, the data contribute to improving the current knowledge on relationship between hepatic Vtg, Zrp and IGF-I and IGF-II, and reproductive endpoints in a teleost fish. In addition, our data point out novel reproductive biomarkers (IGF-I, IGF-II and over-ripening) to assessing

Optimization of protein-protein docking for predicting Fc-protein interactions.

PubMed

Agostino, Mark; Mancera, Ricardo L; Ramsland, Paul A; Fernández-Recio, Juan

2016-11-01

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc-protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc-protein complexes are available, numerous others have not yet been determined. Protein-protein docking could be used to investigate Fc-protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc-protein complexes. Based on the available set of X-ray structures of Fc-protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc-protein complexes, as well as in the design of peptides and proteins that target Fc. Copyright © 2016 John Wiley & Sons, Ltd.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Theoretical studies of protein-protein and protein-DNA binding rates

NASA Astrophysics Data System (ADS)

Alsallaq, Ramzi A.

Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

Electrostatic complementarity at protein/protein interfaces.

PubMed

McCoy, A J; Chandana Epa, V; Colman, P M

1997-05-02

Calculation of the electrostatic potential of protein-protein complexes has led to the general assertion that protein-protein interfaces display "charge complementarity" and "electrostatic complementarity". In this study, quantitative measures for these two terms are developed and used to investigate protein-protein interfaces in a rigorous manner. Charge complementarity (CC) was defined using the correlation of charges on nearest neighbour atoms at the interface. All 12 protein-protein interfaces studied had insignificantly small CC values. Therefore, the term charge complementarity is not appropriate for the description of protein-protein interfaces when used in the sense measured by CC. Electrostatic complementarity (EC) was defined using the correlation of surface electrostatic potential at protein-protein interfaces. All twelve protein-protein interfaces studied had significant EC values, and thus the assertion that protein-protein association involves surfaces with complementary electrostatic potential was substantially confirmed. The term electrostatic complementarity can therefore be used to describe protein-protein interfaces when used in the sense measured by EC. Taken together, the results for CC and EC demonstrate the relevance of the long-range effects of charges, as described by the electrostatic potential at the binding interface. The EC value did not partition the complexes by type such as antigen-antibody and proteinase-inhibitor, as measures of the geometrical complementarity at protein-protein interfaces have done. The EC value was also not directly related to the number of salt bridges in the interface, and neutralisation of these salt bridges showed that other charges also contributed significantly to electrostatic complementarity and electrostatic interactions between the proteins. Electrostatic complementarity as defined by EC was extended to investigate the electrostatic similarity at the surface of influenza virus neuraminidase where the

Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial.

PubMed

Liu, D Y; Johnston, R; Baker, H W

1995-06-01

To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP-binding ratio of test and control spermatozoa averaged 0.82 (range 0.51-1.13).(ABSTRACT TRUNCATED AT 250 WORDS)

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

PubMed

Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

2016-11-15

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Molecular modelling of protein-protein/protein-solvent interactions

NASA Astrophysics Data System (ADS)

Luchko, Tyler

The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

Conformational Heterogeneity of Unbound Proteins Enhances Recognition in Protein-Protein Encounters.

PubMed

Pallara, Chiara; Rueda, Manuel; Abagyan, Ruben; Fernández-Recio, Juan

2016-07-12

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles.

PubMed

Petersen, Claudia G; Vagnini, Laura D; Mauri, Ana L; Massaro, Fabiana C; Silva, Liliane F I; Cavagna, Mario; Baruffi, Ricardo L R; Oliveira, Joao B A; Franco, José G

2011-04-23

This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). The current study demonstrated an absence of

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

PubMed

Hernández-Pichardo, J E; Ducolomb, Y; Romo, S; Kjelland, M E; Fierro, R; Casillas, F; Betancourt, M

2016-01-01

In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents ( P  < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n  = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively ( P  < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) ( P  > 0.05). Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU�

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

UDoNC: An Algorithm for Identifying Essential Proteins Based on Protein Domains and Protein-Protein Interaction Networks.

PubMed

Peng, Wei; Wang, Jianxin; Cheng, Yingjiao; Lu, Yu; Wu, Fangxiang; Pan, Yi

2015-01-01

Prediction of essential proteins which are crucial to an organism's survival is important for disease analysis and drug design, as well as the understanding of cellular life. The majority of prediction methods infer the possibility of proteins to be essential by using the network topology. However, these methods are limited to the completeness of available protein-protein interaction (PPI) data and depend on the network accuracy. To overcome these limitations, some computational methods have been proposed. However, seldom of them solve this problem by taking consideration of protein domains. In this work, we first analyze the correlation between the essentiality of proteins and their domain features based on data of 13 species. We find that the proteins containing more protein domain types which rarely occur in other proteins tend to be essential. Accordingly, we propose a new prediction method, named UDoNC, by combining the domain features of proteins with their topological properties in PPI network. In UDoNC, the essentiality of proteins is decided by the number and the frequency of their protein domain types, as well as the essentiality of their adjacent edges measured by edge clustering coefficient. The experimental results on S. cerevisiae data show that UDoNC outperforms other existing methods in terms of area under the curve (AUC). Additionally, UDoNC can also perform well in predicting essential proteins on data of E. coli.

Aurelia aurita (Cnidaria) Oocytes' Contact Plate Structure and Development

PubMed Central

Adonin, Leonid S.; Shaposhnikova, Tatyana G.; Podgornaya, Olga

2012-01-01

One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the “contact plate”. Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high Mr bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides Mr corresponds to that of mesogleal cells, the other ones' Mr is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida. PMID:23185235

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes

PubMed Central

Trapphoff, T.; Heiligentag, M.; Dankert, D.; Demond, H.; Deutsch, D.; Fröhlich, T.; Arnold, G.J.; Grümmer, R.; Horsthemke, B.; Eichenlaub-Ritter, U.

2016-01-01

ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). MATERIAL AND METHODS GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). MAIN RESULTS AND ROLE OF CHANCE The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

PubMed

Trapphoff, T; Heiligentag, M; Dankert, D; Demond, H; Deutsch, D; Fröhlich, T; Arnold, G J; Grümmer, R; Horsthemke, B; Eichenlaub-Ritter, U

2016-01-01

oocytes were analyzed by selected reaction monitoring (SRM). GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly

Hydrogen-rich Water Exerting a Protective Effect on Ovarian Reserve Function in a Mouse Model of Immune Premature Ovarian Failure Induced by Zona Pellucida 3

PubMed Central

He, Xin; Wang, Shu-Yu; Yin, Cheng-Hong; Wang, Tong; Jia, Chan-Wei; Ma, Yan-Min

2016-01-01

Background: Premature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model. Methods: To set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software. Results: Immune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Protein-protein interaction network-based detection of functionally similar proteins within species.

PubMed

Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

2012-07-01

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

PubMed Central

TAKEO, Shun; SATO, Daichi; KIMURA, Koji; MONJI, Yasunori; KUWAYAMA, Takehito; KAWAHARA-MIKI, Ryoka; IWATA, Hisataka

2013-01-01

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization. PMID:24390595

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: a possible role for cyclin E.

PubMed

Mendonça, Pedro O R de; Lotfi, Claudimara F P

2011-04-10

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Human homolog of the mouse sperm receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chamberlin, M.E.; Dean, J.

1990-08-01

The human zona pellucida, composed of three glycoproteins (ZP1, ZP2, and ZP3), forms an extracellular matrix that surrounds ovulated eggs and mediates species-specific fertilization. The genes that code for at least two of the zona proteins (ZP2 and ZP3) cross-hybridize with other mammalian DNA. The recently characterized mouse sperm receptor gene (Zp-3) was used to isolate its human homolog. The human homolog spans {approx}18.3 kilobase pairs (kbp) (compared to 8.6 kbp for the mouse gene) and contains eight exons, the sizes of which are strictly conserved between the two species. Four short (8-15 bp) sequences within the first 250 bpmore » of the 5{prime} flanking region in the human Zp-3 homolog are also present upstream of mouse Zp-3. These elements may modulate oocyte-specific gene expression. By using the polymerase chain reaction, a full-length cDNA of human ZP3 was isolated from human ovarian poly(A){sup +} RNA and used to deduce the structure of human ZP3 mRNA. Certain features of the human and mouse ZP3 transcripts are conserved. Both have unusually short 5{prime} and 3{prime} untranslated regions, both contain a single open reading frame that is 74% identical, and both code for 424 amino acid polypeptides that are 67% the same. The similarity between the two proteins may define domains that are important in maintaining the structural integrity of the zona pellucida, while the differences may play a role in mediating the species-specific events of mammalian fertilization.« less

Protein-Protein Docking in Drug Design and Discovery.

PubMed

Kaczor, Agnieszka A; Bartuzi, Damian; Stępniewski, Tomasz Maciej; Matosiuk, Dariusz; Selent, Jana

2018-01-01

Protein-protein interactions (PPIs) are responsible for a number of key physiological processes in the living cells and underlie the pathomechanism of many diseases. Nowadays, along with the concept of so-called "hot spots" in protein-protein interactions, which are well-defined interface regions responsible for most of the binding energy, these interfaces can be targeted with modulators. In order to apply structure-based design techniques to design PPIs modulators, a three-dimensional structure of protein complex has to be available. In this context in silico approaches, in particular protein-protein docking, are a valuable complement to experimental methods for elucidating 3D structure of protein complexes. Protein-protein docking is easy to use and does not require significant computer resources and time (in contrast to molecular dynamics) and it results in 3D structure of a protein complex (in contrast to sequence-based methods of predicting binding interfaces). However, protein-protein docking cannot address all the aspects of protein dynamics, in particular the global conformational changes during protein complex formation. In spite of this fact, protein-protein docking is widely used to model complexes of water-soluble proteins and less commonly to predict structures of transmembrane protein assemblies, including dimers and oligomers of G protein-coupled receptors (GPCRs). In this chapter we review the principles of protein-protein docking, available algorithms and software and discuss the recent examples, benefits, and drawbacks of protein-protein docking application to water-soluble proteins, membrane anchoring and transmembrane proteins, including GPCRs.

Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

PubMed Central

Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

2015-01-01

Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

Protein function prediction using neighbor relativity in protein-protein interaction network.

