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1

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT  

EPA Science Inventory

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT. S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2 1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA 2 Reproductive T...

2

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT DURING PREGNANCY  

EPA Science Inventory

Effects of Bromodichloromethane (BDCM) on Ex Vivo Luteal Function In the Pregnant F344 Rat Susan R. Bielmeier1, Ashley S. Murr2, Deborah S. Best2, Jerome M. Goldman2, and Michael G. Narotsky2 1Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC 27599,...

3

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT  

EPA Science Inventory

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT. S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2 1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA 2 Reproductive T...

4

Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat  

Microsoft Academic Search

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo

S. R. Bielmeier; A. S. Murr; D. S. Best; R. A. Harrison; R. A. Pegram; J. M. Goldman; M. G. Narotsky

2007-01-01

5

Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat.  

PubMed

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo and in vitro approaches. For the ex vivo study (i.e., in vivo exposure followed by in vitro assessment), dams were dosed by gavage on gestation days (GD) 6-9 (plug day=GD 0) at 0 or 100 mg/kg/d. One hour after the GD-9 dose, rats were killed, blood was collected, and tissue concentrations of BDCM were assessed. Corpora lutea (CL) were incubated with or without hCG, an LH agonist, to stimulate P secretion. For the in vitro study, CL were pooled from untreated F344 rats on GD 9 and cultured with BDCM at 0, 0.01, 0.10 or 3.0 mM. BDCM was found at highest concentrations in adrenal, ovarian, adipose, and hypothalamic tissues. BDCM treatment decreased serum P and LH levels in vivo. Ex vivo, however, BDCM-exposed CL showed >2-fold increases in P secretion relative to controls. Both control and BDCM-exposed CL displayed a 2.4-fold increase in P secretion in response to hCG challenge. In contrast, in vitro exposures reduced CL responsiveness in a dose-related fashion while baseline levels were unaffected. It is unclear if the ex vivo 'rebound' reflects the removal of the CL from a possible direct inhibitory influence of BDCM, or a response to diminished LH stimulation in vivo. Thus, these data suggest that BDCM disrupts pregnancy in F344 rats via two modes: disruption of LH secretion, and disruption of the CL's ability to respond to LH. PMID:17344021

Bielmeier, S R; Murr, A S; Best, D S; Harrison, R A; Pegram, R A; Goldman, J M; Narotsky, M G

2007-08-01

6

The effect of metritis on luteal function in dairy cows  

PubMed Central

Background Disturbed uterine involution impairs ovarian function in the first weeks after calving. This study analyzed the long-term effect of metritis on luteal function of 47 lactating Holstein-Friesian cows during the first four postpartum estrous cycles. Cows with abnormal uterine enlargement and malodorous lochia were classified as having metritis (group M, n?=?18), and all others were considered healthy (group H, n?=?29). Luteal size was measured once between days 9 and 13 of the first (group H, n?=?11; group M, n?=?12), second (group H, n?=?23; group M, n?=?18) and fourth (group H, n?=?11; group M, n?=?7) postpartum luteal phases. Serum progesterone concentration was measured at the same time. Sixteen cows (group H, n?=?9; group M, n?=?7) underwent transvaginal luteal biopsy for gene expression analysis of steroidogenic regulatory proteins during the second and fourth cycles. Cows with persistence of the corpus luteum (CL) underwent determination of luteal size, luteal biopsy and serum progesterone measurement once between days 29 and 33, followed by prostaglandin treatment to induce luteolysis. The same procedures were repeated once between days 9 and 13 of the induced cycle. Results The cows in group M had smaller first-cycle CLs than the cows in group H (p?=?0.04), but progesterone concentrations did not differ between groups. Luteal size, progesterone concentration and gene expression did not differ between the two groups during the second and fourth cycles. Compared with healthy cows (10%), there was a trend (p?=?0.07) toward a higher prevalence of persistent CLs in cows with metritis (33%). Persistent CLs were limited to the first cycle. Persistent CLs and the induced cyclic CLs did not differ with regard to the variables investigated. Conclusions An effect of metritis on luteal activity was apparent in the first postpartum estrous cycle. However, after the first postpartum cycle, no differences occurred in analyzed parameters between metritis and control cows. Therefore, a metritis is able to impair luteal activity transiently, but does not seem to have a long-term effect on luteal function. PMID:24304943

2013-01-01

7

Assessment of luteal function in goats by ultrasonographic image attribute analysis  

Microsoft Academic Search

The aim of this study was to evaluate the potential of luteal echotexture (mean pixel value and heterogeneity), as a tool for assessing luteal function during different phases of the estrous cycle in Toggenburg goats. Sonographic evaluations of the ovaries were performed daily in nulliparous goats (n=21), using a 5MHz linear rectal probe, commencing at estrus (day 0). Blood samples

E. K. Arashiro; J. F. Fonseca; L. G. B. Siqueira; C. A. Fernandes; F. Z. Brandao; E. Oba; J. H. Viana

2010-01-01

8

RESUMPTION OF POSTPARTUM LUTEAL FUNCTION OF PRIMIPAROUS, SUCKLED BEEF COWS EXPOSED CONTINUOUSLY TO BULL URINE  

Technology Transfer Automated Retrieval System (TEKTRAN)

We tested the hypotheses that interval from urine exposure to resumption of luteal activity and proportions of cows that resume luteal activity by the end of the urine-exposure period do not differ between cows exposed to mature bull urine or steer urine. Thirty-eight Angus x Hereford cows, four mat...

9

Failure to maintain luteal function: a possible cause of early embryonic loss in a cow.  

PubMed Central

The effect of early pregnancy failure on the release of prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin (Ot) was examined in an abnormal breeder (AB) heifer that was not able to maintain a pregnancy beyond 21 days. This animal was used in three experiments: 1) She received one intravenous injection of 100 IU Ot 17 days after the onset of oestrus (Day 0). Frequent blood samples were taken for the measurement of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) by radioimmunoassay. Daily samples for progesterone (P4) determinations were taken to monitor luteal function. This was then repeated using the same animal at either day 17 or 18 or 19 (day 17-19) of pregnancy. 2) Embryos from superovulated normal breeder (NB) donors were transferred at day 7 to the AB heifer as well as to NB control animals. 3) Seven day old embryos from the superovulated AB heifer were transferred to NB recipient animals. At day 17-19 of pregnancy all the recipient heifers (experiments 2 and 3) were subjected to the same protocol as in experiment 1. The results showed that the ability of Ot to stimulate PGF2 alpha release was reduced in the NB recipients bearing viable embryos when compared to cyclic animals. However, for the AB heifer, Ot stimulated PGF2 alpha release to the same extent whether the animal was cyclic or pregnant. Furthermore, the AB animal did not have the extended luteal function associated with removal of viable embryos on day 17-19. The data suggest that the embryonic loss might have been caused by failure of the embryos to prevent the luteolytic release of PGF2 alpha. PMID:2766148

Lafrance, M; Goff, A K; Guay, P; Harvey, D

1989-01-01

10

Structural and functional reserve of steroidogenic membranes in stimulated luteal cells of pregnant rats.  

PubMed

A comparison was made of the ultrastructural features of the remaining corpora lutea (CL) of five unilaterally ovariectomised and five control pregnant rats. Unilateral ovariectomy (ULO) was carried out on Day 8 of gestation; this treatment has been shown to double the normal rate of progesterone secretion by the remaining ovary within 8 days. On Day 16, the ovaries from both ULO and control rats were examined stereologically. The mass of luteal tissue remaining in ULO rats was only 44% of that in the controls, but their plasma progesterone concentration was 81% and not significantly different from the control value. The CL were 10% heavier in ULO rats and the amount of luteal cell cytoplasm per CL was 21% greater. The percentage of the CL occupied by luteal cells was 15% greater in ULO rats but the interstitial space was 50% less. There was no significant change in the percentage of the luteal cell cytoplasm occupied by cytosol, agranular endoplasmic reticulum (AER), mitochondria and electron-dense granules. The surface area per unit cytoplasmic volume of AER and outer and inner mitochondrial membranes (OMM and IMM, respectively) was not affected. However, expressed per CL, both the AER and IMM membrane surface areas were increased by around 30%. These morphometric changes would only account for about 30% of the reported increase in hormone secretion by the CL of ULO rats, and they suggest that the CL of Day 16 pregnant rats may have reached their optimal structural condition. PMID:1769896

Meyer, G T; Bruce, N W

1991-08-01

11

Influence of prostaglandin F?? analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro.  

PubMed

Although prostaglandin (PG) F2? analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF2?. In the first of two related experiments, the effects of different analogues of PGF2? (aPGF2?) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24h with naturally-occurring PGF2? or aPGF2? (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca(2+)]i mobilisation, as well as cell viability and apoptosis were measured. Naturally-occurring PGF2? and dinoprost stimulated P4 secretion (P<0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P<0.001). The greatest effect on [Ca(2+)]i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P<0.001). In a second experiment, the influence of naturally-occurring PGF2? and aPGF2? on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF2? were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF2?. PMID:24268486

Korzekwa, A J; Lukasik, K; Pilawski, W; Piotrowska-Tomala, K K; Jaroszewski, J J; Yoshioka, S; Okuda, K; Skarzynski, D J

2014-01-01

12

Effect of feeding level on luteal function and progesterone concentration in the vena cava during early pregnancy in gilts.  

PubMed

This study assessed the effect of feeding level on progesterone concentration in the caudal vena cava during early pregnancy in gilts. Twenty-four Landrace gilts were allocated to either a high (2.8±0.02) or a low (1.5±0.01 kg day?¹) feeding level at Day 0 of pregnancy. Serial blood samples were collected every 15 min for 3 h before and 3 h after feeding on Days 6 and 9 of pregnancy. Embryo survival and development as well as in vitro luteal progesterone production were assessed at Day 10 of pregnancy. Progesterone concentration in the vena cava was pulsatile with gilts on the high feeding level having more pulses compared with Low gilts on Day 9 of pregnancy (P<0.05). On Day 6 the number of pulses did not differ significantly between treatments; however, the average progesterone concentration in the vena cava tended to be higher in the gilts on the high feeding level (P<0.10). Embryo survival at Day 10 was 92±3% for High gilts compared with 77±3% for Low gilts (P<0.05). No difference in embryo development between the treatments was seen. There was no difference between treatments in in vitro secretion of progesterone by luteal tissue. In conclusion, a high plane of nutrition positively affects progesterone secretion by the ovaries in early pregnancy. PMID:23464500

Athorn, R Z; Stott, P; Bouwman, E G; Chen, T Y; Kennaway, D J; Langendijk, P

2013-01-01

13

Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.  

PubMed

The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2-8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P(4)) were evaluated. The production of P(4) was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P(4), PGF(2?), and PGE(2) compared to fresh cell cultures. This enables the use of pools of frozen-thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P(4) quantified in the medium, but had no effect on PGF(2?) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed. PMID:23054443

Batista, M; Torres, A; Diniz, P; Mateus, L; Lopes-da-Costa, L

2012-10-01

14

The effect of ram exposure on uterine involution and luteal function during the postpartum period of hair sheep ewes in the tropics.  

PubMed

St. Croix White hair sheep ewes lambing in July (n = 20) or November (n = 26) were used to evaluate the effect of ram exposure on uterine involution and postpartum luteal function. Ewes were exposed to an epididymectomized ram (EXPOSED) beginning on d 7 after lambing (d 0) or kept isolated from rams (CONTROL) through d 63. The width of each uterine horn was measured using transrectal ultrasonography at 3.5-d intervals beginning within 3 d after lambing. Jugular blood samples were also collected at these times, and plasma was harvested for progesterone (P4) analysis. Days to first estrus postpartum was not different (P > .10) between EXPOSED ewes that lambed in July or November (39.3 +/- 3.1 vs 44.2 +/- 3.8 d, respectively). Cross-sectional area of uterine horns was not different (P > .10) between EXPOSED and CONTROL ewes, ewes bearing one or two lambs, or ewes that lambed in November or July. Cross-sectional area of uterine horns in EXPOSED and CONTROL ewes had decreased to < 30% of initial values by 28 d postpartum (P < .0001). Ewes exposed to rams had a P4 concentration greater than 1 ng/mL sooner postpartum (P < .006) than CONTROL ewes (32.4 +/- 2.4 vs 42.1 +/- 2.3 d, respectively). The P4 concentration in the first sample greater than 1 ng/mL was greater (P < .06) in EXPOSED ewes than in CONTROL ewes (3.3 +/- .4 vs 2.3 +/- .4 ng/mL, respectively). In July, ewes exposed to rams had greater (P < .03) P4 concentrations than CONTROL ewes during the 63 d after parturition, but this difference was not apparent (P > .10) in ewes that lambed in November. Ram exposure did not hasten uterine involution in hair sheep ewes in the tropics. Luteal function, determined by plasma P4 concentrations, was enhanced by ram exposure during July but not during November. The lack of seasonality of hair sheep in the tropics does not seem to totally inhibit the response of ewes to ram exposure. PMID:9928614

Godfrey, R W; Gray, M L; Collins, J R

1998-12-01

15

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases  

PubMed Central

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20?-hydroxysteroid dehydrogenase (20?-HSD) catabolizes P4 into its biologically inactive form, 20?-hydroxyprogesterone (20?-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20?-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3?-hydroxysteroid dehydrogenase (3?-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3?-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20?-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares. PMID:24492656

KOZAI, Keisuke; HOJO, Takuo; TOKUYAMA, Shota; SZÓSTEK, Anna Z; TAKAHASHI, Masashi; SAKATANI, Miki; NAMBO, Yasuo; SKARZYNSKI, Dariusz J; OKUDA, Kiyoshi

2014-01-01

16

Cantharidin and norcantharidin inhibit caprine luteal cell steroidogenesis in vitro.  

PubMed

Cantharidin and its analog norcantharidin are active constituents of Mylabris, have been demonstrated to ailments for a variety of cancers. But several reports of cantharidin's natural or accidental toxicoses in field animals and humans showed a strong connection between cantharidin and its abortifacient and aphrodisiac properties. However, their exact cellular mechanisms in steroidogenesis remains poorly understood. Thus this study was aimed to explore the effects of cantharidin on luteal cell steroidogensis and to compare its effect with that of norcantharidin. For this purpose, luteal cells isolated from corpora lutea of native Taiwan goats were maintained in vitro and treated for 4 and 24 h with cantharidin and norcantharidin (0.1, 1.0, and 10 ?g ml(-1)) to assess their steroidogenic effects. Progesterone (P(4)) levels and steroidogenic enzyme expression were assessed by enzyme immunoassay and Western blot methods, respectively. In caprine luteal cells, cantharidin and norcantharidin repressed basal P(4) production, as well as that mediated by ovine luteinizing hormone (oLH), 8-bromo-cyclic AMP (8-Br-cAMP), 22R-hydroxycholesterol (22R-OHC) and pregnenolone (P(5)). They also inhibited the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, and 3?-hydroxysteroid dehydrogenase (3?-HSD) enzyme. Additionally, the greater inhibitory effect was detected using cantharidin, when it is compared with that of norcantharidin. Our results suggest that ingestion of cantharidin may decrease luteal steroidogenesis, and the decline in luteal P(4) levels may disrupt reproductive functions in humans as well as animals. PMID:20594813

Twu, Nae-Fang; Srinivasan, Ramanujam; Chou, Chung-Hsi; Wu, Leang-Shin; Chiu, Chih-Hsien

2012-01-01

17

Prostaglandin- and thromboxane-producing activity of isolated luteal cells from pseudopregnant rabbits.  