PubMed

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein Structure Prediction by Protein Threading

NASA Astrophysics Data System (ADS)

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

Predicting protein-protein interactions from protein domains using a set cover approach.

PubMed

Huang, Chengbang; Morcos, Faruck; Kanaan, Simon P; Wuchty, Stefan; Chen, Danny Z; Izaguirre, Jesús A

2007-01-01

One goal of contemporary proteome research is the elucidation of cellular protein interactions. Based on currently available protein-protein interaction and domain data, we introduce a novel method, Maximum Specificity Set Cover (MSSC), for the prediction of protein-protein interactions. In our approach, we map the relationship between interactions of proteins and their corresponding domain architectures to a generalized weighted set cover problem. The application of a greedy algorithm provides sets of domain interactions which explain the presence of protein interactions to the largest degree of specificity. Utilizing domain and protein interaction data of S. cerevisiae, MSSC enables prediction of previously unknown protein interactions, links that are well supported by a high tendency of coexpression and functional homogeneity of the corresponding proteins. Focusing on concrete examples, we show that MSSC reliably predicts protein interactions in well-studied molecular systems, such as the 26S proteasome and RNA polymerase II of S. cerevisiae. We also show that the quality of the predictions is comparable to the Maximum Likelihood Estimation while MSSC is faster. This new algorithm and all data sets used are accessible through a Web portal at http://ppi.cse.nd.edu.

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed Central

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-01-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. PMID:21666777

Detection of protein complex from protein-protein interaction network using Markov clustering

NASA Astrophysics Data System (ADS)

Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

2017-05-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

Syringyl lignin production in conifers: Proof of concept in a Pine tracheary element system

DOE PAGES

Wagner, Armin; Tobimatsu, Yuki; Phillips, Lorelle; ...

2015-04-20

Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformationmore » with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production. In conclusion, our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.« less

Viability of cumulus cells is associated with basal AMH levels in assisted reproduction.

PubMed

Ebner, Thomas; Shebl, Omar; Holzer, Sandra; Oppelt, Peter; Petek, Erwin; Schappacher-Tilp, Gudrun; Mayer, Richard B

2014-12-01

An interesting non-invasive approach to select embryos for transfer is analyzing the health state of somatic granulosa cells surrounding the oocyte addressing their mutual dependence. This prospective study was set up to analyse whether the DNA integrity of cumulus cells correlates with preimplantation development and basal AMH levels. Therefore, 56 patients who gave written consent were enrolled. Sequential denudation of the cumulus-oocyte-complexes was performed in order to separate corona radiata from outer cumulus cells. DNA integrity of both cell types was analysed using a modified chromatin dispersion test. The percentage of viable corona radiata cells per patient showed a linear correlation to blastulation (P<0.05). These innermost cells showed significantly lower rates of strand breaks (P<0.01) as compared to outer cumulus cells. Age-corrected AMH was significantly associated with the DNA integrity of outer cumulus cells (P<0.05). For the first time it could be shown that in fact clinical embryologists deal with two different entities of cumulus cells, inner and outer ones. It seems that any protective mechanism of the female gamete follows an outward gradient, so that negative effects, e.g. apoptosis, may impair outer cumulus cells first. Age-corrected AMH reflects quality of these outer cumulus cells. AMH; Corona radiata cells; DNA fragmentation; Outer cumulus cells; SCD test. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

PubMed Central

Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

2013-01-01

While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface

GDF9 and BMP15 Expressions and Fine Structure Changes During Folliculogenesis in Polycystic Ovary Syndrome.

PubMed

Karagül, Meryem İlkay; Aktaş, Savaş; Coşkun Yılmaz, Banu; Yılmaz, Mustafa; Orekici Temel, Gülhan

2018-01-20

Polycystic ovary syndrome is the most frequently seen endocrine disorder in women of reproductive age with a prevalence of about 10%. To investigate the efficiency of growth differentiation factor 9 and bone morphogenetic protein 15 during folliculogenesis in a dehydroepiandrosterone-induced mouse Polycystic ovary syndrome model. Animal experimentation. Mice were divided into 3 groups: control, vehicle and Polycystic ovary syndrome. Polycystic ovary syndrome model mice were developed by the injection of dehydroepiandrosterone dissolved in 0.1 mL of sesame oil. Ovarian tissues were examined for growth differentiation factor 9 and bone morphogenetic protein 15 using immunofluorescent labelling and electron microscopic examinations. The immunoreactivity of growth differentiation factor 9 and bone morphogenetic protein 15 proteins decreased (p<0.05) in the Polycystic ovary syndrome group (27.73±8.43 and 24.85±7.03, respectively) compared with the control group (33.72±11.22 and 31.12±11.05, respectively) and vehicle group (33.95±10.75 and 29.99±10.72, respectively). Apoptotic changes were observed in granulosa cells, lipid vacuoles increased in Theca cells and thickening and irregularities were noted in the basal lamina of granulosa cells. An increased electron density in the zona pellucida in some of the multilaminar primary and secondary follicles in the Polycystic ovary syndrome model was also observed at the ultrastructural level. These results suggest that the decrease in the growth differentiation factor 9 and bone morphogenetic protein 15 expression initiated at the primary follicle stage effect the follicle development and zona pellucida structure and may cause subfertility or infertility in Polycystic ovary syndrome.

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-06-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. © 2011 Elsevier Ltd. All rights reserved.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Assessment of the reliability of protein-protein interactions and protein function prediction.

PubMed

Deng, Minghua; Sun, Fengzhu; Chen, Ting

2003-01-01

As more and more high-throughput protein-protein interaction data are collected, the task of estimating the reliability of different data sets becomes increasingly important. In this paper, we present our study of two groups of protein-protein interaction data, the physical interaction data and the protein complex data, and estimate the reliability of these data sets using three different measurements: (1) the distribution of gene expression correlation coefficients, (2) the reliability based on gene expression correlation coefficients, and (3) the accuracy of protein function predictions. We develop a maximum likelihood method to estimate the reliability of protein interaction data sets according to the distribution of correlation coefficients of gene expression profiles of putative interacting protein pairs. The results of the three measurements are consistent with each other. The MIPS protein complex data have the highest mean gene expression correlation coefficients (0.256) and the highest accuracy in predicting protein functions (70% sensitivity and specificity), while Ito's Yeast two-hybrid data have the lowest mean (0.041) and the lowest accuracy (15% sensitivity and specificity). Uetz's data are more reliable than Ito's data in all three measurements, and the TAP protein complex data are more reliable than the HMS-PCI data in all three measurements as well. The complex data sets generally perform better in function predictions than do the physical interaction data sets. Proteins in complexes are shown to be more highly correlated in gene expression. The results confirm that the components of a protein complex can be assigned to functions that the complex carries out within a cell. There are three interaction data sets different from the above two groups: the genetic interaction data, the in-silico data and the syn-express data. Their capability of predicting protein functions generally falls between that of the Y2H data and that of the MIPS protein complex

Building protein-protein interaction networks for Leishmania species through protein structural information.

PubMed

Dos Santos Vasconcelos, Crhisllane Rafaele; de Lima Campos, Túlio; Rezende, Antonio Mauro

2018-03-06

Systematic analysis of a parasite interactome is a key approach to understand different biological processes. It makes possible to elucidate disease mechanisms, to predict protein functions and to select promising targets for drug development. Currently, several approaches for protein interaction prediction for non-model species incorporate only small fractions of the entire proteomes and their interactions. Based on this perspective, this study presents an integration of computational methodologies, protein network predictions and comparative analysis of the protozoan species Leishmania braziliensis and Leishmania infantum. These parasites cause Leishmaniasis, a worldwide distributed and neglected disease, with limited treatment options using currently available drugs. The predicted interactions were obtained from a meta-approach, applying rigid body docking tests and template-based docking on protein structures predicted by different comparative modeling techniques. In addition, we trained a machine-learning algorithm (Gradient Boosting) using docking information performed on a curated set of positive and negative protein interaction data. Our final model obtained an AUC = 0.88, with recall = 0.69, specificity = 0.88 and precision = 0.83. Using this approach, it was possible to confidently predict 681 protein structures and 6198 protein interactions for L. braziliensis, and 708 protein structures and 7391 protein interactions for L. infantum. The predicted networks were integrated to protein interaction data already available, analyzed using several topological features and used to classify proteins as essential for network stability. The present study allowed to demonstrate the importance of integrating different methodologies of interaction prediction to increase the coverage of the protein interaction of the studied protocols, besides it made available protein structures and interactions not previously reported.

Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

PubMed

He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

2009-12-31

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production.

PubMed

Lane, V M; Liu, I K M; Casey, K; vanLeeuwen, E M G; Flanagan, D R; Murata, K; Munro, C

2007-01-01

The present 2-year study investigated the feasibility of using porcine zona pellucidae (pZP) as antigen for immunocontraception in American black bears. Sows, 3-6 years of age, were administered either two doses of 250 microg pZP with Freund's adjuvant (n = 10) or adjuvant alone (n = 5), one in April and one in May, and were kept away from the boars until June. Serum samples were collected before injections and before denning (November). The presence of sows with cubs at side was observed during premature emergence from denning. First-year results indicated that anti-pZP antibody titres in vaccinated sows were 2.5-9.0-fold (range) higher compared with non-vaccinated sows and that the vaccinated sows were threefold less likely to become pregnant (P = 0.167). Control and vaccinated bears produced 1.6 and 0.2 cubs per sow, respectively (P = 0.06). The second-year study investigated the feasibility of using pZP sequestered in a controlled-release pellet and a water-soluble adjuvant (QS-21) to avoid regulatory problems associated with Freund's adjuvant. Sows in the treatment group (n = 22) were administered a single dose of an emulsion of 250 microg pZP and 150 microg QS-21 plus a pellet containing 70-90 microg pZP for delayed release as booster dose. Control sows (n = 5) received the QS-21 adjuvant in pellet alone. Serum samples were collected before inoculations (April) and before denning (November). Seven cubs were born to the five control sows, but none was born to the 22 vaccinated sows (P < 0.001). Anti-pZP antibody mean absorbance ratios in control sows remained at background levels, whereas vaccinated sows had ratios fourfold higher than controls. Two-dimensional polyacrylamide gel electrophoresis and immunohistochemical localisation confirmed immunoreactivity of sera from inoculated bears. We conclude that cub production in the American black bear can be effectively limited with either two injections of 250 microg pZP or a single inoculation of partially

The evaluation of xenotransplantation of feline ovarian tissue vitrified by needle immersed vitrification technique into male immunodeficient mice.