PubMed

The pseudopregnant rabbit is proposed as a suitable model for studies on physiology and endocrinology of the luteal phase. Pseudopregnancy is defined by corpus luteum function from day 1 to day 12 post hCG, as demonstrated by peripheral progesterone concentrations. The corpus luteum is highly vascularized which is extremely important to ensure progesterone secretion. Prostaglandins are potent vasoactive compounds and may be involved in controlling the ovarian/luteal blood flow. The present studies were designed to investigate the capacity of luteal cells for their intracellular production rates of prostaglandins in the early luteal phase. Pseudopregnancy was induced with a subcutaneous injection of FSH/LH, followed two days later by an intravenous injection of hCG, on days 0, 1 to 4 of pseudopregnancy luteal cells were isolated and incubated for 4 days. Media were collected every 24 h and analyzed for prostaglandins. The luteal cells were characterized by immunocytochemistry and progesterone measurements. Cultured luteal cells were able to convert exogenously applied arachidonic acid into PGI2, PGE2, PGF2alpha, and TXA2. The major compound that could be detected in the culture medium and in the cells was PGI2. The absolute values of the production pattern varied in all experiments in the ranges PGI2 > PGE2 > PGF2alpha > TXA2 with the greatest difference on day 3. In view of this fact the corpus luteum may contribute locally synthesized prostaglandins to regulate its own function. The physiological meaning of these findings should now be studied in a more optimal environment such as organ culture. PMID:9228209

Schlegel, W; Ammermann, D; John, H

1997-05-01

18

Programmable nanoparticle functionalization for in vivo targeting  

PubMed Central

The emerging demand for programmable functionalization of existing base nanocarriers necessitates development of an efficient approach for cargo loading that avoids nanoparticle redesign for each individual application. Herein, we demonstrate in vivo a postformulation strategy for lipidic nanocarrier functionalization with the use of a linker peptide, which rapidly and stably integrates cargos into lipidic membranes of nanocarriers after simple mixing through a self-assembling process. We exemplified this strategy by generating a VCAM-1-targeted perfluorocarbon nanoparticle for in vivo targeting in atherosclerosis (ApoE-deficient) and breast cancer (STAT-1-deficient) models. In the atherosclerotic model, a 4.1-fold augmentation in binding to affected aortas was observed for targeted vs. nontargeted nanoparticles (P<0.0298). Likewise, in the breast cancer model, a 4.9-fold increase in the nanoparticle signal from tumor vasculature was observed for targeted vs. nontargeted nanoparticles (P<0.0216). In each case, the nanoparticle was registered with fluorine (19F) magnetic resonance spectroscopy of the nanoparticle perfluorocarbon core, yielding a quantitative estimate of the number of tissue-bound nanoparticles. Because other common nanocarriers with lipid coatings (e.g., liposomes, micelles, etc.) can employ this strategy, this peptide linker postformulation approach is applicable to more than half of the available nanosystems currently in clinical trials or clinical uses.—Pan, H., Myerson, J. W., Hu, L., Marsh, J. N., Hou K., Scott, M. J., Allen, J. S., Hu, G., San Roman, S., Lanza, G. M., Schreiber, R. D., Schlesinger, P. H., Wickline, S. A. Programmable nanoparticle functionalization for in vivo targeting. PMID:23047896

Pan, Hua; Myerson, Jacob W.; Hu, Lingzhi; Marsh, Jon N.; Hou, Kirk; Scott, Michael J.; Allen, John S.; Hu, Grace; San Roman, Susana; Lanza, Gregory M.; Schreiber, Robert D.; Schlesinger, Paul H.; Wickline, Samuel A.

2013-01-01

19

Clinostat rotation induces apoptosis in luteal cells of the pregnant rat  

NASA Technical Reports Server (NTRS)

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

Yang, Hyunwon; Bhat, Ganapathy K.; Sridaran, Rajagopala

2002-01-01

20

Effects of IL8 and immune cells on the regulation of luteal progesterone secretion.  

PubMed

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2? (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of interleukin 8 (IL8) on specific luteal cell types in vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5-4?h, and expression of chemokine was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum. PMID:24686456

Talbott, Heather; Delaney, Abigail; Zhang, Pan; Yu, Yangsheng; Cushman, Robert A; Cupp, Andrea S; Hou, Xiaoying; Davis, John S

2014-07-01

21

Gestating for 22 months: luteal development and pregnancy maintenance in elephants  

PubMed Central

The corpus luteum, a temporally established endocrine gland, formed on the ovary from remaining cells of the ovulated follicle, plays a key role in maintaining the early mammalian pregnancy by secreting progesterone. Despite being a monovular species, 2–12 corpora lutea (CLs) were found on the elephant ovaries during their long pregnancy lasting on average 640 days. However, the function and the formation of the additional CLs and their meaning remain unexplained. Here, we show from the example of the elephant, the close relationship between the maternally determined luteal phase length, the formation of multiple luteal structures and their progestagen secretion, the timespan of early embryonic development until implantation and maternal recognition. Through three-dimensional and Colour Flow ultrasonography of the ovaries and the uterus, we conclude that pregnant elephants maintain active CL throughout gestation that appear as main source of progestagens. Two LH peaks during the follicular phase ensure the development of a set of 5.4 ± 2.7 CLs. Accessory CLs (acCLs) form prior to ovulation after the first luteinizing hormone (LH) peak, while the ovulatory CL (ovCL) forms after the second LH peak. After five to six weeks (the normal luteal phase lifespan), all existing CLs begin to regress. However, they resume growing as soon as an embryo becomes ultrasonographically apparent on day 49 ± 2. After this time, all pregnancy CLs grow significantly larger than in a non-conceptive luteal phase and are maintained until after parturition. The long luteal phase is congruent with a slow early embryonic development and luteal rescue only starts ‘last minute’, with presumed implantation of the embryo. Our findings demonstrate a highly successful reproductive solution, different from currently described mammalian models. PMID:22719030

Lueders, Imke; Niemuller, Cheryl; Rich, Peter; Gray, Charlie; Hermes, Robert; Goeritz, Frank; Hildebrandt, Thomas B.

2012-01-01

22

In Vivo Imaging of Tissue Physiological Function  

Cancer.gov

The National Cancer Institute's Radiation Biology Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize methods for in vivo imaging.

23

In Vivo Calcium Imaging of Neural Network Function  

NSDL National Science Digital Library

Spatiotemporal activity patterns in local neural networks are fundamental to brain function. Network activity can now be measured in vivo using two-photon imaging of cell populations that are labeled with fluorescent calcium indicators. In this review, we discuss basic aspects of in vivo calcium imaging and highlight recent developments that will help to uncover operating principles of neural circuits.

2007-12-01

24

EFFECTS OF BROMODICHLOROMETHANE ON EX VIVO AND IN VITRO LUTEAL FUNCTION AND BROMODICHLOROMETHANE TISSUE DOSIMETRY IN THE PREGNANT F344 RAT  

EPA Science Inventory

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH...

25

ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression†  

PubMed Central

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2?(PGF) for 0–4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone. PMID:24196350

Mao, Dagan; Hou, Xiaoying; Talbott, Heather; Cushman, Robert; Cupp, Andrea

2013-01-01

26

Luteal phase support in infertility treatment: a meta-analysis of the randomized trials  

Microsoft Academic Search

BACKGROUND: The addition of GnRH agonist to the treatment regimen in women undergoing IVF cycles is thought to create a luteal phase defect. In an attempt to correct for this, many practitioners supplement with a variety of steroid hormones in the luteal phase. METHODS: To determine whether luteal phase support increases reproductive success in modern IVF cycles, a systematic review

E. A. Pritts; A. K. Atwood

2002-01-01

27

Biophotonics techniques for structural and functional imaging, in vivo  

PubMed Central

In vivo optical imaging is being conducted in a variety of medical applications, including optical breast cancer imaging, functional brain imaging, endoscopy, exercise medicine, and monitoring the photodynamic therapy and progress of neoadjuvant chemotherapy. In the past three decades, in vivo diffuse optical breast cancer imaging has shown promising results in cancer detection, and monitoring the progress of neoadjuvant chemotherapy. The use of near infrared spectroscopy for functional brain imaging has been growing rapidly. In fluorescence imaging, the difference between autofluorescence of cancer lesions compared to normal tissues were used in endoscopy to distinguish malignant lesions from normal tissue or inflammation and in determining the boarders of cancer lesions in surgery. Recent advances in drugs targeting specific tumor receptors, such as AntiBodies (MAB), has created a new demand for developing non-invasive in vivo imaging techniques for detection of cancer biomarkers, and for monitoring their down regulations during therapy. Targeted treatments, combined with new imaging techniques, are expected to potentially result in new imaging and treatment paradigms in cancer therapy. Similar approaches can potentially be applied for the characterization of other disease-related biomarkers. In this chapter, we provide a review of diffuse optical and fluorescence imaging techniques with their application in functional brain imaging and cancer diagnosis. PMID:22433452

Ardeshirpour, Yasaman; Gandjbakhche, Amir H.; Najafizadeh, Laleh

2014-01-01

28

New models for analyzing mast cell functions in vivo  

PubMed Central

In addition to their well-accepted role as critical effector cells in anaphylaxis and other acute IgE-mediated allergic reactions, mast cells have been implicated in a wide variety of process that contribute to disease or help to maintain health. While some of these roles were first suggested by analyses of mast cell products or functions in vitro, it is critical to determine whether, and under which circumstances, such potential roles actually can be performed by mast cells in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of mast cells and mast cell-associated products during biological responses in vivo, and comments on some of the similarities and differences in the results obtained with these newer versus older models of mast cell deficiency. PMID:23127755

Reber, Laurent L.; Marichal, Thomas; Galli, Stephen J.

2013-01-01

29

Cyclin D1 Determines Mitochondrial Function In Vivo†  

PubMed Central

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function. PMID:16809779

Sakamaki, Toshiyuki; Casimiro, Mathew C.; Ju, Xiaoming; Quong, Andrew A.; Katiyar, Sanjay; Liu, Manran; Jiao, Xuanmao; Li, Anping; Zhang, Xueping; Lu, Yinan; Wang, Chenguang; Byers, Stephen; Nicholson, Robert; Link, Todd; Shemluck, Melvin; Yang, Jianguo; Fricke, Stanley T.; Novikoff, Phyllis M.; Papanikolaou, Alexandros; Arnold, Andrew; Albanese, Christopher; Pestell, Richard

2006-01-01

30

Neurovascular coupling: in vivo optical techniques for functional brain imaging  

PubMed Central

Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology. PMID:23631798

2013-01-01

31

Functional regionalization of the teleost cerebellum analyzed in vivo  

PubMed Central

There has been accumulating evidence for a regionalized organization of the cerebellum, which was mostly deduced from anatomical mapping of axonal projections of cerebellar afferents. A likewise regionalization of the cerebellar output has been suggested from lesion studies and dye-tracer experiments, but its physiological targets as well as the functional relevance of such an output regionalization are less clear. Ideally, such functional regionalization should be proven noninvasively in vivo. We here provide evidence for such a regionalization of the output from the cerebellar cortex by genetically encoded transneuronal mapping of efferent circuits of zebrafish Purkinje neurons. These identified circuits correspond to distinct regionalized Purkinje cell activity patterns in freely behaving zebrafish larvae during the performance of cerebellar-dependent behaviors. Furthermore, optogenetic interrogation of selected Purkinje cell regions during animal behavior confirms the functional regionalization of Purkinje cell efferents and reveals their contribution to behavior control as well as their function in controlling lateralized behavioral output. Our findings reveal how brain compartments serve to fulfill a multitude of functions by dedicating specialized efferent circuits to distinct behavioral tasks. PMID:25002482

Matsui, Hideaki; Namikawa, Kazuhiko; Babaryka, Andreas; Köster, Reinhard W.

2014-01-01

32

Considerations when investigating lncRNA function in vivo.  

PubMed

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. PMID:25124674

Bassett, Andrew R; Akhtar, Asifa; Barlow, Denise P; Bird, Adrian P; Brockdorff, Neil; Duboule, Denis; Ephrussi, Anne; Ferguson-Smith, Anne C; Gingeras, Thomas R; Haerty, Wilfried; Higgs, Douglas R; Miska, Eric A; Ponting, Chris P

2014-01-01

33

POLYMERSOMES: MULTI-FUNCTIONAL TOOLS FOR IN VIVO CANCER THERANOSTIC APPLICATIONS.  

E-print Network

??ABSTRACT POLYMERSOMES: MULTI-FUNCTIONAL TOOLS FOR IN VIVO CANCER THERANOSTIC APPLICATIONS Dalia Hope Levine Dr. Daniel A. Hammer Nanoparticles are currently being developed as delivery vehicles… (more)

Levine, Dalia H

2010-01-01

34

Considerations when investigating lncRNA function in vivo  

PubMed Central

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. DOI: http://dx.doi.org/10.7554/eLife.03058.001 PMID:25124674

Bassett, Andrew R; Akhtar, Asifa; Barlow, Denise P; Bird, Adrian P; Brockdorff, Neil; Duboule, Denis; Ephrussi, Anne; Ferguson-Smith, Anne C; Gingeras, Thomas R; Haerty, Wilfried; Higgs, Douglas R; Miska, Eric A; Ponting, Chris P

2014-01-01

35

Influence of genetic background on ex vivo and in vivo cardiac function in several commonly used inbred mouse strains  

PubMed Central

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease. PMID:20627938

Barnabei, Matthew S.; Palpant, Nathan J.

2010-01-01

36

In vitro gene regulatory networks predict in vivo function of liver  

PubMed Central

Background Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. Results Here we use the nitroaromatic 2,4,6-trinitrotoluene (TNT) as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. We found 341 transcripts differentially expressed in common among in vitro and in vivo assays in response to TNT. The major functional term corresponding to these transcripts was cell cycle. Similarly modulated common pathways were identified between in vitro and in vivo. Furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to TNT exposure, which mainly contain 2 subnetwork modules: PTTG1 and PIR centered networks. Interestingly, all 7 genes in the PTTG1 module were involved in cell cycle and downregulated by TNT both in vitro and in vivo. Conclusions The results of our investigation of TNT effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. Inhibiting PTTG1 and its targeted cell cyle related genes could be key machanism for TNT induced liver toxicity. PMID:21073692

2010-01-01

37

The impact of luteal supplement on pregnancy outcome following stimulated IVF cycles.  

PubMed

This is a prospeve randomised study designed to clarify the impact of various luteal support regimes (HCG and progesterone) on progesterone profiles and pregnancy outcomes. This study involved subjects undergone down regulated. stimulated IVF cycles using various types of luteal support, namely: Cyclogest (n=35). Crinone gel (n=36), various doses of Utrogestan (n=55) and HCG (n=35). Various doses of Utrogestan (administered vaginally), Crinone gel (progesterone administered vaginally) and Cyclogest (progesterone administered rectally) supplementation induced similar end plasma progesterone concentrations ranging from 26 to 32 mmnl/l. These progesterone regimes produced no significant differences. Hence, the impact of exogenous proge,terone supplement was relatively trivial and did not 'stabilise' the sub-optimal luteal phase. In contrast, two small HCG injections during the early and mid-luteal phase possessed a much greater ability to 'stabilise' progesterone profiles. Despite this additional advantage, implantation and pregnancy rates with either HCG or progesterone supplements were similar. Although none of these forms of luteal support adequately 'normalised' luteal progesterone profiles, this did not appear to be detrimental to the process of implantation. PMID:16114155

Tay, P Y S; Lenton, E A

2005-06-01

38

Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells.  

PubMed

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function. PMID:19253104

Liu, Jianqi; Bützow, Ralf; Hydén-Granskog, Christel; Voutilainen, Raimo

2009-02-01

39

Rat parotid cell function in vitro following x irradiation in vivo  

SciTech Connect

The effect of X irradiation on rat parotid acinar cell function was evaluated in vitro 1, 3, and 7 days following in vivo exposure to 2000 R. Several cellular functions were followed: protein secretion (amylase release), ion movement (K/sup +/ efflux and reuptake), amino acid transport (..cap alpha..-amino(/sup 14/C)isobutyric acid), and an intermediary metabolic response ((/sup 14/C)glucose oxidation). In addition both the morphologic appearance and in vivo saliva secretory ability of parotid cells were assessed. Our results demonstrate that surviving rat parotid acinar cells, isolated and studied in vitro 1-7 days following 2000 R, remain functionally intact despite in vivo diminution of secretory function.

Bodner, L.; Kuyatt, B.L.; Hand, A.R.; Baum, B.J.

1984-02-01

40

The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (Macaca radiata)  

PubMed Central

Background In higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles. Methods To identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy. Results Based on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined. Conclusion These results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function. PMID:21291521

2011-01-01

41

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear  

E-print Network

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear Tony compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2

Goodrich, Lisa V.

42

Analysis of in vitro and in vivo function of total knee replacements using dynamic contact models  

Microsoft Academic Search

Despite the high incidence of osteoarthritis in human knee joint, its causes remain unknown. Total knee replacement (TKR) has been shown clinically to be effective in restoring the knee function. However, wear of ultra-high molecular weight polyethylene has limited the longevity of TKRs. To address these important issues, it is necessary to investigate the in vitro and in vivo function

Dong Zhao

2006-01-01

43

The Luteal Phase after GnRHa Trigger-Understanding An Enigma  

PubMed Central

The luteal phase of all stimulated in vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI) cycles is disrupted, which makes luteal phase support (LPS) mandatory. The cause of the disruption is thought to be the multifollicular development achieved during ovarian stimulation which results in supraphysiological concentrations of steroids se- creted by a high number of corpora lutea during the early luteal phase. This will directly inhibit luteinizing hormone (LH) secretion by the pituitary via negative feedback at the level of the hypothalamic-pituitary axis, leading to a luteal phase defect. With the intro- duction of the gonadotropin-releasing hormone (GnRH) antagonist protocol, it became feasible to trigger final oocyte maturation and ovulation with a single bolus of GnRH agonist (GnRHa) as an alternative to human chorionic gonadotropin (hCG). GnRHa trig- gering presents several advantages, including the reduction in or even elimination of ovarian hyperstimulation syndrome. Despite the potential advantages of GnRHa trig- gering, previous randomized controlled trials reported a poor clinical outcome with high rates of early pregnancy losses, despite supplementation with a standard LPS in the form of progesterone and estradiol. Following these disappointing results, several studies now report a luteal phase rescue after modifications of the LPS, resulting in a reproductive outcome comparable to that seen after hCG triggering. We herein review luteal phase dif- ferences between the natural cycle, hCG trigger and GnRHa trigger and present the most recent data on handling the luteal phase after GnRHa triggering. PMID:25379149

Leth-Moller, Kathrine; Hammer Jagd, Sandra; Humaidan, Peter

2014-01-01

44

The Luteal Phase after GnRHa Trigger-Understanding An Enigma.  

PubMed

The luteal phase of all stimulated in vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI) cycles is disrupted, which makes luteal phase support (LPS) mandatory. The cause of the disruption is thought to be the multifollicular development achieved during ovarian stimulation which results in supraphysiological concentrations of steroids se- creted by a high number of corpora lutea during the early luteal phase. This will directly inhibit luteinizing hormone (LH) secretion by the pituitary via negative feedback at the level of the hypothalamic-pituitary axis, leading to a luteal phase defect. With the intro- duction of the gonadotropin-releasing hormone (GnRH) antagonist protocol, it became feasible to trigger final oocyte maturation and ovulation with a single bolus of GnRH agonist (GnRHa) as an alternative to human chorionic gonadotropin (hCG). GnRHa trig- gering presents several advantages, including the reduction in or even elimination of ovarian hyperstimulation syndrome. Despite the potential advantages of GnRHa trig- gering, previous randomized controlled trials reported a poor clinical outcome with high rates of early pregnancy losses, despite supplementation with a standard LPS in the form of progesterone and estradiol. Following these disappointing results, several studies now report a luteal phase rescue after modifications of the LPS, resulting in a reproductive outcome comparable to that seen after hCG triggering. We herein review luteal phase dif- ferences between the natural cycle, hCG trigger and GnRHa trigger and present the most recent data on handling the luteal phase after GnRHa triggering. PMID:25379149

Leth-Moller, Kathrine; Hammer Jagd, Sandra; Humaidan, Peter

2014-10-01

45

Inhibition of progesterone secretion by oestradiol administered in the luteal phase of assisted conception cycles.  

PubMed

A prospective randomised study was done to assess the effect of supplemental oestradiol in addition to progesterone on the luteal steroid profiles and pregnancy outcome in stimulated cycles with and without pituitary down regulation. Women undergoing stimulated cycle IVF with GnRH-a and FSH (Group A, n = 63) or stimulated intrauterine insemination using CC and FSH (Group B, n = 55) were studied. These subjects were randomly allocated to receive either 400 mg daily of vaginally administrated Cyclogest (progesterone) alone or in combination with 2 mg daily of oral Oestradiol Valerate (E2V) during the luteal phase. Significant lower concentrations of plasma progesterone were observed in those subjects supplemented with both E2V and progesterone compared to those in whom progesterone only was given during the luteal phase (P < 0.05). Exogenous E2V had a minimal impact on plasma oestradiol concentrations and did not disguise the characterised mid luteal decline in oestradiol secretion. The suppressive effect of E2V on plasma progesterone was lost if implantation occurred normally because any small change in steroid concentrations was reversed by the rapidly increasing concentrations of HCG. Similar pregnancy rates were observed among subjects supplemented with or without oestradiol. The addition of oestradiol to the luteal supplement suppresses endogenous corpus luteum progesterone secretion irrespective of the type of assisted conception cycle and that its use is unlikely to be beneficial to the process of implantation. PMID:14569738

Tay, P Y; Lenton, E A

2003-06-01

46

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo.  

PubMed

Clonal deletion and/or inactivation establishes tolerance to self antigens. Endogenous and exogenous (bacterial) superantigens, like the staphylococcal enterotoxins, induce ligand-specific clonal anergy in vivo and thus are believed to mirror aspects of post-thymic tolerance mechanisms in mature peripheral T cells. Here we analyzed the level of anergy of ligand-responsive V beta 8+ T cells from staphylococcal enterotoxin B (SEB)-primed mice in vivo and in vitro. Upon in vitro restimulation with SEB, CD4+V beta 8+ and CD8+V beta 8+ T cells failed to produce IL-2. However, functional IL-2 receptors were triggered, since supplementation with IL-2 induced clonal growth in virtually all CD4+V beta 8+ and CD8+V beta 8+ T cells as determined by limiting dilution analyses. Thus in vitro unresponsiveness of lymphocytes from SEB-primed mice reflects the inability of SEB-reactive V beta 8+ T cells to produce IL-2. Surprisingly, anergy as defined in vitro was at variance with that in vivo. Following further challenge with SEB, systemic and acute lymphokine production (including IL-2 and tumor necrosis factor) occurred with almost identical peak values and kinetics to primary in vivo responses, and D-galactosamine-sensitized mice succumbed to lethal shock. Polymerase chain reaction analyses revealed that CD4+V beta 8+ expressed IL-2-specific mRNA in vivo upon restimulation with SEB. While lymphokine production and expression of the IL-2 receptor was similar to the response to in vivo primary stimulation, only CD8+V beta 8+ T cells expanded clonally upon reintroduction of SEB in vivo. Hence primed V beta 8+ T cells challenged with SEB display in vitro anergy yet in vivo responsiveness, at least in part. We conclude that the state of anergy is reversible, dependent upon the quality of activation signals provided in in vivo rather than in in vitro culture conditions. PMID:7718507

Heeg, K; Gaus, H; Griese, D; Bendigs, S; Miethke, T; Wagner, H

1995-01-01

47

Inflammation modulates human HDL composition and function in vivo  

Technology Transfer Automated Retrieval System (TEKTRAN)

Inflammation may directly impair HDL functions, in particular reverse cholesterol transport (RCT), but limited data support this concept in humans. Our study was designed to investigate this relationship. We employed low-dose human endotoxemia to assess the effects of inflammation on HDL and RCT-rel...

48

Nicotine Pretreatment Increases Dysphoric Effects of Alcohol in Luteal-Phase Female Volunteers  

PubMed Central

The present report shows that nicotine enhances some of alcohol’s positive and negative effects in women and that these effects are most pronounced during the luteal phase of the menstrual cycle. Ten low progesterone and 10 high progesterone/luteal-phase women received nicotine patch pretreatments (placebo or 21 mg) 3 hours before an alcohol challenge (0.4 g/kg). Subjective effects were recorded on mood adjective scales and the Addiction Research Center Inventory (ARCI). Heart rate and skin temperature were recorded. Luteal-phase women reported peak positive (e.g. “stimulated”) and peak negative effects (e.g. “clumsy”, “dizzy”) almost twice as great as low progesterone women. PMID:19440397

Penetar, David M.; Kouri, Elena M.; McCarthy, Elissa M.; Lilly, Michelle M.; Peters, Erica N.; Juliano, Trisha M.; Lukas, Scott E.

2009-01-01

49

Defense cells profile of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo  

PubMed Central

The aim of this study was to evaluate the defense cells changes of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo. Reproductive organs of the adult and apparently healthy female buffaloes were collected from the slaughterhouse. By visual investigation of both the ovaries for presence of corpus luteum and growing follicles, the luteal and follicular phase of each buffalo was specified. Cervical discharge samples were collected by sterile swabs and then spread over the glass slides, dried and fixed with methanol. The specimens were undergone Giemsa staining. The percentage of lymphocytes, neutrophils, monocytes (macrophages), eosinophils and basophils in each case (for both the follicular and luteal phases) were obtained at 20 microscopic fields. The percentage of lymphocytes, neutrophils and basophils in luteal phase were higher than the follicular phase. The percentage of eosinophils in follicular phase was higher than the luteal phase. The percentage of monocytes (macrophages) in luteal and follicular phases was nearly equal. The statistical analysis showed that the differences of all cells between follicular and luteal phase were not significant (P > 0.05). The most defense cells in discharges of external os of cervix (both follicular and luteal phases) were neutrophils and lymphocytes.

Ayen, Esmail; Hasanzadeh, Shapour; Tabatabaei, Saleh

2012-01-01

50

Effect of Processing and Storage on RBC function in vivo  

PubMed Central

Red Blood Cell (RBC) transfusion is indicated to improve oxygen delivery to tissue, and for no other purpose. We have come to appreciate that donor RBCs are fundamentally altered during processing and storage, in a fashion that both impairs oxygen transport efficacy and introduces additional risk by perturbing both immune and coagulation systems. The protean biophysical and physiologic changes in RBC function arising from storage are termed the ‘storage lesion’; many have been understood for some time; for example, we know that the oxygen affinity of stored blood rises during the storage period1 and that intracellular allosteric regulators, notably 2,3-bisphosphoglyceric acid (DPG) and ATP, are depleted during storage. Our appreciation of other storage lesion features has emerged with improved understanding of coagulation, immune and vascular signaling systems. Herein we review key features of the ‘storage lesion’. Additionally, we call particular attention to the newly appreciated role of RBCs in regulating linkage between regional blood flow and regional O2 consumption by regulating the bioavailability of key vasoactive mediators in plasma, as well as discuss how processing and storage disturbs this key signaling function and impairs transfusion efficacy. PMID:22818545

Doctor, Allan; Spinella, Phil

2012-01-01

51

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation  

E-print Network

In vivo neuronal function of the fragile X mental retardation protein is regulated November 7, 2011 Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene,7). The X-linked neurodevelopmental disorder is caused by the loss of fragile X mental retardation 1 (FMR1

Broadie, Kendal S.

52

In vivo evidence of the targeting of cartilaginous tissue by pyridinium functionalized nanoparticles.  

PubMed

Ultrasmall gadolinium based particles have been functionalized with positively charged pyridinium quaternary ammonium and labelled with (111)In. Evidence of their active targeting properties towards proteoglycans has been demonstrated in vivo after intravenous injection into rats opening thus a route to cancer imaging and therapy. PMID:23467614

Morlieras, Jessica; Chezal, Jean-Michel; Miot-Noirault, Elizabeth; Vidal, Aurélien; Besse, Sophie; Kryza, David; Truillet, Charles; Mignot, Anna; Antoine, Rodolphe; Dugourd, Philippe; Redini, Françoise; Sancey, Lucie; Lux, François; Perriat, Pascal; Janier, Marc; Tillement, Olivier

2013-04-14

53

Effects of ACL Reconstruction on In-Vivo, Dynamic Knee Function  

PubMed Central

Synopsis The purposes of this article are to discuss key factors for assessing joint function, to present some recent findings and to address the future directions for evaluating the function of the ACL-injured/reconstructed knees. Well-designed studies, using state-of-the art tools to assess knee kinematics under in vivo, dynamic, high-loading conditions, are necessary to evaluate the relative performance of different procedures for restoring normal joint motion. PMID:23177461

Tashman, Scott; Araki, Daisuke

2012-01-01

54

In vivo, high-throughput imaging for functional characterization of the embryonic zebrafish heart  

Microsoft Academic Search

High-throughput imaging allows characterizing the phenotype of large populations of cells, organs or organisms. Studying the function of organs in similar ways remains a challenge. Here, we present a semi-automatic, in vivo, high-throughput imaging and analysis method to characterize cardiac function in the embryonic heart of developing zebrafish larvae subjected to an increase of breeding temperature. We sequentially acquire high-speed

Jungho Ohn; Michael Liebling

2011-01-01

55

Comparison of the steroidogenic capacity of bovine follicular and luteal cells, and corpora lutea originating from dominant follicles of the first or second follicular wave.  