PubMed

Demirel, Mürşide Ayşe; Acar, Duygu Baki; Ekim, Burcu; Çelikkan, Ferda Topal; Alkan, Kübra Karakaş; Salar, Seçkin; Erdemli, Esra Atabenli; Özkavukçu, Sinan; Yar, Seda Sağlam; Kanca, Halit; Baştan, Ayhan

2018-03-01

In this study, the efficiency of the "Needle Immersed Vitrification" technique was tested on cryopreserved feline ovarian tissue. For vitrification, ovarian fragments (0.5-1.5 mm 2 ) from each ovary were collected; the grafts were exposed to 7.5-15% ethylene glycol and 7.5-15% dimethyl sulfoxide at room temperature and stored in liquid nitrogen at least 1 week. Morphologic examinations, expression of genes such as B cell lymphoma 2, B-cell lymphoma-2-associated X protein, Bone morphogenetic protein 15, zone of polarizing activity, zona pellucida C protein and DNA (cytosine-5)-methyltransferase 1, ultrastructural analysis and viability tests were carried out from collected grafts. Light microscopy examinations revealed the percentage of morphologically normal primordial follicles in a fresh group which was significantly higher than the treatment groups (p < 0.001). Terminal deoxynucleotidyl transferase dUTP nick end labeling and anti-caspase-3 staining observed in oocytes, follicle cells, interstitial tissue showed higher rates of apoptosis for post-vitrification and -transplantation groups than freshly grafted ovarian tissues. Furthermore, we observed significant downregulation of zone of polarizing activity and zona pellucida C protein gene expression in vitrified ovarian tissue grafts than in the fresh grafts (p < 0.05). In conclusion, we suggest that the needle immersed vitrification method is a convenient, cheap, and feasible vitrification method for cat ovarian tissues. However, further studies need to be performed to determine more optimal vitrification solutions and equilibration times for the needle immersed vitrification method in order to adapt it for cat ovaries.

PREFACE: Protein protein interactions: principles and predictions

NASA Astrophysics Data System (ADS)

Nussinov, Ruth; Tsai, Chung-Jung

2005-06-01

Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

Protein complex prediction in large ontology attributed protein-protein interaction networks.

PubMed

Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng; Xu, Bo

2013-01-01

Protein complexes are important for unraveling the secrets of cellular organization and function. Many computational approaches have been developed to predict protein complexes in protein-protein interaction (PPI) networks. However, most existing approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology (GO) annotation information. In this paper, we constructed ontology attributed PPI networks with PPI data and GO resource. After constructing ontology attributed networks, we proposed a novel approach called CSO (clustering based on network structure and ontology attribute similarity). Structural information and GO attribute information are complementary in ontology attributed networks. CSO can effectively take advantage of the correlation between frequent GO annotation sets and the dense subgraph for protein complex prediction. Our proposed CSO approach was applied to four different yeast PPI data sets and predicted many well-known protein complexes. The experimental results showed that CSO was valuable in predicting protein complexes and achieved state-of-the-art performance.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

C-Myc Protein-Protein and Protein-DNA Interactions: Targets for Therapeutic Intervention.

DTIC Science & Technology

1997-09-01

including those of the Myc family. In fact, members of different bHLH protein subgroups, including the Myc proteins, are characterized by conserved BR...important functional consequences, and they provide insights into how different bHLH proteins can act on different targets. The zinc finger protein...roles for a number of BR residues which do not contact bases, yet are conserved within different bHLH protein sub- families (Benezra et al. 1990), and

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

PubMed Central

Vendruscolo, Michele; Knowles, Tuomas P.J.; Dobson, Christopher M.

2011-01-01

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility. PMID:21825020

Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

PubMed

Yang, Lei; Zhao, Xudong; Tang, Xianglong

2014-01-01

Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

Regulation of protein turnover by heat shock proteins.

PubMed

Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

2014-12-01

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

Principles of Protein Recognition and Properties of Protein-protein Interfaces

NASA Astrophysics Data System (ADS)

Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth

In this chapter we address two aspects - the static physical interactions which allow the information transfer for the function to be performed; and the dynamic, i.e. how the information is transmitted between the binding sites in the single protein molecule and in the network. We describe the single protein molecules and their complexes; and the analogy between protein folding and protein binding. Eventually, to fully understand the interactome and how it performs the essential cellular functions, we have to put all together - and hierarchically progress through the system.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009–31 January 2010

USDA-ARS?s Scientific Manuscript database

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primiti...

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Predicting permanent and transient protein-protein interfaces.

PubMed

La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

2013-05-01

Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. Copyright © 2013 Wiley Periodicals, Inc.

Molecular simulations of lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

2008-08-01

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.

An Aspergillus aculateus strain was capable of producing agriculturally useful nanoparticles via bioremediation of iron ore tailings.

PubMed

Bedi, Ankita; Singh, Braj Raj; Deshmukh, Sunil K; Adholeya, Alok; Barrow, Colin J

2018-06-01

Mining waste such as iron ore tailing is environmentally hazardous, encouraging researchers to develop effective bioremediation technologies. Among the microbial isolates collected from iron ore tailings, Aspergillus aculeatus (strain T6) showed good leaching efficiency and produced iron-containing nanoparticles under ambient conditions. This strain can convert iron ore tailing waste into agriculturally useful nanoparticles. Fourier-transform Infrared Spectroscopy (FT-IR analysis) established the at the particles are protein coated, with energy dispersive X-ray Spectroscopy (EDX analysis) showing strong signals for iron. Transmission Electron Microscopy (TEM analysis) showed semi-quasi spherical particles having average size of 15 ± 5 nm. These biosynthesized nanoparticles when tested for their efficacy on seed emergence activity of mungbean (Vigna radiata) seeds, and enhanced plant growth at 10 and 20 ppm. Copyright © 2018 Elsevier Ltd. All rights reserved.

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome.

PubMed

Meczekalski, B; Nawrot, R; Nowak, W; Czyzyk, A; Kedzia, H; Gozdzicka-Jozefiak, A

2015-07-01

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte. We analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes. The expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices. Data from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Nature and consequences of protein-protein interactions in high protein concentration solutions.

PubMed

Saluja, Atul; Kalonia, Devendra S

2008-06-24

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein-protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.

Computer Simulation of Protein-Protein and Protein-Peptide Interactions

DTIC Science & Technology

1983-12-08

a full molecular dynamic z simulation is performed, with resulting dipolar re - laxation. However, this is prohibitive when a large . number of...1993 Dr. Mike Marron Program Manager Molecular Biology Office of Naval Research 800 N. Quincy Street Arlington, VA 22217 Dear Mike, Here is the...rztnbutior is unLi--ited. , 93-30630 98 12 � 12/08/93 01/0/92-;03/31/93: Final Report, Computer Simulation of Protein-Protein and Protein-Peptide

Prediction of physical protein protein interactions

NASA Astrophysics Data System (ADS)

Szilágyi, András; Grimm, Vera; Arakaki, Adrián K.; Skolnick, Jeffrey

2005-06-01

Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. In recent years, new experimental techniques have been developed to discover the protein-protein interaction networks of several organisms. However, the accuracy and coverage of these techniques have proven to be limited, and computational approaches remain essential both to assist in the design and validation of experimental studies and for the prediction of interaction partners and detailed structures of protein complexes. Here, we provide a critical overview of existing structure-independent and structure-based computational methods. Although these techniques have significantly advanced in the past few years, we find that most of them are still in their infancy. We also provide an overview of experimental techniques for the detection of protein-protein interactions. Although the developments are promising, false positive and false negative results are common, and reliable detection is possible only by taking a consensus of different experimental approaches. The shortcomings of experimental techniques affect both the further development and the fair evaluation of computational prediction methods. For an adequate comparative evaluation of prediction and high-throughput experimental methods, an appropriately large benchmark set of biophysically characterized protein complexes would be needed, but is sorely lacking.

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

PubMed

Paulmurugan, R; Gambhir, S S

2003-04-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu,P.

2007-01-01

The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behaviormore » and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.« less

CONVERSION OF PLASMA PROTEIN TO TISSUE PROTEIN WITHOUT EVIDENCE OF PROTEIN BREAKDOWN

PubMed Central

Yuile, C. L.; Lamson, B. G.; Miller, L. L.; Whipple, G. H.

1951-01-01

Labeled plasma proteins obtained from donor dogs, previously fed ε-C14-dl-lysine, have been given intravenously to recipient dogs. The disappearance of labeled globulin from the plasma at a rate considerably faster than albumin has been confirmed. Evidence suggesting that the mass of protein in solution in the extravascular, extracellular fluid is approximately equal to the plasma proteins in circulation has been derived from a study of the dilution of labeled plasma protein by repeated injections of non-labeled plasma protein. In a period of 7 days the transfer of C14 from plasma to tissue proteins amounted to between 30 and 40 per cent of the activity in the labeled plasma protein injected intravenously. The conversion was accompanied by a very small loss of activity in the urine and expired air and the activity remained in the lysine residue of the liver and probably of other tissues. The data presented favor the view that plasma proteins are utilized in the body economy after partial catabolism within the cell area and provide no evidence of complete breakdown to the amino acid level. PMID:14832401

Benchmarking protein-protein interface predictions: why you should care about protein size.

PubMed

Martin, Juliette

2014-07-01

A number of predictive methods have been developed to predict protein-protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over-estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein-protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. © 2014 Wiley Periodicals, Inc.

Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

PubMed Central

Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

2011-01-01

Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

Rigid-Docking Approaches to Explore Protein-Protein Interaction Space.

PubMed

Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ohue, Masahito; Akiyama, Yutaka

Protein-protein interactions play core roles in living cells, especially in the regulatory systems. As information on proteins has rapidly accumulated on publicly available databases, much effort has been made to obtain a better picture of protein-protein interaction networks using protein tertiary structure data. Predicting relevant interacting partners from their tertiary structure is a challenging task and computer science methods have the potential to assist with this. Protein-protein rigid docking has been utilized by several projects, docking-based approaches having the advantages that they can suggest binding poses of predicted binding partners which would help in understanding the interaction mechanisms and that comparing docking results of both non-binders and binders can lead to understanding the specificity of protein-protein interactions from structural viewpoints. In this review we focus on explaining current computational prediction methods to predict pairwise direct protein-protein interactions that form protein complexes.