PubMed

This study, compared the endocrine function of dominant follicles of the first and second follicular waves (DF1 and DF2, respectively) and the corpora lutea that were subsequently formed. In the experiments conducted in vitro, ovaries were collected from dairy cows on day 6.1 +/- 0.2 or day 14.8 +/- 0.2 of the oestrous cycle to obtain steroidogenically active DF1 (n = 8) and DF2 (n = 7). Granulosa and thecal cells were isolated, dispersed and incubated for 16 h with testosterone (granulosa cells) or forskolin or bLH (thecal cells). Both types of cell were subsequently cultured for 9 days with forskolin and insulin. The viability of the granulosa cells was similar in DF1 and DF2, but the concentration of oestradiol in the follicular fluid was higher in DF1 than in DF2. Production of oestradiol and progesterone by granulosa cells was similar in DF1 and DF2, but androstenedione and progesterone production by thecal cells were 3.5-6.5-fold higher in DF1 than in DF2. During the 9 days of luteinization, progesterone production was similar in DF1- and DF2-derived granulosa cells, but was two- to three-fold higher in DF1- than in DF2-derived thecal cells. Experiments were also conducted in vivo. In Expt 1 in vivo, lactating cows that were assigned to ovulate DF1 or DF2 (n = 9 and 13 in replicate 1 and 2, respectively) were injected with PGF2 alpha on days 6 and 7 or on days 14 and 15 of the oestrous cycle, respectively. A wave by replicate interaction was detected for plasma progesterone concentration in the subsequent cycle: in the first replicate, progesterone production was approximately 40% higher in cows that ovulated DF1; in the second replicate, progesterone production was similar in cows that ovulated DF1 or DF2. In Expt 2, pooled plasma progesterone in the mid-luteal phase (days 12-15) after insemination of pregnant and non-pregnant cows was approximately 30% higher in cows that had ovulated DF1 (n = 32) than in cows that had ovulated DF2 (n = 22). This study showed DF1 had a higher steroidogenic capacity compared with DF2, which may be related to the hormonal environment in which the follicles developed. PMID:10690191

Wolfenson, D; Sonego, H; Shaham-Albalancy, A; Shpirer, Y; Meidan, R

1999-11-01

56

Defining Uremic Arterial Functional Abnormalities in Patients Recently Started on Haemodialysis: Combined In Vivo and Ex Vivo Assessment  

PubMed Central

Endothelial dysfunction is a key initiating event in vascular disease in chronic kidney disease (CKD) patients and haemodialysis (HD) patients exhibit significant vascular abnormalities. To understand this further, we examined how ex vivo intrinsic function in isolated arteries correlates with in vivo assessments of cardiovascular status in HD patients. Abdominal fat biopsies were obtained from 11 HD patients and 26 non-uremic controls. Subcutaneous arteries were dissected and mounted on a wire myograph, and cumulative concentration-response curves to noradrenalin, endothelin-1, a thromboxane A2 agonist (U46619), angiotensin II, vasopressin, bradykinin (BK), acetylcholine (ACh) and sodium nitroprusside (SNP) were constructed. Pulse wave velocity and blood pressure were measured in HD patients. Enhanced (P<0.05?0.0001) maximal contractile responses (Rmax) to all spasmogens (particularly vasopressin) were observed in arteries from HD patients compared to controls, and this effect was more pronounced in arteries with an internal diameter>600 µm. The potency (pEC50) of U46619 (P<0.01) and vasopressin (P<0.001) was also increased in arteries>600 µm of HD patients. The maximal relaxant response to the endothelium-dependent dilators ACh and BK were lower in HD patients (P<0.01-P<0.0001) (worse for ACh than BK); however the endothelium-independent dilator SNP was similar in both groups. PWV was significantly correlated with the vasoconstrictor response to vasopressin (P?=?0.042) in HD patients. HD patients are primed for hypertension and end organ demand ischaemia by a highly sensitised pressor response. The failure of arterial relaxation is mediated by endothelial dysfunction. Intrinsic vascular abnormalities may be important in sensitising HD patients to recurrent cumulative ischaemic end organ injury. PMID:25546407

Abushufa, Adil M.; Eldehni, Mohamed T.; Odudu, Aghogho; Evans, Philip D.; O?Sullivan, Saoirse E.; McIntyre, Chris W.

2014-01-01

57

How Much In Vitro Cholesterol Reducing Activity of Lactobacilli Predicts Their In Vivo Cholesterol Function?  

PubMed Central

Background: Based on literature, in vitro cholesterol removal of lactic acid bacteria has been accounted for their in vivo cholesterol reduction. But recently it has been proposed that such in vitro characteristic may not be directly relevant to their in vivo activity. The objective of this study was to find how much in vitro cholesterol reducing potential of Lactobacillus plantarum A7 (LA7), a native strain isolated from an infant fecal flora, reflects its in vivo efficiency. LA7 previously showed serum cholesterol reducing capability in mice subjected to fatty diet. Here, we investigate whether the given strain is capable of in vitro cholesterol assimilation or consumption. Method: LA7 was cultured in whole milk and de-Man–Rogosa–Sharpe (MRS) added with water-soluble cholesterol. Colorimetric method was adopted for cholesterol determination in both cultured media during incubation period. Results: No cholesterol assimilation was detected by growth and incubation of the active culture in either of the medium. Thus, in vivo cholesterol function of LA7 was not caused by cholesterol consumption. A comprehensive review of literature on the related studies also showed that there are other documented studies which evidenced the uncertainty of the direct relation between in vitro and in vivo studies. Conclusion: Cholesterol removal from the cultured media may not be considered as an appropriate integral index for selection of Lactobacillus strains with cholesterol-lowering activity. PMID:23671771

Madani, Golnoush; Mirlohi, Maryam; Yahay, Mahmoud; Hassanzadeh, Akbar

2013-01-01

58

Ex vivo lung function measurements in precision-cut lung slices (PCLS) from chemical allergen-sensitized mice represent a suitable alternative to in vivo studies.  

PubMed

A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation. PMID:18775882

Henjakovic, M; Martin, C; Hoymann, H G; Sewald, K; Ressmeyer, A R; Dassow, C; Pohlmann, G; Krug, N; Uhlig, S; Braun, A

2008-12-01

59

Luteal Phase Support in the Intrauterine Insemination (IUI) Cycles: A Randomized Double Blind, Placebo Controlled Study  

PubMed Central

Objective: To evaluate the impact of luteal phase support with vaginal progesterone on pregnancy rates in the intrauterine insemination (IUI) cycles, stimulated with clomiphene citrate and human menopausal gonadotropin (hMG), in sub fertile couples. Materials and methods: This prospective, randomized, double blind study was performed in a tertiary infertility center from March 2011 to January 2012. It consisted of 253 sub fertile couples undergoing ovarian stimulation for IUI cycles. They underwent ovarian stimulation with clomiphene citrate (100 mg) and hMG (75 IU) in preparation for the IUI cycle. Study group (n = 127) received luteal phase support in the form of vaginal progesterone (400 mg twice a day), and control group (n = 126) received placebo. Clinical pregnancy and abortion rates were assessed and compared between the two groups. Results: The clinical pregnancy rate was not significantly higher for supported cycles than that for the unsupported ones (15.75% vs. 12.69%, p = 0.3). The abortion rate in the patients with progesterone luteal support compared to placebo group was not statistically different (10% vs. 18.75%, p = 0.45). Conclusion: It seems that luteal phase support with vaginal progesterone was not enhanced the success of IUI cycles outcomes, when clomiphene citrate and hMG were used for ovulation stimulation. PMID:25530766

Hossein Rashidi, Batool; Davari Tanha, Fatemeh; Rahmanpour, Haleh; Ghazizadeh, Mahya

2014-01-01

60

Functionalized gold nanoparticles: a detailed in vivo multimodal microscopic brain distribution study  

NASA Astrophysics Data System (ADS)

In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex.In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex. Electronic supplementary information (ESI) available: Fig. S1-S6. See DOI: 10.1039/c0nr00345j

Sousa, Fernanda; Mandal, Subhra; Garrovo, Chiara; Astolfo, Alberto; Bonifacio, Alois; Latawiec, Diane; Menk, Ralf Hendrik; Arfelli, Fulvia; Huewel, Sabine; Legname, Giuseppe; Galla, Hans-Joachim; Krol, Silke

2010-12-01

61

In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8[W  

PubMed Central

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the ?-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function. PMID:23012433

O’Hara, Andrew; Jenkins, Gareth I.

2012-01-01

62

Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo  

Microsoft Academic Search

Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in

Richard Benton; Silke Sachse; Stephen W. Michnick; Leslie B. Vosshall

2006-01-01

63

GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo  

PubMed Central

Summary Rho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family. PMID:17373658

Bustelo, Xosé R.; Sauzeau, Vincent; Berenjeno, Inmaculada M.

2007-01-01

64

Non-invasive in vivo imaging of pancreatic ?-cell function and survival – a perspective  

PubMed Central

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/?-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic ?-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional ?-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus. PMID:21477063

Leibiger, I. B.; Caicedo, A.; Berggren, P.-O.

2012-01-01

65

An In Vivo Functional Analysis System for Renal Gene Discovery in Drosophila Pericardial Nephrocytes  

PubMed Central

The difficulty in accessing mammalian nephrons in vivo hinders the study of podocyte biology. The Drosophila nephrocyte shares remarkable similarities to the glomerular podocyte, but the lack of a functional readout for nephrocytes makes it challenging to study this model of the podocyte, which could potentially harness the power of Drosophila genetics. Here, we present a functional analysis of nephrocytes and establish an in vivo system to screen for renal genes. We found that nephrocytes efficiently take up secreted fluorescent protein, and therefore, we generated a transgenic line carrying secreted fluorescent protein and combined it with a nephrocyte-specific driver for targeted gene knockdown, allowing the identification of genes required for nephrocyte function. To validate this system, we examined the effects of knocking down sns and duf, the Drosophila homologs of nephrin and Neph1, respectively, in pericardial nephrocytes. Knockdown of sns or duf completely abolished the accumulation of the fluorescent protein in pericardial nephrocytes. Examining the ultrastructure revealed that the formation of the nephrocyte diaphragm and lacunar structure, which is essential for protein uptake, requires sns. Our preliminary genetic screen also identified Mec2, which encodes the homolog of mammalian Podocin. Taken together, these data suggest that the Drosophila pericardial nephrocyte is a useful in vivo model to help identify genes involved in podocyte biology and facilitate the discovery of renal disease genes. PMID:23291470

Zhang, Fujian; Zhao, Ying

2013-01-01

66

In vivo investigations on luteotropic activity of prostaglandins during early diestrus in nonpregnant bitches.  

PubMed

The aim of this study was to test for the postulated luteotropic effect of prostaglandin E2 during early diestrus in the dog in an in vivo study. This study was performed on 30 bitches which were randomly assigned to a treatment group (TG) and a control group. Starting on the day of ovulation (Day 0), dogs of the TG were treated for 5, 10, 20, or 30 days with 10 mg firocoxib/kg body weight per day (Previcox, a selective PTGS2 inhibitor) and ovariohysterectomized for collection of corpora lutea on the last day of treatment. Similarly, dogs of the control group were ovariohysterectomized on Days 0, 5, 10, 20, and 30. Blood samples for progesterone measurement were collected every second day; additionally, the area of luteal cell nuclei and the expression of 3?-hydroxysteroid-dehydrogenase at the mRNA and the protein levels were assessed. Mean P4 concentrations were lower in TGs; however, a significant difference was only observed on Day 10. This observation is in line with the finding that treatment with firocoxib reduced expression of 3?-hydroxysteroid-dehydrogenase mRNA and protein (P < 0.05) and the area of luteal cell nuclei (P < 0.05). The results of this study further point to the postulated luteotropic function of prostaglandin E2. PMID:25115648

Janowski, Tomasz; Fingerhut, Julia; Kowalewski, Mariusz P; Zdu?czyk, S?awomir; Domos?awska, Anna; Jurczak, Andrzej; Boos, Alois; Schuler, Gerhard; Hoffmann, Bernd

2014-10-01

67

Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions  

SciTech Connect

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

2005-01-01

68

Study of Brain Function and Bioenergetics using fMRI and In Vivo MRS at High Fields  

Microsoft Academic Search

The greatest merit of magnetic resonance (MR) methodology applied to medicine is its capabilities of measuring a variety of physiological parameters in vivo. MR imaging (MRI) with unique imaging contrasts can provide vital information which tightly links to brain functions at both normal and diseased states. In contrast, in vivo MR spectroscopy (MRS) is capable of determining metabolites, bioenergetics and

Wei Chen

2005-01-01

69

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo.  

PubMed

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ''PTM'' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels. PMID:16737711

Berry, Katherine L; Hobert, Oliver

2006-06-23

70

In vivo cardiac anatomical and functional effects of wheel running in mice by magnetic resonance imaging.  

PubMed

Physical activity is frequently used as a strategy to decrease pathogenesis and improve outcomes in chronic pathologies such as metabolic or cardiac diseases. In mice, it has been shown that voluntary wheel running (VWR) could induce an aerobic training effect and may provide a means of exploring the relationship between physical activity and the progression of pathology, or the effect of a drug on locomotor activity. To the best of our knowledge, in vivo magnetic resonance imaging (MRI) and other non-invasive methods had not been investigated for training evaluation in mice; therefore, it was proposed to test an MRI method coupled with a cardiorespiratory gating system on C57Bl/6 mice for in vivo heart anatomical and functional characterization in both trained and untrained animals. Twenty mice were either assigned to a 12-week VWR program or to a control group (CON - no wheel in the cage). At week 12, MRI scans showed an increase in the left ventricular (LV) wall mass in the VWR group compared with the CON group. The ex vivo measurements also found an increase in the heart and LV weight, as well as an increase in oxidative enzyme activities (i.e. cytochrome c oxidase [COx] in the soleus). In addition, correlations have been observed between ex vivo LV/body weight ratio, COx activity in the soleus and in vivo MRI LV wall mass/body weight. In conclusion, mouse cardiac MRI methods coupled with a cardio-respiratory gating system are sufficiently effective and feasible for non-invasive, training-induced heart hypertrophy characterization, and may be used for longitudinal training level follow-up in mouse models of cardiovascular and metabolic diseases. PMID:22328593

Aufradet, Emeline; Bessaad, Amine; Alsaid, Hasan; Schäfer, Florian; Sigovan, Monica; De Souza, Geneviève; Chirico, Erica; Martin, Cyril; Canet-Soulas, Emmanuelle

2012-03-01

71

Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo  

PubMed Central

Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown Norway rats were dosed (8 mg/kg, i.t.) 24 hr prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed. PMID:21385100

Lord, Kevin; Moll, David; Lindsey, John K.; Mahne, Sarah; Raman, Girija; Dugas, Tammy; Cormier, Stephania; Troxlair, Dana; Lomnicki, Slawo; Dellinger, Barry; Varner, Kurt

2011-01-01

72

Structural determinants of Arabidopsis thaliana Hyponastic leaves 1 function in vivo.  

PubMed

MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity. PMID:25409478

Burdisso, Paula; Milia, Fernando; Schapire, Arnaldo L; Bologna, Nicolás G; Palatnik, Javier F; Rasia, Rodolfo M

2014-01-01

73

Structural Determinants of Arabidopsis thaliana Hyponastic Leaves 1 Function In Vivo  

PubMed Central

MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity. PMID:25409478

Burdisso, Paula; Milia, Fernando; Schapire, Arnaldo L.; Bologna, Nicolás G.; Palatnik, Javier F.; Rasia, Rodolfo M.