Reverse Nearest Neighbor Search on a Protein-Protein Interaction Network to Infer Protein-Disease Associations.

PubMed

Suratanee, Apichat; Plaimas, Kitiporn

2017-01-01

The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse k -nearest neighbor (R k NN) search. The R k NN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the R k NN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases.

Molecular identity and gene expression of aldosterone synthase cytochrome P450

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamoto, Mitsuhiro; Nonaka, Yasuki; Takemori, Hiroshi

11{beta}-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11{beta}-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolatedmore » from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.« less

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Bolamba, D.; Patino, R.; Yoshizaki, G.; Thomas, P.

2003-01-01

Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-??m segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P<0.05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. ?? 2003 Elsevier Science (USA). All rights reserved.

Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

PubMed

Solano, Jaime; Anabalón, Leonardo; Encina, Francisco

2016-03-01

Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Delayed recovery of the affected finger extensors at chronic stage in a stroke patient: A case report.

PubMed

Jang, Sung Ho; Lee, Han Do

2017-10-01

A 33-year-old male presented with complete weakness of the right extremities due to corona radiata infarct. The main concerns of the patient is recovery of hand function especially related to finger extension. Right corona radiata infarct. He underwent physical therapy and occupational therapy at the outpatient clinic of the rehabilitation department of the same university hospital until 2 years after onset. In addition, he underwent neuromuscular electrical stimulation for the right finger extensors continuously until 4 years after onset. At 6 months after onset, the weakness of his right side recovered to subnormal state except for the right finger extensors which were completely weak. At 1.5 years after onset, the right finger extensors began to show slow and continuous recovery. At 4 years after onset, the patient showed motor recovery in the right finger extensors to the extent that he was able to move against gravity. Discontinuation of the left corticospinal tract was observed on 2-month diffusion tensor tractography (DTT); however, the integrity of this discontinuation had recovered to the primary motor cortex on 4-year DTT. On 2-month transcranial magnetic stimulation (TMS), no motor-evoked potential was evoked; in contrast, motor-evoked potentials were obtained at the right-hand muscle on 4-year TMS study. We demonstrated unusual delayed and long-term recovery of the affected finger extensors in a patient with corona radiata infarct using DTT and TMS.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

PubMed

Jaremko, Matt J; Lee, D John; Patel, Ashay; Winslow, Victoria; Opella, Stanley J; McCammon, J Andrew; Burkart, Michael D

2017-10-10

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Protein-Protein Interface and Disease: Perspective from Biomolecular Networks.

PubMed

Hu, Guang; Xiao, Fei; Li, Yuqian; Li, Yuan; Vongsangnak, Wanwipa

Protein-protein interactions are involved in many important biological processes and molecular mechanisms of disease association. Structural studies of interfacial residues in protein complexes provide information on protein-protein interactions. Characterizing protein-protein interfaces, including binding sites and allosteric changes, thus pose an imminent challenge. With special focus on protein complexes, approaches based on network theory are proposed to meet this challenge. In this review we pay attention to protein-protein interfaces from the perspective of biomolecular networks and their roles in disease. We first describe the different roles of protein complexes in disease through several structural aspects of interfaces. We then discuss some recent advances in predicting hot spots and communication pathway analysis in terms of amino acid networks. Finally, we highlight possible future aspects of this area with respect to both methodology development and applications for disease treatment.

Protein-protein recognition control by modulating electrostatic interactions.

PubMed

Han, Song; Yin, Shijin; Yi, Hong; Mouhat, Stéphanie; Qiu, Su; Cao, Zhijian; Sabatier, Jean-Marc; Wu, Yingliang; Li, Wenxin

2010-06-04

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Biophysics of protein evolution and evolutionary protein biophysics

PubMed Central

Sikosek, Tobias; Chan, Hue Sun

2014-01-01

The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

Protein and protein hydrolysates in sports nutrition.

PubMed

van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

2007-08-01

With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

A modified Lowry protein test for dilute protein solutions

Treesearch

Garold F. Gregory; Keith F. Jensen

1971-01-01

A modified Lowry protein test for dilute protein solutions modified Lowry protein test was compared with the standard Lowry protein test. The modified test was found to give estimates of protein concentration that were as good as the standard test and has the advange that proteins can be measured in very dilute solutions.

The Role of Shape Complementarity in the Protein-Protein Interactions

NASA Astrophysics Data System (ADS)

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-11-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody-antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity.

Electrostatic design of protein-protein association rates.

PubMed

Schreiber, Gideon; Shaul, Yossi; Gottschalk, Kay E

2006-01-01

De novo design and redesign of proteins and protein complexes have made promising progress in recent years. Here, we give an overview of how to use available computer-based tools to design proteins to bind faster and tighter to their protein-complex partner by electrostatic optimization between the two proteins. Electrostatic optimization is possible because of the simple relation between the Debye-Huckel energy of interaction between a pair of proteins and their rate of association. This can be used for rapid, structure-based calculations of the electrostatic attraction between the two proteins in the complex. Using these principles, we developed two computer programs that predict the change in k(on), and as such the affinity, on introducing charged mutations. The two programs have a web interface that is available at www.weizmann.ac.il/home/bcges/PARE.html and http://bip.weizmann.ac.il/hypare. When mutations leading to charge optimization are introduced outside the physical binding site, the rate of dissociation is unchanged and therefore the change in k(on) parallels that of the affinity. This design method was evaluated on a number of different protein complexes resulting in binding rates and affinities of hundreds of fold faster and tighter compared to wild type. In this chapter, we demonstrate the procedure and go step by step over the methodology of using these programs for protein-association design. Finally, the way to easily implement the principle of electrostatic design for any protein complex of choice is shown.

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

PubMed

Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

2018-01-15

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

A Method for Predicting Protein Complexes from Dynamic Weighted Protein-Protein Interaction Networks.

PubMed

Liu, Lizhen; Sun, Xiaowu; Song, Wei; Du, Chao

2018-06-01

Predicting protein complexes from protein-protein interaction (PPI) network is of great significance to recognize the structure and function of cells. A protein may interact with different proteins under different time or conditions. Existing approaches only utilize static PPI network data that may lose much temporal biological information. First, this article proposed a novel method that combines gene expression data at different time points with traditional static PPI network to construct different dynamic subnetworks. Second, to further filter out the data noise, the semantic similarity based on gene ontology is regarded as the network weight together with the principal component analysis, which is introduced to deal with the weight computing by three traditional methods. Third, after building a dynamic PPI network, a predicting protein complexes algorithm based on "core-attachment" structural feature is applied to detect complexes from each dynamic subnetworks. Finally, it is revealed from the experimental results that our method proposed in this article performs well on detecting protein complexes from dynamic weighted PPI networks.

A protein interaction network analysis for yeast integral membrane protein.

PubMed

Shi, Ming-Guang; Huang, De-Shuang; Li, Xue-Ling

2008-01-01

Although the yeast Saccharomyces cerevisiae is the best exemplified single-celled eukaryote, the vast number of protein-protein interactions of integral membrane proteins of Saccharomyces cerevisiae have not been characterized by experiments. Here, based on the kernel method of Greedy Kernel Principal Component analysis plus Linear Discriminant Analysis, we identify 300 protein-protein interactions involving 189 membrane proteins and get the outcome of a highly connected protein-protein interactions network. Furthermore, we study the global topological features of integral membrane proteins network of Saccharomyces cerevisiae. These results give the comprehensive description of protein-protein interactions of integral membrane proteins and reveal global topological and robustness of the interactome network at a system level. This work represents an important step towards a comprehensive understanding of yeast protein interactions.

Unresolved issues in mammalian fertilization.

PubMed

Olds-Clarke, Patricia

2003-01-01

This review considers the role of the sperm in fertilization, addressing areas of misunderstanding and unfounded assumptions and taking particular advantage of the large body of data resulting from work with rodent species in vitro. Considerable attention is given to the appropriate use and interpretation of assays for capacitation, acrosomal exocytosis, hyperactivation, and sperm protein phosphorylation, as well as tests for sperm-zona and sperm-oocyte membrane interactions. The lack of general agreement on the means of sperm adhesion to and penetration of the zona pellucida is addressed, and the need for new approaches to this problem is pointed out. Some molecular advances in our understanding of specific steps in the process of fertilization are discussed in the context of intact cell-matrix and cell-cell interaction. This review should provide practical information for researchers just beginning the study of fertilization and interesting but not widely known observations to stimulate new ideas in experienced scientists.

Milestones in contraceptive vaccines development and hurdles in their application

PubMed Central

Gupta, Satish Kumar; Shrestha, Abhinav; Minhas, Vidisha

2014-01-01

Contraceptive vaccines have been proposed for controlling the growing human population and wildlife population management. Multiple targets such as gonadotropin releasing hormone (GnRH), luteinizing hormone, follicle stimulating hormone, gonadotropin receptors, sperm-specific proteins and zona pellucida glycoproteins have been exploited to develop contraceptive vaccine and their efficacy investigated and shown in various experimental animal models. Vaccines based on GnRH have found application in immuno-castration of male pigs for prevention of boar-taint. Vaccines based on zona pellucida glycoproteins have shown promising results for population management of wild horses and white-tailed deer. Phase II clinical trials in women with β-human chorionic gonadotropin (β-hCG)-based contraceptive vaccine established proof of principle that these can be developed for human application. Block in fertility by β-hCG contraceptive vaccine was reversible. Further research inputs are required to establish the safety of contraceptive vaccines, improve their immunogenicity and to develop novel vaccine delivery platforms for providing long lasting immunity. PMID:24262991

Protein kinesis: The dynamics of protein trafficking and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

The Role of Shape Complementarity in the Protein-Protein Interactions

PubMed Central

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-01-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody–antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity. PMID:24253561

FRODOCK 2.0: fast protein-protein docking server.

PubMed

Ramírez-Aportela, Erney; López-Blanco, José Ramón; Chacón, Pablo

2016-08-01

The prediction of protein-protein complexes from the structures of unbound components is a challenging and powerful strategy to decipher the mechanism of many essential biological processes. We present a user-friendly protein-protein docking server based on an improved version of FRODOCK that includes a complementary knowledge-based potential. The web interface provides a very effective tool to explore and select protein-protein models and interactively screen them against experimental distance constraints. The competitive success rates and efficiency achieved allow the retrieval of reliable potential protein-protein binding conformations that can be further refined with more computationally demanding strategies. The server is free and open to all users with no login requirement at http://frodock.chaconlab.org pablo@chaconlab.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Computational prediction of host-pathogen protein-protein interactions.