2014-01-01

74

In vivo functional microangiography by visible-light optical coherence tomography  

PubMed Central

Although hemoglobin oxygen saturation (sO2) in the microvasculature is an essential physiological parameter of local tissue functions, non-invasive measurement of microvascular sO2 is still challenging. Here, we demonstrated that visible-light optical coherence tomography (vis-OCT) can simultaneously provide three-dimensional anatomical tissue morphology, visualize microvasculature at the capillary level, and measure sO2 from the microvasculature in vivo. We utilized speckle contrast caused by the moving blood cells to enhance microvascular imaging. We applied a series of short-time inverse Fourier transforms to obtain the spectroscopic profile of blood optical attenuation, from which we quantified sO2. We validated the sO2 measurement in mouse ears in vivo through hypoxia and hyperoxia challenges. We further demonstrated that vis-OCT can continuously monitor dynamic changes of microvascular sO2. PMID:25360376

Yi, Ji; Chen, Siyu; Backman, Vadim; Zhang, Hao F.

2014-01-01

75

In vivo functional microangiography by visible-light optical coherence tomography.  

PubMed

Although hemoglobin oxygen saturation (sO2) in the microvasculature is an essential physiological parameter of local tissue functions, non-invasive measurement of microvascular sO2 is still challenging. Here, we demonstrated that visible-light optical coherence tomography (vis-OCT) can simultaneously provide three-dimensional anatomical tissue morphology, visualize microvasculature at the capillary level, and measure sO2 from the microvasculature in vivo. We utilized speckle contrast caused by the moving blood cells to enhance microvascular imaging. We applied a series of short-time inverse Fourier transforms to obtain the spectroscopic profile of blood optical attenuation, from which we quantified sO2. We validated the sO2 measurement in mouse ears in vivo through hypoxia and hyperoxia challenges. We further demonstrated that vis-OCT can continuously monitor dynamic changes of microvascular sO2. PMID:25360376

Yi, Ji; Chen, Siyu; Backman, Vadim; Zhang, Hao F

2014-10-01

76

Homeostasis and function of regulatory T cells (Tregs) in vivo: lessons from TCR-transgenic Tregs  

PubMed Central

The identification of CD25 and subsequently Forkhead box protein 3 (Foxp3) as markers for regulatory T cells (Tregs) has revolutionized our ability to explore this population experimentally. In a similar vein, our understanding of antigen-specific Treg responses in vivo owes much to the fortuitous generation of T-cell receptor (TCR)-transgenic Tregs. This has permitted tracking of Tregs with a defined specificity in vivo, facilitating analysis of how encounter with cognate antigen shapes Treg homeostasis and function. Here, we review the key lessons learned from a decade of analysis of TCR-transgenic Tregs and set this in the broader context of general progress in the field. Use of TCR-transgenic Tregs has led to an appreciation that Tregs are a highly dynamic proliferative population in vivo, rather than an anergic population as they were initially portrayed. It is now clear that Treg homeostasis is positively regulated by encounter with self-antigen expressed on peripheral tissues, which is likely to be relevant to the phenomenon of peripheral repertoire reshaping that has been described for Tregs and the observation that the Treg TCR specificities vary by anatomical location. Substantial evidence has also accumulated to support the role of CD28 costimulation and interleukin-2 in Treg homeostasis. The availability of TCR-transgenic Tregs has enabled analysis of Treg populations that are sufficient or deficient in particular genes, without the comparison being confounded by repertoire alterations. This approach has yielded insights into genes required for Treg function in vivo, with particular progress being made on the role of ctla-4 in this context. As the prospect of manipulating Treg populations in the clinic becomes reality, a full appreciation of the rules governing their homeostasis will prove increasingly important. PMID:24712457

Attridge, Kesley; Walker, Lucy S K

2014-01-01

77

Relationship between in vivo activity and in vitro measures of function and stability of a protein  

SciTech Connect

The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein. 54 refs., 3 figs., 2 tabs.

Sandberg, W.S.; Schlunk, P.M.; Zabin, H.G. [Univ. of Chicago, IL (United States)] [and others

1995-09-19

78

Magnetic stimulation of mammalian peripheral nerves in vivo: An alternative to functional electrical stimulation.  

PubMed

Functional electrical stimulation is the current gold standard for stimulating neuronal interfaces for functional neuromuscular and cortical applications, but it is not without its drawbacks. One such fault is the need to have direct electrical contact with the nerve tissue, and any side effects this causes. Functional magnetic stimulation, which works though electromagnetic induction, does not require electrical contact and may be a viable alternative to functional electrical stimulation. We are investigating the capabilities of magnetic stimulation with centimeter scale (<; 2.5 cm) coils in feline and rodent sciatic nerves in vivo. We have shown that magnetic stimulation can consistently produce the same levels of neuromuscular activation as electrical stimulation. Additionally, the position of the coil relative to the nerve influences neuromuscular activation, suggesting the possibility of selective muscle activation. PMID:25570516

Kagan, Zachary B; RamRakhyani, Anil Kumar; Khan, Faisal; Lazzi, Gianluca; Normann, Richard A; Warren, David J

2014-08-01

79

In Vivo Imaging of the Photoreceptor Mosaic in Retinal Dystrophies and Correlations with Visual Function  

PubMed Central

Purpose To relate in vivo microscopic retinal changes to visual function in patients who have various forms of retinal dystrophy. Methods The UC Davis Adaptive Optics (AO) fundus camera was used to acquire in vivo retinal images at the cellular level. Visual function tests consisting of visual fields, multifocal electroretinography (mfERG), and contrast sensitivity were measured in all subjects by using stimuli that were coincident with areas imaged. Five patients with different forms of retinal dystrophy and three control subjects were recruited. Cone densities were quantified for all retinal images. Results In all images of diseased retinas, there were extensive areas of dark space between groups of photoreceptors, where no cone photoreceptors were evident. These irregular features were not seen in healthy retinas, but were apparent in patients with retinal dystrophy. There were significant correlations between functional vision losses and the extent to which these irregularities, quantified by cone density, occurred in retinal images. Conclusions AO fundus imaging is a reliable technique for assessing and quantifying the changes in the photoreceptor layer as disease progresses. Furthermore, this technique can be useful in cases where visual function tests provide borderline or ambiguous results, as it allows visualization of individual photoreceptors. PMID:16639019

Choi, Stacey S.; Doble, Nathan; Hardy, Joseph L.; Jones, Steven M.; Keltner, John L.; Olivier, Scot S.; Werner, John S.

2008-01-01

80

Gain-of-function mutations indicate that Escherichia coli Kch forms a functional K+ conduit in vivo.  

PubMed

Although Kch of Escherichia coli is thought to be a K(+) channel by sequence homology, there is little evidence that it actually conducts K(+) ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria specifically sensitive to K(+) ions. Millimolar added K(+), but not Na(+) or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K(+)-filter sequence rescue the GOF mutant. The apparent K(+)-specific conduction through the 'loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K(+) concentration. Possible implications on the normal function of Kch are discussed. PMID:12912904

Kuo, Mario Meng-Chiang; Saimi, Yoshiro; Kung, Ching

2003-08-15

81

Toxic effects of cypermethrin and methamidophos on bovine corpus luteal cells and progesterone production.  

PubMed

This study was planned and executed with the aims to explore corpus luteal primary cell culture as an "animal alternate testing system" in toxicity studies and in vitro toxic effects of cypermethrin (CY) 90% (pyrethroid) and methamidophos (MTP) 73% (organophosphate) on morphology and progesterone secretory activity of bovine corpus luteal cells and tissue. For this purpose, primary cell cultures of bovine corpus luteum (CL) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal calf serum (FCS). Toxicity evaluation were based on viable CL cell counts, morphological changes in CL cells, ability of CL cells to produce progesterone and histological changes in CL tissue at different hours post exposure to CY and MTP. The changes induced by both the insecticides were time and dose dependant. Viable cell counts and progesterone concentration decreased significantly (P<0.05) with the treatment of CY and MTP when compared to control. Corpus luteal cells exposed to CY showed more severe toxic effects as compared to MTP, though the difference was non-significant. Cellular or tissue alterations included degenerative changes in luteal cells, pleomorphic changes, nuclear degeneration and vacuolation, cell shrinkage and rupture, cloudy swelling and hydropic degeneration, less cytoplasmic granulation, cell elongation, hyalinization and cytoplasmic haziness and stripling and necrosis. It was concluded that both the insecticides induce toxic effects in terms of viable counts, morphological and histological changes and progesterone production of bovine CL cells. Cypermethrin exhibited more adverse toxic changes in viable cell counts, progesterone production and histological findings as compared to methamidophos. PMID:19942419

Gill, Shahid Afzal; Rizvi, Farzana; Khan, Muhammad Zargham; Khan, Ahrar

2011-01-01

82

Histological and endocrine characterisation of the annual luteal activity in Eurasian lynx (Lynx lynx).  

PubMed

Lynx presents a unique sexual cycle with persistent corpora lutea (CLs) and elevated serum progesterone (P?) throughout parturition and lactation. In other mammals, CLs normally disintegrate after parturition, therefore the aim of our study was to characterise the annual life cycle of lynx CLs. Ovaries from Eurasian lynxes were obtained from the National Veterinary Institute in Sweden, where tissues from killed lynx were stored at -20?°C. Ovaries from 66 animals were weighed; each corpus luteum was segmented for histology and hormone analysis. Ovary and CLs weights were constant throughout the year, peaking during pregnancy. In non-pregnant lynxes, the seasonal level of intraluteal steroids was steady for P? (3.2±1.9 s.d. ?g/g, n=53) and total oestrogens (18.3±15.5 s.d. ng/g, n=53). Within histology slides, structurally intact luteal cells were found throughout the year with the highest incidence in March/April; evidence of luteal regression was predominantly found in post-breeding season. Ovaries from pregnant animals contained two types of CLs. Group A was bigger in size with large luteal cells (P?, 72.3±65.4 s.d. ?g/g; oestrogen, 454.0±52.4 s.d. ng/g). In contrast, group B were smaller, with greater luteal regression and lower steroid concentrations (P?, 8.3±2.9 s.d. ?g/g; oestrogen, 31.5±20.4 s.d. ng/g). Our results suggest that structural luteolysis proceeds throughout the year and into next breeding cycle, resulting in two CLs types on the same ovary. PMID:22829688

Carnaby, Kim; Painer, Johanna; Söderberg, Arne; Gavier-Widèn, Dolores; Göritz, Frank; Dehnhard, Martin; Jewgenow, Katarina

2012-10-01

83

Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: Kinetic studies  

Microsoft Academic Search

Ovine luteal cells grown in suspensions and\\/or monolayer culture were used to study the rates of internalization and degradation of (¹²⁵I)hCG. At specified times after a 5- to 7-min exposure to (¹²⁵I)hCG, cells were treated with acidic buffer (pH 3.9) to elute membrane-bound hormone, which left the internalized radioactivity associated with the cell pellet. Radioactivity released into the medium during

C. E. Ahmed; H. R. Sawyer; G. D. Niswender

1981-01-01

84

N-acetylcysteine impairs survival of luteal cells through mitochondrial dysfunction.  

PubMed

N-acetylcysteine (NAC) is known as an antioxidant and used for mucus viscosity reduction. However, this drug prevents or induces cell death depending on the cell type. The response of steroidogenic luteal cells to NAC is unknown. Our data shows that NAC can behave as an antioxidant or prooxidant in dependency on the concentration and mitochondrial energization. NAC elevated the flowcytometric-measured portion of hypodiploid (dying) cells. This rise was completely abolished by aurintricarboxylic acid, an inhibitor of topoisomerase II. NAC increased the secretion of nitric oxide and cellular nitrotyrosine. An image analysis indicated that cells pretreated with NAC and loaded with DHR showed a fluorescent structure probably elicited by the oxidative product of DHR, rhodamine 123 that sequesters mitochondrially. Pretreating luteal cells with NAC or adding NAC directly to mitochondrial fractions followed by assessing the mitochondrial transmembrane potential difference (Deltapsi) by the JC-1 technique demonstrated a marked decrease in Deltapsi. A protonophore restored Deltapsi and rotenone (an inhibitor of respiratory chain complex I) inhibited mitochondrial recovering. Thus, in steroidogenic luteal cells from healthy mature corpus luteum, NAC impairs cellular survival by interfering with mitochondrial metabolism. The protonophore-induced recovering of NAC-provoked decrease in Deltapsi indicates that an ATP synthase-favored route of H(+) re-entry to the matrix is essentially switched off by NAC while other respiratory chain complexes remain intact. These data may be important for therapeutic timing of treatments with NAC. (c) 2010 International Society for Advancement of Cytometry. PMID:20151456

Löhrke, Berthold; Xu, Jinxian; Weitzel, Joachim M; Krüger, Burkhard; Goldammer, Tom; Viergutz, Torsten

2010-04-01

85

Stress and memory retrieval in women: no strong impairing effect during the luteal phase.  

PubMed

Stress has been shown to impair delayed memory retrieval, but so far no study has been conducted solely with naturally cycling women. In a crossover design, 36 women (all in the luteal phase) participated in two experimental conditions (stress vs. control). Delayed memory retrieval of a wordlist learned 24 hours earlier was tested after stress or control treatment. Although stressed subjects showed a strong cortisol increase following stress, no influence on memory retrieval occurred. In an additional data analysis, subjects were split up into a cortisol responder and a cortisol nonresponder group. However, again no evidence for a stress-induced retrieval impairment became apparent. Similarly, no correlation was observed between the stress-induced cortisol increase and memory. This study failed to find an influence of stress on memory retrieval in women tested in the luteal phase. The findings are in contrast to our previous results obtained with men. Evidence is discussed that the luteal phase, which is characterized by elevated gonadal steroids, is associated with reduced glucocorticoid sensitivity. This might underlie the missing impact of stress on memory. PMID:19485561

Schoofs, Daniela; Wolf, Oliver T

2009-06-01

86

Luteal changes after treatment with sub-luteolytic doses of prostaglandin (cloprostenol sodium) in cattle.  

PubMed

This study characterizes the physiological and morphological changes related to partial luteolysis in bovine corpus luteum (CL) after challenges with sub-doses of cloprostenol sodium on Day 6 (D6) of the estrous cycle. Cows (n=12/treatment) were treated as follows: Control (2mL, saline, i.m.); 2XPGF (two treatments i.m. 500?g of cloprostenol sodium 2h apart) and 1/6PGF (83.3?g of cloprostenol sodium, i.m., once). Plasma progesterone (P4) concentration, CL volume and blood flow were measured immediately before the treatments, then every 8h (h) for 48h. In the Control, P4 concentrations were higher at 48h than at 0h. P4 decreased 8h after 2XPGF treatment (P<0.05), and remained low until the end of the trial. P4 decreased in 1/6PGF between 8 and 16h (P<0.05), then began to rebound at 24h. Luteal volume was higher in Controls at 48h than at 0h. Under 1/6PGF, luteal volume decreased at 24h (P<0.05) and began to rebound at 32h. Luteal volume and blood flow were reduced starting at 24 and 32h, respectively, after 2XPGF treatment (P<0.05). In this study, we were able to describe the partial luteolysis phenomenon, induced by a treatment of a D6CL with cloprostenol sub-dose. PMID:25578505

Trevisol, Eduardo; Ferreira, Jair Camargo; Ackermann, Camila Louise; Destro, Flavia Caroline; Marques Filho, Wolff Camargo; Carmagos, Aline Souza; Biehl, Marcos Vinicius; do Amaral, Jackson Barros; de Figueiredo Pantoja, José Carlos; Sartori, Roberto; Ferreira, João Carlos Pinheiro

2015-02-01

87

Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo  

PubMed Central

Presently, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures which provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high resolution images, it is also safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically-specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically-relevant depths, ideal for soft tissue imaging. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, enabling multi-modality imaging with complementary contrast. Here, we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and demonstrate its ability to image internal organs in vivo, illustrating its potential clinical application. PMID:22797808

Yang, Joon-Mo; Favazza, Christopher; Chen, Ruimin; Yao, Junjie; Cai, Xin; Maslov, Konstantin; Zhou, Qifa; Shung, K. Kirk; Wang, Lihong V.