PubMed

Dyer, Matthew D; Murali, T M; Sobral, Bruno W

2007-07-01

Infectious diseases such as malaria result in millions of deaths each year. An important aspect of any host-pathogen system is the mechanism by which a pathogen can infect its host. One method of infection is via protein-protein interactions (PPIs) where pathogen proteins target host proteins. Developing computational methods that identify which PPIs enable a pathogen to infect a host has great implications in identifying potential targets for therapeutics. We present a method that integrates known intra-species PPIs with protein-domain profiles to predict PPIs between host and pathogen proteins. Given a set of intra-species PPIs, we identify the functional domains in each of the interacting proteins. For every pair of functional domains, we use Bayesian statistics to assess the probability that two proteins with that pair of domains will interact. We apply our method to the Homo sapiens-Plasmodium falciparum host-pathogen system. Our system predicts 516 PPIs between proteins from these two organisms. We show that pairs of human proteins we predict to interact with the same Plasmodium protein are close to each other in the human PPI network and that Plasmodium pairs predicted to interact with same human protein are co-expressed in DNA microarray datasets measured during various stages of the Plasmodium life cycle. Finally, we identify functionally enriched sub-networks spanned by the predicted interactions and discuss the plausibility of our predictions. Supplementary data are available at http://staff.vbi.vt.edu/dyermd/publications/dyer2007a.html. Supplementary data are available at Bioinformatics online.

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

PubMed

Kilani, Suha S; Cooke, Simon; Kan, Andrew K; Chapman, Michael G

2006-02-01

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

The De Novo Design of Protein-Protein Interfaces

DTIC Science & Technology

it was our intention to add to this body by engineering de novo (from scratch) protein/protein complexes. Using this inverse approach we have furthered...key physical features needed to drive specific protein/protein interactions. It is considered inverse because, instead of studying natural complexes

Influence of Protein Abundance on High-Throughput Protein-Protein Interaction Detection

DTIC Science & Technology

2009-06-05

the interaction data sets we determined, via comparisons with strict randomized simulations , the propensity for essential proteins to selectively...and analysis of high- quality PPI data sets. Materials and Methods We analyzed protein interaction networks for yeast and E. coli determined from Y2H...we reinvestigated the centrality-lethality rule, which implies that proteins having more interactions are more likely to be essential. From analysis

Allozyme differentiation and biosystematics of the Californian closed-cone pines (Pinus subsect. Oocarpae)

Treesearch

Constance I. Millar; Steven H. Strauss; M. Thompson Conkle; Robert D. Westfall

1988-01-01

Allozyme differentiation at 32 loci was studied in the three Californian species of Pinus subsect. Oocarpae: P. attenuata, P. muricata, and P. radiata, and in a small sample of a Latin American species of the subsection, P. oocarpa. The Californian species...

ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

PubMed

Cheng, Yiming; Perocchi, Fabiana

2015-07-01

ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The NMR contribution to protein-protein networking in Fe-S protein maturation.

PubMed

Banci, Lucia; Camponeschi, Francesca; Ciofi-Baffoni, Simone; Piccioli, Mario

2018-03-22

Iron-sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe-S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe-2S], [3Fe-4S] and [4Fe-4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe-S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe-S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of "Fe-S interactomics". This contribution was particularly effective when protein-protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.

Investigation of process and product parameters for physicochemical properties of rice and mung bean (Vigna radiata) flour based extruded snacks.

PubMed

Sharma, Chetan; Singh, Baljit; Hussain, Syed Zameer; Sharma, Savita

2017-05-01

PR 106 and SML 668 cultivars of rice and mung bean respectively, were studied for their potential to serve as a nutritious snack with improved protein quality and quantity. The effect of extrusion conditions, including feed moisture content (14-18%), screw speed (400-550 rpm) and barrel temperature (130-170°C) on the physicochemical properties (bulk density, water absorption index (WAI), water solubility index (WSI) and hardness) was investigated. The replacement of rice flour at 30% level with mung bean flour for making extruded snacks was evaluated. Pasting temperature increased (84-93 °C) while peak viscosity (2768-408 cP), hold viscosity (2018-369 cP), breakdown (750-39 cP), setback (2697-622 cP) and final viscosity (4715-991 cP) decreased with increasing mung bean flour addition. Increasing feed moisture lowered the specific mechanical energy (SME), WAI and WSI of extrudates whereas increased bulk density and hardness. Higher screw speed had linear positive effect on SME of extruder and negative linear effect on WAI. Positive curvilinear quadratic effect of screw speed was also observed on WSI and density. Higher barrel temperature linearly decreased the SME, density and hardness of extrudates. Developed extrusion cooked rice-mung bean snacks with increased protein content and improved protein quality along with higher dietary fibre and minerals have good potential in effectively delivering the nutrition to the population.

Molecular tweezers modulate 14-3-3 protein-protein interactions

NASA Astrophysics Data System (ADS)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Populations of subplate and interstitial neurons in fetal and adult human telencephalon.

PubMed

Judaš, Miloš; Sedmak, Goran; Pletikos, Mihovil; Jovanov-Milošević, Nataša

2010-10-01

In the adult human telencephalon, subcortical (gyral) white matter contains a special population of interstitial neurons considered to be surviving descendants of fetal subplate neurons [Kostovic & Rakic (1980) Cytology and the time of origin of interstitial neurons in the white matter in infant and adult human and monkey telencephalon. J Neurocytol9, 219]. We designate this population of cells as superficial (gyral) interstitial neurons and describe their morphology and distribution in the postnatal and adult human cerebrum. Human fetal subplate neurons cannot be regarded as interstitial, because the subplate zone is an essential part of the fetal cortex, the major site of synaptogenesis and the 'waiting' compartment for growing cortical afferents, and contains both projection neurons and interneurons with distinct input-output connectivity. However, although the subplate zone is a transient fetal structure, many subplate neurons survive postnatally as superficial (gyral) interstitial neurons. The fetal white matter is represented by the intermediate zone and well-defined deep periventricular tracts of growing axons, such as the corpus callosum, anterior commissure, internal and external capsule, and the fountainhead of the corona radiata. These tracts gradually occupy the territory of transient fetal subventricular and ventricular zones.The human fetal white matter also contains distinct populations of deep fetal interstitial neurons, which, by virtue of their location, morphology, molecular phenotypes and advanced level of dendritic maturation, remain distinct from subplate neurons and neurons in adjacent structures (e.g. basal ganglia, basal forebrain). We describe the morphological, histochemical (nicotinamide-adenine dinucleotide phosphate-diaphorase) and immunocytochemical (neuron-specific nuclear protein, microtubule-associated protein-2, calbindin, calretinin, neuropeptide Y) features of both deep fetal interstitial neurons and deep (periventricular

Predicting Functions of Proteins in Mouse Based on Weighted Protein-Protein Interaction Network and Protein Hybrid Properties

PubMed Central

Shi, Xiaohe; Lu, Wen-Cong; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

Background With the huge amount of uncharacterized protein sequences generated in the post-genomic age, it is highly desirable to develop effective computational methods for quickly and accurately predicting their functions. The information thus obtained would be very useful for both basic research and drug development in a timely manner. Methodology/Principal Findings Although many efforts have been made in this regard, most of them were based on either sequence similarity or protein-protein interaction (PPI) information. However, the former often fails to work if a query protein has no or very little sequence similarity to any function-known proteins, while the latter had similar problem if the relevant PPI information is not available. In view of this, a new approach is proposed by hybridizing the PPI information and the biochemical/physicochemical features of protein sequences. The overall first-order success rates by the new predictor for the functions of mouse proteins on training set and test set were 69.1% and 70.2%, respectively, and the success rate covered by the results of the top-4 order from a total of 24 orders was 65.2%. Conclusions/Significance The results indicate that the new approach is quite promising that may open a new avenue or direction for addressing the difficult and complicated problem. PMID:21283518

TGF-beta signaling proteins and the Protein Ontology.

PubMed

Arighi, Cecilia N; Liu, Hongfang; Natale, Darren A; Barker, Winona C; Drabkin, Harold; Blake, Judith A; Smith, Barry; Wu, Cathy H

2009-05-06

The Protein Ontology (PRO) is designed as a formal and principled Open Biomedical Ontologies (OBO) Foundry ontology for proteins. The components of PRO extend from a classification of proteins on the basis of evolutionary relationships at the homeomorphic level to the representation of the multiple protein forms of a gene, including those resulting from alternative splicing, cleavage and/or post-translational modifications. Focusing specifically on the TGF-beta signaling proteins, we describe the building, curation, usage and dissemination of PRO. PRO is manually curated on the basis of PrePRO, an automatically generated file with content derived from standard protein data sources. Manual curation ensures that the treatment of the protein classes and the internal and external relationships conform to the PRO framework. The current release of PRO is based upon experimental data from mouse and human proteins wherein equivalent protein forms are represented by single terms. In addition to the PRO ontology, the annotation of PRO terms is released as a separate PRO association file, which contains, for each given PRO term, an annotation from the experimentally characterized sub-types as well as the corresponding database identifiers and sequence coordinates. The annotations are added in the form of relationship to other ontologies. Whenever possible, equivalent forms in other species are listed to facilitate cross-species comparison. Splice and allelic variants, gene fusion products and modified protein forms are all represented as entities in the ontology. Therefore, PRO provides for the representation of protein entities and a resource for describing the associated data. This makes PRO useful both for proteomics studies where isoforms and modified forms must be differentiated, and for studies of biological pathways, where representations need to take account of the different ways in which the cascade of events may depend on specific protein modifications. PRO provides

Energy Landscape and Transition State of Protein-Protein Association

NASA Astrophysics Data System (ADS)

Alsallaq, Ramzi; Zhou, Huan-Xiang

2006-11-01

Formation of a stereospecific protein complex is favored by specific interactions between two proteins but disfavored by the loss of translational and rotational freedom. Echoing the protein folding process, we have previously proposed a transition state for protein-protein association. Here we clarify the specification of the transition state by working with two toy models for protein association. The models demonstrate that a sharp transition between the bound state with numerous short-range interactions but restricted translation and rotational freedom and the unbound state with at most a small number of interactions but expanded configurational freedom. This transition sets the outer boundary of the bound state as well as the transition state for association. The energy landscape is funnel-like, with the deep well of the bound state surrounded by a broad shallow basin. This formalism of protein-protein association is applied to four protein-protein complexes, and is found to give accurate predictions for the effects of charge mutations and ionic strength on the association rates.