2013-01-01

88

Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding  

PubMed Central

Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering. PMID:24152813

Qiu, Juhui; Zheng, Yiming; Hu, Jianjun; Liao, Donghua; Gregersen, Hans; Deng, Xiaoyan; Fan, Yubo; Wang, Guixue

2014-01-01

89

Neurofibrillary tangle-bearing neurons are functionally integrated in cortical circuits in vivo  

PubMed Central

Alzheimer's disease (AD) is pathologically characterized by the deposition of extracellular amyloid-? plaques and intracellular aggregation of tau protein in neurofibrillary tangles (NFTs) (1, 2). Progression of NFT pathology is closely correlated with both increased neurodegeneration and cognitive decline in AD (3) and other tauopathies, such as frontotemporal dementia (4, 5). The assumption that mislocalization of tau into the somatodendritic compartment (6) and accumulation of fibrillar aggregates in NFTs mediates neurodegeneration underlies most current therapeutic strategies aimed at preventing NFT formation or disrupting existing NFTs (7, 8). Although several disease-associated mutations cause both aggregation of tau and neurodegeneration, whether NFTs per se contribute to neuronal and network dysfunction in vivo is unknown (9). Here we used awake in vivo two-photon calcium imaging to monitor neuronal function in adult rTg4510 mice that overexpress a human mutant form of tau (P301L) and develop cortical NFTs by the age of 7–8 mo (10). Unexpectedly, NFT-bearing neurons in the visual cortex appeared to be completely functionally intact, to be capable of integrating dendritic inputs and effectively encoding orientation and direction selectivity, and to have a stable baseline resting calcium level. These results suggest a reevaluation of the common assumption that insoluble tau aggregates are sufficient to disrupt neuronal function. PMID:24368848

Kuchibhotla, Kishore V.; Wegmann, Susanne; Kopeikina, Katherine J.; Hawkes, Jonathan; Rudinskiy, Nikita; Andermann, Mark L.; Spires-Jones, Tara L.; Bacskai, Brian J.; Hyman, Bradley T.

2014-01-01

90

Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): methodology and applications  

PubMed Central

Background A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions. Results Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 ?m), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 ?m), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated. Conclusion The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant. PMID:18838008

Hardman, Ron C; Kullman, Seth W; Hinton, David E

2008-01-01

91

In Vivo Evaluation of Vena Caval Filters: Can Function Be Linked to Design Characteristics?  

Microsoft Academic Search

Purpose: To compare the five vena caval filters marketed in the United States and one investigational vena caval filter and to determine\\u000a whether there is an association between their design and their in vivo function.\\u000a \\u000a \\u000a \\u000a \\u000a Methods: Four of each type of filter—Simon Nitinol (SN), Bird's Nest (BN), Vena Tech (VT), Greenfield stainless steel (PSGF), Greenfield\\u000a titanium (TGF), and the investigational

Mary C. Proctor; Kyung J. Cho; Lazar J. Greenfield

2000-01-01

92

Exploring Functional ?-Cell Heterogeneity In Vivo Using PSA-NCAM as a Specific Marker  

PubMed Central

Background The mass of pancreatic ?-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous ?-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of ?-cells and investigated their physiological relevance in increased insulin demand conditions in rats. Methods Two rat ?-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. ?high and ?low-cells. Insulin release, Ca2+ movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, ?high and ?low-cell distribution and functionality were investigated in animal models with decreased or increased ?-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat. Results We show that ?-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike ?low-cells, ?high-cells express functional ?-cell markers and are highly responsive to various insulin secretagogues. Whereas ?low-cells represent the main population in diabetic pancreas, an increase in ?high-cells is associated with gain of function that follows sustained glucose overload. Conclusion Our data show that a functional heterogeneity of ?-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in ?-cell defects in type 2 diabetes. PMID:19440374

Karaca, Melis; Castel, Julien; Tourrel-Cuzin, Cécile; Brun, Manuel; Géant, Anne; Dubois, Mathilde; Catesson, Sandra; Rodriguez, Marianne; Luquet, Serge; Cattan, Pierre; Lockhart, Brian; Lang, Jochen; Ktorza, Alain

2009-01-01

93

High pressure modulated transport and signaling functions of membrane proteins in models and in vivo  

NASA Astrophysics Data System (ADS)

Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

2008-07-01

94

Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function.  

PubMed

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U). The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo. PMID:12070319

Hobart, Sarah A; Ilin, Sergey; Moriarty, Daniel F; Osuna, Robert; Colón, Wilfredo

2002-07-01

95

Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo.  

PubMed

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations. PMID:25151500

Kreiser, Simon; Eckhardt, Jenny; Kuhnt, Christine; Stein, Marcello; Krzyzak, Lena; Seitz, Christine; Tucher, Christine; Knippertz, Ilka; Becker, Christoph; Günther, Claudia; Steinkasserer, Alexander; Lechmann, Matthias

2015-02-01

96

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function  

PubMed Central

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis. PMID:23236142

Rodriguez-Diaz, Rayner; Speier, Stephan; Molano, Ruth Damaris; Formoso, Alexander; Gans, Itai; Abdulreda, Midhat H.; Cabrera, Over; Molina, Judith; Fachado, Alberto; Ricordi, Camillo; Leibiger, Ingo; Pileggi, Antonello; Berggren, Per-Olof; Caicedo, Alejandro

2012-01-01

97

Ubiquitination Regulates the Neuroprotective Function of the Deubiquitinase Ataxin-3 in Vivo*  

PubMed Central

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability. PMID:24106274

Tsou, Wei-Ling; Burr, Aaron A.; Ouyang, Michelle; Blount, Jessica R.; Scaglione, K. Matthew; Todi, Sokol V.

2013-01-01

98

Numerical and In Vivo Validation of Fast Cine DENSE MRI for Quantification of Regional Cardiac Function  

PubMed Central

Quantitative assessment of regional cardiac function can improve the accuracy of detecting wall motion abnormalities due to heart disease. While recently developed fast cine displacement-encoded with stimulated echoes (DENSE) MRI is a promising modality for the quantification of regional myocardial function, it has not been validated for clinical applications. The purpose of this study, therefore, was to validate the accuracy of fast cine DENSE MRI with numerical simulation and in vivo experiments. A numerical phantom was generated to model physiologically relevant deformation of the heart, and the accuracy of fast cine DENSE was evaluated against the numerical reference. For in vivo validation, 12 controls and 13 heart disease patients were imaged using both fast cine DENSE and myocardial tagged MRI. Numerical simulation demonstrated that the echo-combination DENSE reconstruction method is relatively insensitive to clinically relevant resonance frequency offsets. The strain measurements by fast cine DENSE and the numerical reference were strongly correlated and in excellent agreement (mean difference=0.00; 95% limits of agreement were 0.01 and ?0.02). The strain measurements by fast cine DENSE and myocardial tagged MRI were strongly correlated (correlation coefficient = 0.92) and in good agreement (mean difference=0.01; 95% limits of agreement were 0.07 and ?0.04). PMID:19585609

Feng, Li; Donnino, Robert; Babb, James; Axel, Leon; Kim, Daniel

2009-01-01

99

Allele Compensation in Tip60+/? Mice Rescues White Adipose Tissue Function In Vivo  

PubMed Central

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/? mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/? mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/? displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/? mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID:24870614

Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

2014-01-01

100

Proteomics meets genetics: SILAC labeling of Drosophila melanogaster larvae and cells for in vivo functional studies.  

PubMed

Stable isotope labeling by amino acids in cell culture (SILAC) is an established and potent method for quantitative proteomics. When combined with high-resolution mass spectrometry (MS) and efficient algorithms for the analysis of quantitative MS data, SILAC has proven to be the strategy of choice for the in-depth characterization of functional states at the protein level. The fruit fly Drosophila melanogaster is one of the most widely used model systems for studies of genetics and developmental biology. Despite this, a global proteomic approach in Drosophila is rarely considered. Here, we describe an adaptation of SILAC for functional investigation of fruit flies by proteomics: We illustrate how to perform efficient SILAC labeling of cells in culture and whole fly larvae. The combination of SILAC, a highly accurate global protein quantification method, and of the fruit fly, the prime genetics and developmental model, represents a unique opportunity for quantitative proteomic studies in vivo. PMID:25059620

Cuomo, Alessandro; Sanfilippo, Roberta; Vaccari, Thomas; Bonaldi, Tiziana

2014-01-01

101

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.  

PubMed

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology. PMID:24390440

Thestrup, Thomas; Litzlbauer, Julia; Bartholomäus, Ingo; Mues, Marsilius; Russo, Luigi; Dana, Hod; Kovalchuk, Yuri; Liang, Yajie; Kalamakis, Georgios; Laukat, Yvonne; Becker, Stefan; Witte, Gregor; Geiger, Anselm; Allen, Taylor; Rome, Lawrence C; Chen, Tsai-Wen; Kim, Douglas S; Garaschuk, Olga; Griesinger, Christian; Griesbeck, Oliver

2014-02-01

102

Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.  

PubMed

Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics. PMID:25366491

Lee, Kyoung Jin; Lee, Jae Hee; Chung, Hye Kyung; Choi, Jinhyang; Park, Jaesook; Park, Seok Soon; Ju, Eun Jin; Park, Jin; Shin, Seol Hwa; Park, Hye Ji; Ko, Eun Jung; Suh, Nayoung; Kim, InKi; Hwang, Jung Jin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

2015-02-01

103

The effectiveness of luteal phase support with cyclogest in ovarian stimulated intra uterine insemination cycles: A randomized controlled trial  

PubMed Central

Background: Controlled ovarian stimulation combined with intra uterine insemination (IUI) is a convenient treatment of infertility with a success rate of 11%. The clinical observation and pattern of progesterone secretion in this method is suggestive of luteal phase defect and postulated as an implicating factor of treatment failure. Objective: To investigate the efficacy of luteal phase support with intravaginal cyclogest in women undergoing controlled ovarian stimulation combined with intrauterine insemination. Materials and Methods: In this single-blinded clinical trial, 196 consecutively seen women eligible for the study protocol, were randomized to receive either intravaginal progesterone (cyclogest pessary, Actavis) or no medication in luteal phase. Blood samples were collected and serum progesterone level in 7th and 11th day of the cycle, biochemical and clinical pregnancy and luteal phase duration were compared in case and control groups. Results: The mean age in case and control group was 28 and 27.9 years, respectively and the most frequent cause of infertility was unexplained. Additionally, ovulatory dysfunction was the most common cause of female infertility in both groups. Based on these variables, there was no statistically significant difference between the two groups. Mean serum progesterone level in the case group were 48.34 and 34.24nmol/day on day 7 and 11 after insemination, respectively and both values were significantly higher than the control group. There was no difference between the two groups in terms of biochemical and clinical pregnancy. Luteal phase duration in the case group was significantly longer than the control group. Conclusion: Luteal phase support by Cyclogest pessary increases progesterone level and prolongs the luteal phase, but does not affect success rate of IUI cycles in terms of achieving pregnancy. PMID:24639761

Aali, Bibi Shahnaz; Ebrahimipour, Sakineh; Medhdizadeh, Siavash

2013-01-01

104

Protective effects of Zhuyeqing liquor on the immune function of normal and immunosuppressed mice in vivo  

PubMed Central

Background Zhuyeqing Liquor (ZYQL), a well-known Chinese traditional health liquor, has various biological properties, including anti-oxidant, anti-inflammatory, immunoenhancement and cardiovascular protective effects. Methods The protective effects of Zhuyeqing Liquor (ZYQL) on the immune function was investigated in vivo in normal healthy mice and immunosuppressed mice treated with Cyclophosphamide (Cy, 100 mg/kg) by intraperitoneal injection on days 4, 8 and 12. ZYQL (100, 200 and 400 mg/kg) was administered via gavage daily for 14 days. The phagocytotic function of mononuclear phagocytic system was detected with carbon clearance methods, the levels of interleukin-6 (IL-6) and interferon-gamma (IFN-?) in serum were detected with Enzyme linked immunosorbent assay (ELISA). Immune organs were weighed and organ indexes (organ weight/body weight) of thymus and spleen were calculated. Meanwhile, the activity of lysozyme (LSZ) in serum and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) in spleen tissue were measured. Results ZYQL significantly upgrades the K value for clearance of carbon particles in normal mice treated with ZYQL (400 mg/kg) and immunosuppressed mice treated with ZYQL (100, 200 and 400 mg/kg) together with Cy (100 mg/kg) in vivo. The treatment of ZYQL (100, 200 and 400 mg/kg) effectively increased the activity of serum lysozyme as well as promoted the serum levels of IL-6 and IFN-? in normal mice and immunosuppressed mice. Furthermore, ZYQL (100, 200 and 400 mg/kg) had an antioxidant effects in immune system by enhancing the antioxidant enzyme activity of SOD, CAT and GSH-Px in vivo. In addition, ZYQL (100, 200 and 400 mg/kg) effectively elevated the Cy-induced decreased organ index (thymus and spleen). Conclusions The present work shows that the dose-dependent administration of ZYQL is capable of influencing immune responses, which implying that its valuable functional health may be attributed partly to its protective effects for the immune function. PMID:24090456

2013-01-01

105

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo  

PubMed Central

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models. PMID:22140042

Milkovich, Stephanie; Goldman, Daniel; Ellis, Christopher G.

2012-01-01

106

The ex vivo function and expression of function-associated antigens of peripheral blood neutrophils and monocytes.  

PubMed

The availability of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors (rhG-CSF and rhGM-CSF) has prompted many studies of the analysis of antigen expression and function of monocytes and neutrophils from patients receiving these factors as therapeutic agents. Preparatory procedures for leukocytes are known to alter antigen expression and so function. We therefore investigated the use of a novel procedure in which live leukocytes are analyzed by flow cytometry without isolation from blood. The expression levels of CD11b, CD13, CD14, CD16, and CD18 antigens and L-selectin (TQ1 and Leu-8 epitopes) on neutrophils and monocytes from 15 normal individuals were determined and compared with a previously used method in which the leukocytes were fixed and the erythrocytes lysed before analysis. Significant differences for the apparent expression of CD11b, CD18, and L-selectin were observed between the two methods. The reasons for this were investigated. Since the new method allowed analysis of live cells, we also investigated whether modulation of antigen expression could be determined following receptor agonist interaction. This was found to be easily achievable, and we advocate using the new procedure where possible for the ex vivo analysis of function and function-associated antigens on monocytes and neutrophils. PMID:7522183

Macey, M G; McCarthy, D A; Newland, A C

1994-09-01

107

Body adiposity dictates different mechanisms of increased coronary reactivity related to improved in vivo cardiac function  

PubMed Central

Background Saturated fatty acid-rich high fat (HF) diets trigger abdominal adiposity, insulin resistance, type 2 diabetes and cardiac dysfunction. This study was aimed at evaluating the effects of nascent obesity on the cardiac function of animals fed a high-fat diet and at analyzing the mechanisms by which these alterations occurred at the level of coronary reserve. Materials and methods Rats were fed a control (C) or a HF diet containing high proportions of saturated fatty acids for 3 months. Thereafter, their cardiac function was evaluated in vivo using a pressure probe inserted into the cavity of the left ventricle. Their heart was isolated, perfused iso-volumetrically according to the Langendorff mode and the coronary reserve was evaluated by determining the endothelial-dependent (EDV) and endothelial-independent (EIV) vasodilatations in the absence and presence of endothelial nitric oxide synthase and cyclooxygenase inhibitors (L-NAME and indomethacin). The fatty acid composition of cardiac phospholipids was then evaluated. Results Although all the HF-fed rats increased their abdominal adiposity, some of them did not gain body weight (HF- group) compared to the C group whereas other ones had a higher body weight (HF+). All HF rats displayed a higher in vivo cardiac activity associated with an increased EDV. In the HF- group, the improved EDV was due to an increase in the endothelial cell vasodilatation activity whereas in the HF+?group, the enhanced EDV resulted from an improved sensitivity of coronary smooth muscle cells to nitric oxide. Furthermore, in the HF- group the main pathway implicated in the EDV was the NOS pathway while in the HF+?group the COX pathway. Conclusions Nascent obesity-induced improvement of cardiac function may be supported by an enhanced coronary reserve occurring via different mechanisms. These mechanisms implicate either the endothelial cells activity or the smooth muscle cells sensitivity depending on the body adiposity of the animals. PMID:24572210

2014-01-01

108

Segmental in vivo vertebral motion during functional human lumbar spine activities  

PubMed Central

Quantitative data on the range of in vivo vertebral motion is critical to enhance our understanding of spinal pathology and to improve the current surgical treatment methods for spinal diseases. Little data have been reported on the range of lumbar vertebral motion during functional body activities. In this study, we measured in vivo 6 degrees-of-freedom (DOF) vertebral motion during unrestricted weightbearing functional body activities using a combined MR and dual fluoroscopic imaging technique. Eight asymptomatic living subjects were recruited and underwent MRI scans in order to create 3D vertebral models from L2 to L5 for each subject. The lumbar spine was then imaged using two fluoroscopes while the subject performed primary flexion-extension, left-right bending, and left-right twisting. The range of vertebral motion during each activity was determined through a previously described imaging-model matching technique at L2-3, L3-4, and L4-5 levels. Our data revealed that the upper vertebrae had a higher range of flexion than the lower vertebrae during flexion-extension of the body (L2-3, 5.4 ± 3.8°; L3-4, 4.3 ± 3.4°; L4-5, 1.9 ± 1.1°, respectively). During bending activity, the L4-5 had a higher (but not significant) range of left-right bending motion (4.7 ± 2.4°) than both L2-3 (2.9 ± 2.4°) and L3-4 (3.4 ± 2.1°), while no statistical difference was observed in left-right twisting among the three vertebral levels (L2-3, 2.5 ± 2.3°; L3-4, 2.4 ± 2.6°; and L4-5, 2.9 ± 2.1°, respectively). Besides the primary rotations reported, coupled motions were quantified in all DOFs. The coupled translation in left-right and anterior-posterior directions, on average, reached greater than 1 mm, while in the proximal-distal direction this was less than 1 mm. Overall, each vertebral level responds differently to flexion-extension and left-right bending, but similarly to the left-right twisting. This data may provide new insight into the in vivo function of human spines and can be used as baseline data for investigation of pathological spine kinematics. PMID:19301040

Wang, Shaobai; Passias, Peter; Xia, Qun; Li, Gang; Wood, Kirkham

2009-01-01

109

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo  

PubMed Central

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans. PMID:22589735

Misra, Chaitali; Sachan, Nita; McNally, Caryn Rothrock; Koenig, Sara N.; Nichols, Haley A.; Guggilam, Anuradha; Lucchesi, Pamela A.; Pu, William T.; Srivastava, Deepak; Garg, Vidu

2012-01-01

110

In Vivo Evaluation of Vena Caval Filters: Can Function Be Linked to Design Characteristics?  

SciTech Connect

Purpose: To compare the five vena caval filters marketed in the United States and one investigational vena caval filter and to determine whether there is an association between their design and their in vivo function.Methods: Four of each type of filter-Simon Nitinol (SN), Bird's Nest (BN), Vena Tech (VT), Greenfield stainless steel (PSGF), Greenfield titanium (TGF), and the investigational stent cone filter (NGF)-were studied for 60 days in 12 sheep. Radiographic and pathologic outcomes to be assessed included clot capture and resolution, vena caval penetration, position of the filter, thrombogenicity, and vessel wall reaction.Results: Filters differed with respect to the number of clot-trapping levels and the interdependence of the legs. All devices were successfully placed. Intentionally embolized clot was captured. One VT and two SN filters migrated in response to clot capture. Resolution of thrombus was variable, and related to the design of the device. Fibrin webbing was widely present with the VT, BN, and SN filters but limited in the others. The VT and NGF filters demonstrated the most stable filter base diameter.Conclusions: The performance of vena caval filters differs with respect to clot resolution and mechanical stability. Interdependent filter limbs and single-stage conical capture sites appear to result in more favorable performance in in vivo studies.

Proctor, Mary C. [Department of Surgery, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States); Cho, Kyung J. [Department of Radiology, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States); Greenfield, Lazar J. [Department of Surgery, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States)

2000-11-15

111

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function  

PubMed Central

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation. PMID:20160103

Lafont, Chrystel; Desarménien, Michel G.; Cassou, Mathieu; Molino, François; Lecoq, Jérôme; Hodson, David; Lacampagne, Alain; Mennessier, Gérard; El Yandouzi, Taoufik; Carmignac, Danielle; Fontanaud, Pierre; Christian, Helen; Coutry, Nathalie; Fernandez-Fuente, Marta; Charpak, Serge; Le Tissier, Paul; Robinson, Iain CAF; Mollard, Patrice

2010-01-01

112

In Vitro Hematological and In Vivo Vasoactivity Assessment of Dextran Functionalized Graphene  

PubMed Central

The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ? 7?mg/ml, showed a 12–20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1–50?mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect. PMID:24002570

Chowdhury, Sayan Mullick; Kanakia, Shruti; Toussaint, Jimmy D.; Frame, Mary D.; Dewar, Anthony M.; Shroyer, Kenneth R.; Moore, William; Sitharaman, Balaji

2013-01-01

113

Increased 5-HT2A receptor expression and function following central glucocorticoid receptor knockdown in vivo.  

PubMed

Central glucocorticoid receptor function may be reduced in depression. In vivo modelling of glucocorticoid receptor underfunctionality would assist in understanding its role in depressive illness. The role of glucocorticoid receptors in modulating 5-HT(2A) receptor expression and function in the central nervous system (CNS) is presently unclear, but 5-HT(2A) receptor function also appears altered in depression. With the aid of RNAse H accessibility mapping, we have developed a 21-mer antisense oligodeoxynucleotide (5'-TAAAAACAGGCTTCTGATCCT-3', termed GRAS-5) that showed 56% reduction in glucocorticoid receptor mRNA and 80% down-regulation in glucocorticoid receptor protein in rat C6 glioma cells. Sustained delivery to rat cerebral ventricles in slow release biodegradable polymer microspheres produced a marked decrease in glucocorticoid receptor mRNA and protein in hypothalamus (by 39% and 80%, respectively) and frontal cortex (by 26% and 67%, respectively) 5 days after a single injection, with parallel significant up-regulation of 5-HT(2A) receptor mRNA expression (13%) and binding (21%) in frontal cortex. 5-HT(2A) receptor function, determined by DOI-head-shakes, showed a 55% increase. These findings suggest that central 5-HT(2A) receptors are, directly or indirectly, under tonic inhibitory control by glucocorticoid receptor. PMID:15476747

Islam, Aminul; Thompson, Kevin S J; Akhtar, Saghir; Handley, Sheila L

2004-10-19

114

Understanding functional miRNA–target interactions in vivo by site-specific genome engineering  

PubMed Central

MicroRNA (miRNA) target recognition is largely dictated by short ‘seed’ sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA–target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA–MRE interactions at any point during development. PMID:25135198

Bassett, Andrew R.; Azzam, Ghows; Wheatley, Lucy; Tibbit, Charlotte; Rajakumar, Timothy; McGowan, Simon; Stanger, Nathan; Ewels, Philip Andrew; Taylor, Stephen; Ponting, Chris P.; Liu, Ji-Long; Sauka-Spengler, Tatjana; Fulga, Tudor A.

2014-01-01

115

Effects of follicular versus luteal phase-based strength training in young women.  

PubMed

Hormonal variations during the menstrual cycle (MC) may influence trainability of strength. We investigated the effects of a follicular phase-based strength training (FT) on muscle strength, muscle volume and microscopic parameters, comparing it to a luteal phase-based training (LT). Eumenorrheic women without oral contraception (OC) (N?=?20, age: 25.9?±?4.5 yr, height: 164.2?±?5.5 cm, weight: 60.6?±?7.8 kg) completed strength training on a leg press for three MC, and 9 of them participated in muscle biopsies. One leg had eight training sessions in the follicular phases (FP) and only two sessions in the luteal phases (LP) for follicular phase-based training (FT), while the other leg had eight training sessions in LP and only two sessions in FP for luteal phase-based training (LT). Estradiol (E2), progesterone (P4), total testosterone (T), free testosterone (free T) and DHEA-s were analysed once during FP (around day 11) and once during LP (around day 25). Maximum isometric force (Fmax), muscle diameter (Mdm), muscle fibre composition (No), fibre diameter (Fdm) and cell nuclei-to-fibre ratio (N/F) were analysed before and after the training intervention. T and free T were higher in FP compared to LP prior to the training intervention (P?

Sung, Eunsook; Han, Ahreum; Hinrichs, Timo; Vorgerd, Matthias; Manchado, Carmen; Platen, Petra

2014-01-01

116

Women Ornament Themselves for Intrasexual Competition near Ovulation, but for Intersexual Attraction in Luteal Phase  

PubMed Central

The present study examined women's attentional bias toward ornamental objects in relation to their menstrual phase as well as to motivations of intersexual courtship or intrasexual competition. In Experiment 1, 33 healthy heterosexual women were tested in a bias-assessment visual cuing task twice: once on a high-fertility day (during the ovulatory phase) and once on a low-fertility day (during the luteal phase). They paid greater attention to pictures of ornamental objects than to pictures of non-ornamental objects near ovulation, but not during the luteal phase, suggesting an ornamental bias during the high-fertility phase. In Experiment 2, before the visual cuing task, 40 participants viewed 10 same-sex or opposite-sex facial photographs with either high or low attractiveness as priming tasks to activate the intrasexual competition or intersexual courtship motives. Results showed that women's ornamental bias was dependent on the interaction of menstrual phase and mating motive. Specifically, the ornamental bias was observed on the high-fertility day when the subjects were primed with high-attractive same-sex images (intrasexual competition) and was observed on the low-fertility day when they were primed with high-attractive opposite-sex photographs (intersexual courtship). In conclusion, the present findings confirm the hypothesis that, during the high-fertility phase, women have an attentional bias toward ornamental objects and further support the hypothesis that the ornamental bias is driven by intrasexual competition motivation near ovulation, but driven by intersexual courtship motivation during the luteal phase. PMID:25180577

Zhuang, Jin-Ying; Wang, Jia-Xi

2014-01-01

117

In vivo effects of monoclonal antibodies that functionally inhibit complement regulatory proteins in rats  

PubMed Central

The present work was designed to evaluate the effects of functional suppression of complement regulatory proteins in vivo. Male Wistar rats were anesthetized with Nembutal and were intravenously injected with 1 mg/kg of F(ab')2 or Fab fraction of either monoclonal antibody 5I2, which inhibits the function of rat counterpart of mouse Crry/p65, or monoclonal antibody 6D1, which inhibits the rat counterpart of CD59. Mean arterial pressure was continuously measured for 30 min. When 5I2 was injected, there was a biphasic change of mean arterial pressure, namely, the rapid increase immediately after the injection (approximately 2 min, phase 1) and the subsequent fall and slow recovery (approximately 4-30 min, phase 2). These effects were completely abrogated by pretreatment of rats with cobra venom factor. Pretreatment with carboxypeptidase inhibitor, which inhibits inactivation of anaphylatoxins C3a and C5a, induced enhanced reduction of blood pressure. Circulating leukocytes and platelets were rapidly decreased 5 min after antibody injection and became normal by 2 h. Hematocrit and erythrocyte count were continuously increased up to 2 h after injection, suggesting that there was hemoconcentration due to increased vascular permeability. Immunofluorescence study revealed binding of antibody fragments and rat C3 along the capillaries of lung, heart, and liver 5 min after injection. In contrast to 5I2, F(ab')2 fraction of 6D1, though localized to the same areas and in similar amounts, had no significant effect on the parameters measured. These data suggest that the rat counterpart of mouse Crry/p65 plays a vital role in vivo by preventing the activation of autologous complement on vascular endothelium. PMID:7525834

1994-01-01

118

Artemisia scoparia Enhances Adipocyte Development and Endocrine Function In Vitro and Enhances Insulin Action In Vivo  

PubMed Central

Background Failure of adipocytes to expand during periods of energy excess can result in undesirable metabolic consequences such as ectopic fat accumulation and insulin resistance. Blinded screening studies have indicated that Artemisia scoparia (SCO) extracts can enhance adipocyte differentiation and lipid accumulation in cultured adipocytes. The present study tested the hypothesis that SCO treatment modulates fat cell development and function in vitro and insulin sensitivity in adipose tissue in vivo. Methods In vitro experiments utilized a Gal4-PPAR? ligand binding domain (LBD) fusion protein-luciferase reporter assay to examine PPAR? activation. To investigate the ability of SCO to modulate adipogenesis and mature fat cell function in 3T3-L1 cells, neutral lipid accumulation, gene expression, and protein secretion were measured by Oil Red O staining, qRT-PCR, and immunoblotting, respectively. For the in vivo experiments, diet-induced obese (DIO) C57BL/6J mice were fed a high-fat diet (HFD) or HFD containing 1% w/w SCO for four weeks. Body weight and composition, food intake, and fasting glucose and insulin levels were measured. Phospho-activation and expression of insulin-sensitizing proteins in epididymal adipose tissue (eWAT) were measured by immunoblotting. Results Ethanolic extracts of A. scoparia significantly activated the PPAR? LBD and enhanced lipid accumulation in differentiating 3T3-L1 cells. SCO increased the transcription of several PPAR? target genes in differentiating 3T3-L1 cells and rescued the negative effects of tumor necrosis factor ? on production and secretion of adiponectin and monocyte chemoattractant protein-1 in fully differentiated fat cells. DIO mice treated with SCO had elevated adiponectin levels and increased phosphorylation of AMPK? in eWAT when compared to control mice. In SCO-treated mice, these changes were also associated with decreased fasting insulin and glucose levels. Conclusion SCO has metabolically beneficial effects on adipocytes in vitro and adipose tissue in vivo, highlighting its potential as a metabolically favorable botanical supplement. PMID:24915004

Richard, Allison J.; Fuller, Scott; Fedorcenco, Veaceslav; Beyl, Robbie; Burris, Thomas P.; Mynatt, Randall; Ribnicky, David M.; Stephens, Jacqueline M.