An ontology-based search engine for protein-protein interactions.

PubMed

Park, Byungkyu; Han, Kyungsook

2010-01-18

Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

PubMed Central

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Prediction of Heterodimeric Protein Complexes from Weighted Protein-Protein Interaction Networks Using Novel Features and Kernel Functions

PubMed Central

Ruan, Peiying; Hayashida, Morihiro; Maruyama, Osamu; Akutsu, Tatsuya

2013-01-01

Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes. PMID:23776458

Light-scattering studies of protein solutions: role of hydration in weak protein-protein interactions.

PubMed

Paliwal, A; Asthagiri, D; Abras, D; Lenhoff, A M; Paulaitis, M E

2005-09-01

We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B(2), by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B(2) and comparing our results with those derived from light-scattering measurements of B(2) for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25 degrees C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B(2). We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration.

Computational Prediction of Protein-Protein Interactions

PubMed Central

Ehrenberger, Tobias; Cantley, Lewis C.; Yaffe, Michael B.

2015-01-01

The prediction of protein-protein interactions and kinase-specific phosphorylation sites on individual proteins is critical for correctly placing proteins within signaling pathways and networks. The importance of this type of annotation continues to increase with the continued explosion of genomic and proteomic data, particularly with emerging data categorizing posttranslational modifications on a large scale. A variety of computational tools are available for this purpose. In this chapter, we review the general methodologies for these types of computational predictions and present a detailed user-focused tutorial of one such method and computational tool, Scansite, which is freely available to the entire scientific community over the Internet. PMID:25859943

Protein Condensation

NASA Astrophysics Data System (ADS)

Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

2007-09-01

Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

Protein Condensation

NASA Astrophysics Data System (ADS)

Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

2014-07-01

Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

PubMed

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

PubMed Central

Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

Protein subcellular localization assays using split fluorescent proteins

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

An ontology-based search engine for protein-protein interactions

PubMed Central

2010-01-01

Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

Hot-spot analysis for drug discovery targeting protein-protein interactions.

PubMed

Rosell, Mireia; Fernández-Recio, Juan

2018-04-01

Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

Protein-induced satiety: effects and mechanisms of different proteins.

PubMed

Veldhorst, M; Smeets, A; Soenen, S; Hochstenbach-Waelen, A; Hursel, R; Diepvens, K; Lejeune, M; Luscombe-Marsh, N; Westerterp-Plantenga, M

2008-05-23

Relatively high protein diets, i.e. diets that maintain the absolute number of grams of protein ingested as compared to before dieting, are a popular strategy for weight loss and weight maintenance. Research into multiple mechanisms regulating body weight has focused on the effects of different quantities and types of dietary protein. Satiety and energy expenditure are important in protein-enhanced weight loss and weight maintenance. Protein-induced satiety has been shown acutely, with single meals, with contents of 25% to 81% of energy from protein in general or from specific proteins, while subsequent energy intake reduction was significant. Protein-induced satiety has been shown with high protein ad libitum diets, lasting from 1 to 6 days, up to 6 months. Also significantly greater weight loss has been observed in comparison with control. Mechanisms explaining protein-induced satiety are nutrient-specific, and consist mainly of synchronization with elevated amino acid concentrations. Different proteins cause different nutrient related responses of (an)orexigenic hormones. Protein-induced satiety coincides with a relatively high GLP-1 release, stimulated by the carbohydrate content of the diet, PYY release, while ghrelin does not seem to be especially affected, and little information is available on CCK. Protein-induced satiety is related to protein-induced energy expenditure. Finally, protein-induced satiety appears to be of vital importance for weight loss and weight maintenance. With respect to possible adverse events, chronic ingestion of large amounts of sulphur-containing amino acids may have an indirect effect on blood pressure by induction of renal subtle structural damage, ultimately leading to loss of nephron mass, and a secondary increase in blood pressure. The established synergy between obesity and low nephron number on induction of high blood pressure and further decline of renal function identifies subjects with obesity, metabolic syndrome and

A Discontinuous Potential Model for Protein-Protein Interactions.

PubMed

Shao, Qing; Hall, Carol K

2016-01-01

Protein-protein interactions play an important role in many biologic and industrial processes. In this work, we develop a two-bead-per-residue model that enables us to account for protein-protein interactions in a multi-protein system using discontinuous molecular dynamics simulations. This model deploys discontinuous potentials to describe the non-bonded interactions and virtual bonds to keep proteins in their native state. The geometric and energetic parameters are derived from the potentials of mean force between sidechain-sidechain, sidechain-backbone, and backbone-backbone pairs. The energetic parameters are scaled with the aim of matching the second virial coefficient of lysozyme reported in experiment. We also investigate the performance of several bond-building strategies.

Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

PubMed Central

Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

2013-01-01

We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

NMR Studies of Protein Hydration and Protein-Ligand Interactions

NASA Astrophysics Data System (ADS)

Chong, Yuan

Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

DiffSLC: A graph centrality method to detect essential proteins of a protein-protein interaction network.

PubMed

Mistry, Divya; Wise, Roger P; Dickerson, Julie A

2017-01-01

Identification of central genes and proteins in biomolecular networks provides credible candidates for pathway analysis, functional analysis, and essentiality prediction. The DiffSLC centrality measure predicts central and essential genes and proteins using a protein-protein interaction network. Network centrality measures prioritize nodes and edges based on their importance to the network topology. These measures helped identify critical genes and proteins in biomolecular networks. The proposed centrality measure, DiffSLC, combines the number of interactions of a protein and the gene coexpression values of genes from which those proteins were translated, as a weighting factor to bias the identification of essential proteins in a protein interaction network. Potentially essential proteins with low node degree are promoted through eigenvector centrality. Thus, the gene coexpression values are used in conjunction with the eigenvector of the network's adjacency matrix and edge clustering coefficient to improve essentiality prediction. The outcome of this prediction is shown using three variations: (1) inclusion or exclusion of gene co-expression data, (2) impact of different coexpression measures, and (3) impact of different gene expression data sets. For a total of seven networks, DiffSLC is compared to other centrality measures using Saccharomyces cerevisiae protein interaction networks and gene expression data. Comparisons are also performed for the top ranked proteins against the known essential genes from the Saccharomyces Gene Deletion Project, which show that DiffSLC detects more essential proteins and has a higher area under the ROC curve than other compared methods. This makes DiffSLC a stronger alternative to other centrality methods for detecting essential genes using a protein-protein interaction network that obeys centrality-lethality principle. DiffSLC is implemented using the igraph package in R, and networkx package in Python. The python package can be

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

PubMed

Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

2016-10-20

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

PubMed

Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

2016-02-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. © 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A credit-card library approach for disrupting protein-protein interactions.

PubMed

Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Solitary Pulmonary Nodule

DTIC Science & Technology

2010-05-01

also classically has “ popcorn ” calcifications.1 Another benign albeit rarer diagnosis that can be considered in patients from the southern...spiculated border 95% with corona radiata3 Calcifications Diffuse homogenous Central Laminated “ Popcorn ” Any other pattern Density *seldom

77 FR 34059 - Endangered Species; Receipt of Applications for Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-08

... leopard (Uncia uncia) African wild dog (Lycaon pictus) Cheetah (Acinonyx jubatus) Applicant: M. Knudsen... radiata) salmon-crested cockatoo (Cacatua moluccensis) ring-tailed lemur (Lemur catta) black-and-white... (Spheniscus demersus) Andean condor (Vultur gryphus) White-naped crane (Grus vipio) Salmon-crested cockatoo...

Noninvasive imaging of protein-protein interactions in living animals

NASA Astrophysics Data System (ADS)

Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

2002-05-01

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

NASA Technical Reports Server (NTRS)

Basel, L. E.; Cleland, R. E.

1992-01-01

A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

NOXclass: prediction of protein-protein interaction types.

PubMed

Zhu, Hongbo; Domingues, Francisco S; Sommer, Ingolf; Lengauer, Thomas

2006-01-19

Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at http://noxclass.bioinf.mpi-inf.mpg.de/.

Modelling of DNA-Mediated of Two- and -Three dimensional Protein-Protein and Protein-Nanoparticle Self-Assembly

NASA Astrophysics Data System (ADS)

Millan, Jaime; McMillan, Janet; Brodin, Jeff; Lee, Byeongdu; Mirkin, Chad; Olvera de La Cruz, Monica

Programmable DNA interactions represent a robust scheme to self-assemble a rich variety of tunable superlattices, where intrinsic and in some cases non-desirable nano-scale building blocks interactions are substituted for DNA hybridization events. Recent advances in synthesis has allowed the extension of this successful scheme to proteins, where DNA distribution can be tuned independently of protein shape by selectively addressing surface residues, giving rise to assembly properties in three dimensional protein-nanoparticle superlattices dependent on DNA distribution. In parallel to this advances, we introduced a scalable coarse-grained model that faithfully reproduces the previously observed co-assemblies from nanoparticles and proteins conjugates. Herein, we implement this numerical model to explain the stability of complex protein-nanoparticle binary superlattices and to elucidate experimentally inaccessible features such as protein orientation. Also, we will discuss systematic studies that highlight the role of DNA distribution and sequence on two-dimensional protein-protein and protein-nanoparticle superlattices.

Prediction of Protein Aggregation in High Concentration Protein Solutions Utilizing Protein-Protein Interactions Determined by Low Volume Static Light Scattering.

PubMed

Hofmann, Melanie; Winzer, Matthias; Weber, Christian; Gieseler, Henning

2016-06-01

The development of highly concentrated protein formulations is more demanding than for conventional concentrations due to an elevated protein aggregation tendency. Predictive protein-protein interaction parameters, such as the second virial coefficient B22 or the interaction parameter kD, have already been used to predict aggregation tendency and optimize protein formulations. However, these parameters can only be determined in diluted solutions, up to 20 mg/mL. And their validity at high concentrations is currently controversially discussed. This work presents a μ-scale screening approach which has been adapted to early industrial project needs. The procedure is based on static light scattering to directly determine protein-protein interactions at concentrations up to 100 mg/mL. Three different therapeutic molecules were formulated, varying in pH, salt content, and addition of excipients (e.g., sugars, amino acids, polysorbates, or other macromolecules). Validity of the predicted aggregation tendency was confirmed by stability data of selected formulations. Based on the results obtained, the new prediction method is a promising screening tool for fast and easy formulation development of highly concentrated protein solutions, consuming only microliter of sample volumes. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Urinary retention, erectile dysfunction and meningitis due to sacral herpes zoster: a case report and review of the literature.

PubMed

Erol, B; Avci, A; Eken, C; Ozgok, Y

2009-01-01

Zona zoster infection is often associated with painful erythematous vesicular eruptions of the skin or mucous membranes. Varicella zoster virus which stays latent in the sensorial root ganglia causes zona zoster infection. The most recognized feature of zona zoster is the dermatomal distribution of vesicular rashes. In the present case report, we state an unusual presentation of sacral zona zoster with urinary retention, erectile dysfunction and meningitis. Copyright 2009 S. Karger AG, Basel.

Anomalous Protein-Protein Interactions in Multivalent Salt Solution.

PubMed

Pasquier, Coralie; Vazdar, Mario; Forsman, Jan; Jungwirth, Pavel; Lund, Mikael

2017-04-13

The stability of aqueous protein solutions is strongly affected by multivalent ions, which induce ion-ion correlations beyond the scope of classical mean-field theory. Using all-atom molecular dynamics (MD) and coarse grained Monte Carlo (MC) simulations, we investigate the interaction between a pair of protein molecules in 3:1 electrolyte solution. In agreement with available experimental findings of "reentrant protein condensation", we observe an anomalous trend in the protein-protein potential of mean force with increasing electrolyte concentration in the order: (i) double-layer repulsion, (ii) ion-ion correlation attraction, (iii) overcharge repulsion, and in excess of 1:1 salt, (iv) non Coulombic attraction. To efficiently sample configurational space we explore hybrid continuum solvent models, applicable to many-protein systems, where weakly coupled ions are treated implicitly, while strongly coupled ones are treated explicitly. Good agreement is found with the primitive model of electrolytes, as well as with atomic models of protein and solvent.

Emerging Role of Protein-Protein Transnitrosylation in Cell Signaling Pathways

PubMed Central

2013-01-01

Abstract Significance: Protein S-nitrosylation, a covalent reaction of a nitric oxide (NO) group with a critical protein thiol (or more properly thiolate anion), mediates an important form of redox-related signaling as well as aberrant signaling in disease states. Recent Advances: A growing literature suggests that over 3000 proteins are S-nitrosylated in cell systems. Our laboratory and several others have demonstrated that protein S-nitrosylation can regulate protein function by directly inhibiting catalytically active cysteines, by reacting with allosteric sites, or via influencing protein-protein interaction. For example, S-nitrosylation of critical cysteine thiols in protein-disulfide isomerase and in parkin alters their activity, thus contributing to protein misfolding in Parkinson's disease. Critical Issues: However, the mechanism by which specific protein S-nitrosylation occurs in cell signaling pathways is less well investigated. Interestingly, the recent discovery of protein-protein transnitrosylation reactions (transfer of an NO group from one protein to another) has revealed a unique mechanism whereby NO can S-nitrosylate a particular set of protein thiols, and represents a major class of nitrosylating/denitrosylating enzymes in mammalian systems. In this review, we will discuss recent evidence for transnitrosylation reactions between (i) hemoglobin/anion exchanger 1, (ii) thioredoxin/caspase-3, (iii) X-linked inhibitor of apoptosis/caspase-3, (iv) GAPDH-HDAC2/SIRT1/DNA-PK, and (v) Cdk5/dynamin related protein 1 (Drp1). This review also discusses experimental techniques useful in characterizing protein-protein transnitrosylations. Future Directions: Elucidation of additional transnitrosylation cascades will further our understanding of the enzymes that catalyze nitrosation, thereby contributing to NO-mediated signaling pathways. Antioxid. Redox Signal. 18, 239–249. PMID:22657837

Activation of estrogen receptor signaling by the dioxin-like aryl hydrocarbon receptor agonist, 3,3',4,4',5-Pentachlorobiphenyl (PCB126) in salmon in vitro system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mortensen, Anne Skjetne; Arukwe, Augustine

2008-03-01

Available toxicological evidence indicates that environmental contaminants with strong affinity to the aryl hydrocarbon receptor (AhR) have anti-estrogenic properties in both mammalian and non-mammalian in vivo and in vitro studies. The primary objective of the present study was to investigate the interactions between the AhR and estrogen receptor (ER) in salmon in vitro system. Two separate experiments were performed and gene expression patterns were analyzed using real-time PCR, while protein analysis was done by immunoblotting. Firstly, salmon primary hepatocytes were exposed to the dioxin-like PCB126 at 1, 10 and 50 pM and ER agonist nonylphenol (NP) at 5 and 10more » {mu}M, singly or in combination. Our data showed increased levels of ER-mediated gene expression (vitellogenin: Vtg, zona radiata protein: Zr-protein, ER{alpha}, ER{beta} and vigilin) as well as increased cellular ER{alpha} protein levels after treatment with NP and PCB126, singly or in combination. PCB126 treatment alone produced, as expected, increased transcription of AhR nuclear translocator (Arnt), CYP1A1 and AhR repressor (AhRR) mRNA, and these responses were reduced in the presence of NP concentrations. PCB126 exposure alone did not produce significant effect on AhR2{alpha} mRNA but increased (at 1 and 50 pM) and decreased (at 10 pM) AhR2{beta} mRNA below control level. For AhR2{delta} and AhR2{gamma} isotypes, PCB126 (at 1 pM) produced significant decreases (total inhibition for AhR2{gamma}) of mRNA levels but was indifferent at 10 and 50 pM, compared to control. NP exposure alone produced concentration-dependent significant decrease of AhR2{beta} mRNA. In contrast, while 5 {mu}M NP produced an indifferent effect on AhR2{delta} and AhR2{gamma}, 10 {mu}M NP produced significant decrease (total inhibition for AhR2{gamma}) and the presence of NP produced apparent PCB126 concentration-specific modulation of all AhR isotypes. A second experiment was performed to evaluate the involvement of ER

Modular protein switches derived from antibody mimetic proteins.

PubMed

Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

2016-02-01

Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Endocrine-disruptor molecular responses, occurrence of intersex and gonado-histopathological changes in tilapia species from a tropical freshwater dam (Awba Dam) in Ibadan, Nigeria.

PubMed

Adeogun, Aina O; Onibonoje, Kolawole; Ibor, Oju R; Omiwole, Roseline A; Chukwuka, Azubuike V; Ugwumba, Alex O; Ugwumba, Adiaha A A; Arukwe, Augustine

2016-05-01

In the present study, the occurrence of endocrine disruptive responses in Tilapia species from Awba Dam has been investigated, and compared to a reference site (Modete Dam). The Awba Dam is a recipient of effluents from University of Ibadan (Nigeria) and several other anthropogenic sources. A total of 132 Tilapia species (Sarotherodon malenotheron (n=57 and 32, males and females, respectively) and Tilapia guineensis (n=23 and 20, males and females, respectively)) were collected from June to September 2014. At the reference site, samples of adult male and female S. melanotheron (48 males and 47 females) and T. guineensis (84 males and 27 females) were collected. Gonads were morphologically and histologically examined and gonadosomatic index (GSI) was calculated. Hepatic mRNA transcriptions of vitellogenin (Vtg) and zona radiata protein (Zrp) genes were analyzed using validated RT-qPCR. Significant increase in Vtg and Zrp transcripts were observed in male tilapias from Awba Dam, compared to males from the reference site. In addition, male tilapias from Awba Dam produced significantly higher Vtg and Zrp mRNA, compared to females in June and July. However, at the natural peak spawning period in August and September, females produced, significantly higher Vtg and Zrp mRNA, compared to males. Fish gonads revealed varying incidence of intersex with a striking presence of two (2) pairs of testes and a pair of ovary in S. melanotheron from Awba Dam. The entire fish population examined at Awba Dam showed a high prevalence of intersex (34.8%), involving phenotypic males and females of both species. Analysis of sediment contaminant levels revealed that As, Cd, Pb, Hg and Ni (heavy metals), monobutyltin cation, 4-iso-nonyphenol and PCB congeners (138, 153 and 180) were significantly higher in Awba Dam, compared to the reference site. Principal component analysis (PCA) showed that fish variables were positively correlated with sediment contaminant burden at Awba Dam, indicating

Optimizing energy functions for protein-protein interface design.

PubMed

Sharabi, Oz; Yanover, Chen; Dekel, Ayelet; Shifman, Julia M

2011-01-15

Protein design methods have been originally developed for the design of monomeric proteins. When applied to the more challenging task of protein–protein complex design, these methods yield suboptimal results. In particular, they often fail to recapitulate favorable hydrogen bonds and electrostatic interactions across the interface. In this work, we aim to improve the energy function of the protein design program ORBIT to better account for binding interactions between proteins. By using the advanced machine learning framework of conditional random fields, we optimize the relative importance of all the terms in the energy function, attempting to reproduce the native side-chain conformations in protein–protein interfaces. We evaluate the performance of several optimized energy functions, each describes the van der Waals interactions using a different potential. In comparison with the original energy function, our best energy function (a) incorporates a much “softer” repulsive van der Waals potential, suitable for the discrete rotameric representation of amino acid side chains; (b) does not penalize burial of polar atoms, reflecting the frequent occurrence of polar buried residues in protein–protein interfaces; and (c) significantly up-weights the electrostatic term, attesting to the high importance of these interactions for protein–protein complex formation. Using this energy function considerably improves side chain placement accuracy for interface residues in a large test set of protein–protein complexes. Moreover, the optimized energy function recovers the native sequences of protein–protein interface at a higher rate than the default function and performs substantially better in predicting changes in free energy of binding due to mutations.

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

PubMed Central

Meitzler, Jennifer L.; Hinde, Sara; Bánfi, Botond; Nauseef, William M.; Ortiz de Montellano, Paul R.

2013-01-01

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

Structure and Protein-Protein Interaction Studies on Chlamydia trachomatis Protein CT670 (YscO Homolog)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzini, Emily; Singer, Alexander; Singh, Bhag

2010-07-28

Comparative genomic studies have identified many proteins that are found only in various Chlamydiae species and exhibit no significant sequence similarity to any protein in organisms that do not belong to this group. The CT670 protein of Chlamydia trachomatis is one of the proteins whose genes are in one of the type III secretion gene clusters but whose cellular functions are not known. CT670 shares several characteristics with the YscO protein of Yersinia pestis, including the neighboring genes, size, charge, and secondary structure, but the structures and/or functions of these proteins remain to be determined. Although a BLAST search withmore » CT670 did not identify YscO as a related protein, our analysis indicated that these two proteins exhibit significant sequence similarity. In this paper, we report that the CT670 crystal, solved at a resolution of 2 {angstrom}, consists of a single coiled coil containing just two long helices. Gel filtration and analytical ultracentrifugation studies showed that in solution CT670 exists in both monomeric and dimeric forms and that the monomer predominates at lower protein concentrations. We examined the interaction of CT670 with many type III secretion system-related proteins (viz., CT091, CT665, CT666, CT667, CT668, CT669, CT671, CT672, and CT673) by performing bacterial two-hybrid assays. In these experiments, CT670 was found to interact only with the CT671 protein (YscP homolog), whose gene is immediately downstream of ct670. A specific interaction between CT670 and CT671 was also observed when affinity chromatography pull-down experiments were performed. These results suggest that CT670 and CT671 are putative homologs of the YcoO and YscP proteins, respectively, and that they likely form a chaperone-effector pair.« less

[Chromosomal proteins: histones and acid proteins].

PubMed

Salvini, M; Gabrielli, F

1976-01-01

Experimental data about the chemistry and the biology of chromosomal proteins are reviewed. Paragraphs include: aminoacid sequential data and post-translational covalent modications of histones, histone chemical differences in different tissues of the same species and in homologous organs of different species, histone synthesis subcellular localization and its association with DNA synthesis, histone synthesis transcriptional and translational control, histone synthesis during meiosis, oogenesis and early embryogenesis. The possible role of histones as controllers of gene expression is discussed and a model of primary structure of chromatine is proposed. The "acidic proteins" data concern the high tissue eterogenity of these proteins and their role in the steroid-hormon-controlled gene expression. The possible role of acidic proteins as general controllers of gene expression in eucariotic cells is discussed.

A structural view of egg coat architecture and function in fertilization.

PubMed

Monné, Magnus; Jovine, Luca

2011-10-01

Species-restricted interaction between gametes at the beginning of fertilization is mediated by the extracellular coat of the egg, a matrix of cross-linked glycoprotein filaments called the zona pellucida (ZP) in mammals and the vitelline envelope in nonmammals. All egg coat subunits contain a conserved protein-protein interaction module-the "ZP domain"-that allows them to polymerize upon dissociation of a C-terminal propeptide containing an external hydrophobic patch (EHP). Recently, the first crystal structures of a ZP domain protein, sperm receptor ZP subunit zona pellucida glycoprotein 3 (ZP3), have been reported, giving a glimpse of the structural organization of the ZP at the atomic level and the molecular basis of gamete recognition in vertebrates. The ZP module is divided in two related immunoglobulin-like domains, ZP-N and ZP-C, that contain characteristic disulfide bond patterns and, in the case of ZP-C, also incorporate the EHP. This segment lies at the interface between the two domains, which are connected by a long loop carrying a conserved O-glycan important for binding to sperm in vitro. The structures explain several apparently contradictory observations by reconciling the variable disulfide bond patterns found in different homologues of ZP3 as well as the multiple ZP3 determinants alternatively involved in gamete interaction. These findings have implications for our understanding of ZP subunit biogenesis; egg coat assembly, architecture, and interaction with sperm; structural rearrangements leading to postfertilization hardening of the ZP and the block to sperm binding; and the evolutionary origin of egg coats.

Protein-protein interactions and cancer: targeting the central dogma.

PubMed

Garner, Amanda L; Janda, Kim D

2011-01-01

Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

PubMed

He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

2012-07-01

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Dynamic imaging of protein-protein interactions by MP-FLIM

NASA Astrophysics Data System (ADS)

Ameer-Beg, Simon M.; Peter, Marion; Keppler, Melanie D.; Prag, Soren; Barber, Paul R.; Ng, Tony C.; Vojnovic, Borivoj

2005-03-01

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

Interactions affect hyphal growth and enzyme profiles in combinations of coniferous wood-decaying fungi of Agaricomycetes

PubMed Central

Mali, Tuulia; Kuuskeri, Jaana; Shah, Firoz

2017-01-01

Fomitopsis pinicola is a species of Polyporales frequently encountered in Nordic temperate and boreal forests. In nature, the fungus causes destructive brown rot in wood, colonizing tree trunks often occupied by other Basidiomycota species. We mimicked these species-species interactions by introducing F. pinicola to five white rot species, all common saprotrophs of Norway spruce. Hyphal interactions and mycelial growth in various combinations were recorded, while activities of lignocellulose-acting CAZymes and oxidoreductases were followed in co-cultures on two different carbon-source media. Of the species, Phlebia radiata and Trichaptum abietinum were the strongest producers of lignin-modifying oxidoreductases (laccase, manganese peroxidase) when evaluated alone, as well as in co-cultures, on the two different growth media (low-nitrogen liquid medium containing ground coniferous wood, and malt extract broth). F. pinicola was an outstanding producer of oxalic acid (up to 61 mM), whereas presence of P. radiata prevented acidification of the growth environment in the liquid malt-extract cultures. When enzyme profiles of the species combinations were clustered, time-dependent changes were observed on wood-supplemented medium during the eight weeks of growth. End-point acidity and production of mycelium, oxalic acid and oxidoreductase activities, in turn clustered the fungal combinations into three distinct functional groups, determined by the presence of F. pinicola and P. radiata, by principal component analysis. Our findings indicate that combinations of wood-decay fungi have dramatic dynamic effects on the production of lignocellulose-active enzymes, which may lead to divergent degradative processes of dead wood and forest litter. PMID:28953947

Reproductive characteristics and population decline of four species of skate (Rajidae) off the eastern coast of Canada.

PubMed

Mcphie, R P; Campana, S E

2009-07-01

Four of the most common species of skate (Rajidae) were studied off eastern Canada to determine if their reproductive characteristics were linked to their population trajectories. The fecundity of the winter skate Leucoraja ocellata, the little skate Leucoraja erinacea, the thorny skate Amblyraja radiata and the smooth skate Malacoraja senta averaged between 41 and 56 egg cases per year for each species. For all species but L. ocellata, males matured at larger sizes and at later ages than females. Theoretical rates of population increase for non-equilibrium populations of L. ocellata (c. 0.07), M. senta (c. 0.14) and L. erinacea and A. radiata (c. 0.20) were low compared to most fishes, indicating that north-west Atlantic skates are intrinsically unproductive, yet are theoretically capable of supporting low-level fisheries. Nevertheless, the results of 36 years of research surveys indicate that the abundance of mature L. ocellata, A. radiata and M. senta all decreased by >90% since 1970, indicating that past fishing mortality (both directed and undirected) has outstripped the net productivity of the skate populations on the eastern Scotian Shelf. The relationship between maximum age (t(max)) and age of maturity (t(mat)) was a better predictor of population growth rate than was body size, with the species exhibiting the highest ratios of t(mat) :t(max) (L. ocellata = 0.68, M. senta = 0.66) having the lowest predicted population growth rates. L. ocellata appears to have the lowest productivity and has experienced the greatest population decline, thus raising concerns over its future status.

Interactions affect hyphal growth and enzyme profiles in combinations of coniferous wood-decaying fungi of Agaricomycetes.

PubMed

Mali, Tuulia; Kuuskeri, Jaana; Shah, Firoz; Lundell, Taina Kristina

2017-01-01

Fomitopsis pinicola is a species of Polyporales frequently encountered in Nordic temperate and boreal forests. In nature, the fungus causes destructive brown rot in wood, colonizing tree trunks often occupied by other Basidiomycota species. We mimicked these species-species interactions by introducing F. pinicola to five white rot species, all common saprotrophs of Norway spruce. Hyphal interactions and mycelial growth in various combinations were recorded, while activities of lignocellulose-acting CAZymes and oxidoreductases were followed in co-cultures on two different carbon-source media. Of the species, Phlebia radiata and Trichaptum abietinum were the strongest producers of lignin-modifying oxidoreductases (laccase, manganese peroxidase) when evaluated alone, as well as in co-cultures, on the two different growth media (low-nitrogen liquid medium containing ground coniferous wood, and malt extract broth). F. pinicola was an outstanding producer of oxalic acid (up to 61 mM), whereas presence of P. radiata prevented acidification of the growth environment in the liquid malt-extract cultures. When enzyme profiles of the species combinations were clustered, time-dependent changes were observed on wood-supplemented medium during the eight weeks of growth. End-point acidity and production of mycelium, oxalic acid and oxidoreductase activities, in turn clustered the fungal combinations into three distinct functional groups, determined by the presence of F. pinicola and P. radiata, by principal component analysis. Our findings indicate that combinations of wood-decay fungi have dramatic dynamic effects on the production of lignocellulose-active enzymes, which may lead to divergent degradative processes of dead wood and forest litter.

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

NDR proteins

PubMed Central

Jones, Alan M

2010-01-01

N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

The interface of protein structure, protein biophysics, and molecular evolution

PubMed Central

Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

2012-01-01

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

Evolutionary diversification of protein-protein interactions by interface add-ons.

PubMed

Plach, Maximilian G; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H; Merkl, Rainer; Sterner, Reinhard

2017-10-03

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

Identification of PDC-109-like protein(s) in buffalo seminal plasma.

PubMed

Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

2009-10-01

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

PubMed

Kent, Stephen Bh

2018-04-04

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

General protein-protein cross-linking.

PubMed

Alegria-Schaffer, Alice

2014-01-01

This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). © 2014 Elsevier Inc. All rights reserved.

Surface energetics and protein-protein interactions: analysis and mechanistic implications

PubMed Central

Peri, Claudio; Morra, Giulia; Colombo, Giorgio

2016-01-01

Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828