2014-01-01

119

Development of functional in vivo imaging of cerebral lenticulostriate artery using novel synchrotron radiation angiography.  

PubMed

The lenticulostriate artery plays a vital role in the onset and development of cerebral ischemia. However, current imaging techniques cannot assess the in vivo functioning of small arteries such as the lenticulostriate artery in the brain of rats. Here, we report a novel method to achieve a high resolution multi-functional imaging of the cerebrovascular system using synchrotron radiation angiography, which is based on spatio-temporal analysis of contrast density in the arterial cross section. This method provides a unique tool for studying the sub-cortical vascular elasticity after cerebral ischemia in rats. Using this technique, we demonstrated that the vascular elasticity of the lenticulostriate artery decreased from day 1 to day 7 after transient middle cerebral artery occlusion in rats and recovered from day 7 to day 28 compared to the controls (p < 0.001), which paralleled with brain edema formation and inversely correlated with blood flow velocity (p < 0.05). Our results demonstrated that the change of vascular elasticity was related to the levels of brain edema and the velocity of focal blood flow, suggesting that reducing brain edema is important for the improvement of the function of the lenticulostriate artery in the ischemic brain. PMID:25632958

Lin, Xiaojie; Miao, Peng; Mu, Zhihao; Jiang, Zhen; Lu, Yifan; Guan, Yongjing; Chen, Xiaoyan; Xiao, Tiqiao; Wang, Yongting; Yang, Guo-Yuan

2015-02-21

120

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering.  

PubMed

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor ? and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy. PMID:24602616

Takagi, Soichi; Shimizu, Tatsuya; Kuramoto, Goro; Ishitani, Ken; Matsui, Hideo; Yamato, Masayuki; Okano, Teruo

2014-03-28

121

Duration of luteal support after IVF is important, so why is there no consistency in practice? The results of a dynamic survey of practice in the United Kingdom.  

PubMed

Luteal support is considered as an essential component of IVF treatment following ovarian stimulation and embryo transfer. Several studies have consistently demonstrated a benefit of luteal support compared with no treatment and whilst a number of preparations are available, no product has been demonstrated as superior. There is an emerging body of evidence which suggests that extension of luteal support beyond biochemical pregnancy does not confer a benefit in terms of successful pregnancy outcome. We performed two surveys separated by 5 years of practice evolution, with the latter reporting on the use of luteal support in all IVF clinics in the UK. All clinics reported utilising luteal support with the majority favouring the use of Cyclogest 400 mg twice daily. In contrast, there was no consensus on the optimal duration of luteal support. Whilst 24% of clinics withdrew luteal support at biochemical confirmation of pregnancy, 40% continued treatment until 12 weeks gestation. Several clinics even extended luteal support beyond 12 weeks gestation. We observed no difference in practice based on the size of the IVF unit or treatment funding source. Although there was some change in practice between surveys in many clinics, there was no uniformity in the direction of change. PMID:25116191

Russell, Richard; Kingsland, Charles; Alfirevic, Zarko; Gazvani, Rafet

2014-08-13

122

Functional Changes in Neocortical Activity in Huntington's Disease Model Mice: An in vivo Intracellular Study  

PubMed Central

Studies of animal models of Huntington's disease (HD) have revealed that neocortical and neostriatal neurons of these animals in vitro exhibit a number of morphological and physiological changes, including increased input resistance and changes in neocortical synaptic inputs. We measured the functional effects of polyglutamate accumulation in neocortical neurons in R6/2 mice (8–14 weeks of age) and their age-matched non-transgenic littermates using in vivo intracellular recordings. All neurons showed spontaneous membrane potential fluctuations. The current/voltage and the firing properties of the HD neocortical neurons were significantly altered, especially in the physiologically relevant current range around and below threshold. As a result, membrane potential transitions from the Down state to Up state were evoked with smaller currents in HD neocortical neurons than in controls. The excitation-to-frequency curves of the HD mice were significantly steeper than those of controls, indicating a smaller input–output dynamic range for these neurons. Increased likelihood of Down to Up state transitions could cause pathological recruitment of corticostriatal assemblies by increasing correlated neuronal activity. We measured coherence of the in vivo intracellular recordings with simultaneously recorded electrocorticograms. We found that the peak of the coherence at <5?Hz was significantly smaller in the HD animals, indicating that the amount of coherence in the state transitions of single neurons is less correlated with global activity than non-transgenic controls. We propose that decreased correlation of neocortical inputs may be a major physiological cause underlying the errors in sensorimotor pattern generation in HD. PMID:21720524

Stern, Edward A.

2011-01-01

123

Vascular Endothelial Growth Factor Modulates the Function of the Retinal Pigment Epithelium In Vivo  

PubMed Central

Purpose. Retinal edema, the accumulation of extracellular fluid in the retina is usually attributed to inner blood retina barrier (BRB) leakage. Vascular endothelial growth factor plays an important role in this process. The effects of VEGF on the outer BRB, the RPE, however, have received limited attention. Here, we present a methodology to assess how VEGF modulates the integrity of the RPE barrier in vivo. Methods. Control subretinal blebs (1–5 ?L) and blebs containing VEGF (1–100 ?g/mL), placental growth factor (PlGF; 100 ?g/mL), or albumin (100–1000 ?g/mL) were injected into New Zealand White or Dutch Belted rabbits with IOP maintained at 10, 15, or 20 mm Hg. One-hour intravitreal pretreatment with ZM323881 (10 ?M/L) was used to inhibit the VEGF response. Fluid resorption was followed by optical coherence tomography for 1 hour. Retinal pigment epithelium leakage was assessed by fluorescein angiography. Results. Increasing IOP resulted in an elevated rate of bleb resorption, while increasing albumin concentration in the bleb decreased the rate of resorption. Vascular endothelial growth factor, but not PlGF, caused a significant, concentration-dependent decrease in the rate of fluid resorption, which was reversed by ZM323881. Compared with albumin-filled blebs, VEGF-filled blebs showed accelerated early-phase leakage from the choroid. Conclusions. Consistent with a localized modulation of RPE function, VEGF induced a significant reduction in fluid resorption and an increase in hydraulic conductivity. Our results establish VEGF as a major cytokine regulating RPE barrier properties in vivo and indicate that the RPE is a principal factor in the pathogenesis of retinal edema. PMID:24550368

Dahrouj, Mohammad; Alsarraf, Oday; McMillin, Jake C.; Liu, Yueying; Crosson, Craig E.; Ablonczy, Zsolt

2014-01-01

124

Impact of hydrogel nanoparticle size and functionalization on in vivo behavior for lung imaging and therapeutics  

PubMed Central

Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg9), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1–5 µm diameter) and nanoparticles (20–40 nm diameter) without and with Arg9 showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg9 and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages, and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg9. Whereas microparticles may be advantageous for short-term applications, nano-sized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg9. Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling. PMID:19852512

Liu, Yongjian; Ibricevic-Richardson, Aida; Cohen, Joel A.; Cohen, Jessica L.; Gunsten, Sean P.; Fréchet, Jean M. J.; Walter, Michael J.; Welch, Michael J.; Brody, Steven L.

2009-01-01

125

Polyglycerolsulfate Functionalized Gold Nanorods as Optoacoustic Signal Nanoamplifiers for In Vivo Bioimaging of Rheumatoid Arthritis  

PubMed Central

We have synthesized a targeted imaging agent for rheumatoid arthritis based on polysulfated gold nanorods. The CTAB layer on gold nanorods was first replaced with PEG-thiol and then with dendritic polyglycerolsulfate at elevated temperature, which resulted in significantly reduced cytotoxicity compared to polyanionic gold nanorods functionalized by non-covalent approaches. In addition to classical characterization methods, we have established a facile UV-VIS based BaCl2 agglomeration assay to confirm a quantitative removal of unbound ligand. With the help of a competitive surface plasmon resonance-based L-selectin binding assay and a leukocyte adhesion-based flow cell assay, we have demonstrated the high inflammation targeting potential of the synthesized gold nanorods in vitro. In combination with the surface plasmon resonance band of AuNRs at 780 nm, these findings permitted the imaging of inflammation in an in vivo mouse model for rheumatoid arthritis with high contrast using multispectral optoacoustic tomography. The study offers a robust method for otherwise difficult to obtain covalently functionalized polyanionic gold nanorods, which are suitable for biological applications as well as a low-cost, actively targeted, and high contrast imaging agent for the diagnosis of rheumatoid arthritis. This paves the way for further research in other inflammation associated pathologies, in particular, when photothermal therapy can be applied. PMID:24723984

Vonnemann, Jonathan; Beziere, Nicolas; Böttcher, Christoph; Riese, Sebastian B.; Kuehne, Christian; Dernedde, Jens; Licha, Kai; von Schacky, Claudio; Kosanke, Yvonne; Kimm, Melanie; Meier, Reinhard; Ntziachristos, Vasilis; Haag, Rainer

2014-01-01

126

A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen  

PubMed Central

A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of ?-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents. PMID:25627011

Tikhonova, Elena B.; Ethayathulla, Abdul S.; Su, Yue; Hariharan, Parameswaran; Xie, Shicong; Guan, Lan

2015-01-01

127

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation  

PubMed Central

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. PMID:22294727

Psaila, Bethan; Bussel, James B.; Linden, Matthew D.; Babula, Bracken; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L.

2012-01-01

128

in vivo analysis of Drosophila deoxyribonucleoside kinase function in cell cycle, cell survival and anti-cancer drugs resistance  

E-print Network

1 in vivo analysis of Drosophila deoxyribonucleoside kinase function in cell cycle, cell survival Drosophila, deoxyribonucleoside kinase, dNK, antifolate resistance, apoptosis, proliferation, growth, dE2F1. Knecht and S.Carroll and the Bloomington Drosophila stock center for fly strains and antibodies

Boyer, Edmond

129

Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions  

PubMed Central

Carbon sequestration, infrastructure rehabilitation, brownfields clean-up, hazardous waste disposal, water resources protection and global warming—these twenty-first century challenges can neither be solved by the high-energy consumptive practices that hallmark industry today, nor by minor tweaking or optimization of these processes. A more radical, holistic approach is required to develop the sustainable solutions society needs. Most of the above challenges occur within, are supported on, are enabled by or grown from soil. Soil, contrary to conventional civil engineering thought, is a living system host to multiple simultaneous processes. It is proposed herein that ‘soil engineering in vivo’, wherein the natural capacity of soil as a living ecosystem is used to provide multiple solutions simultaneously, may provide new, innovative, sustainable solutions to some of these great challenges of the twenty-first century. This requires a multi-disciplinary perspective that embraces the science of biology, chemistry and physics and applies this knowledge to provide multi-functional civil and environmental engineering designs for the soil environment. For example, can native soil bacterial species moderate the carbonate cycle in soils to simultaneously solidify liquefiable soil, immobilize reactive heavy metals and sequester carbon—effectively providing civil engineering functionality while clarifying the ground water and removing carbon from the atmosphere? Exploration of these ideas has begun in earnest in recent years. This paper explores the potential, challenges and opportunities of this new field, and highlights one biogeochemical function of soil that has shown promise and is developing rapidly as a new technology. The example is used to propose a generalized approach in which the potential of this new field can be fully realized. PMID:20829246

DeJong, Jason T.; Soga, Kenichi; Banwart, Steven A.; Whalley, W. Richard; Ginn, Timothy R.; Nelson, Douglas C.; Mortensen, Brina M.; Martinez, Brian C.; Barkouki, Tammer

2011-01-01

130

Quantifying long-term microelectrode array functionality using chronic in vivo impedance testing  

NASA Astrophysics Data System (ADS)

Long-term acquisition of high-quality neural recordings is a cornerstone of neuroprosthetic system design. Mitigating the experimental variability of chronically implanted arrays has been a formidable task because the sensor recording sites can be influenced by biotic and abiotic responses. Several studies have implicated changes in electrical interface impedance as a preliminary marker to infer electrode viability. Microelectrode impedance plays an important role in the monitoring of low amplitude and high-resolution extracellular neural signals. In this work, we seek to quantify long-term microelectrode array functionality and derive an impedance-based predictor for electrode functionality that correlates the recording site electrical properties with the functional neuronal recordings in vivo. High temporal resolution metrics of this type would allow one to assess, predict, and improve electrode performance in the future. In a large cohort of animals, we performed daily impedance measurements and neural signal recordings over long periods (up to 21 weeks) of time in rats using tungsten microwire arrays implanted into the somatosensory cortex. This study revealed that there was a time-varying trend in the modulation of impedance that was related to electrode performance. Single units were best detected from electrodes at time points when the electrode entered into the 40-150 K? impedance range. This impedance trend was modeled across the full cohort of animals to predict future electrode performance. The model was tested on data from all animals and was able to provide predictions of electrode performance chronically. Insight from this study can be combined with knowledge of electrode materials and histological analysis to provide a more comprehensive predictive model of electrode failure in the future.

Prasad, Abhishek; Sanchez, Justin C.

2012-04-01

131

Function of dopamine transporter is compromised in DYT1 transgenic animal model in vivo.  

PubMed

Early onset torsion dystonia (DYT1), the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein, torsinA. We previously examined the effect of the human mutant torsinA on striatal dopaminergic function in a conventional transgenic mouse model of DYT1 dystonia (hMT1), in which human mutant torsinA is expressed under the cytomegalovirus promotor. Systemic administration of amphetamine did not increase dopamine (DA) release as efficiently in these mice as compared with wild-type transgenic and non-transgenic mice. We, now, studied the contribution of the DA transporter (DAT) to amphetamine-induced DA release in hMT1 transgenic mice using in vivo no-net flux microdialysis. This method applies different concentrations of DA through the microdialysis probe and measures DA concentration at the output of the probe following an equilibrium period. The slope (extraction fraction) is the measure of the DAT activity in vivo. The slope for hMT1 transgenic mice was 0.58 +/- 0.07 and for non-transgenic animals, 0.87 +/- 0.06 (p < 0.05). We further investigated the efficacy of nomifensine (a specific DAT inhibitor) in inhibiting amphetamine-induced DA release. Local application of nomifensine 80 min before the systemic application of amphetamine inhibited DA release in both transgenic mice and their non-transgenic littermates. The efficiency of the inhibition appeared to be different, with mean values of 48% for hMT1 transgenic mice versus 84% for non-transgenic littermates. Moreover, we have evaluated basal and amphetamine-induced locomotion in hMT1 transgenic mice compared with their non-transgenic littermates, using an O-maze behavioral chamber. Basal levels of locomotion in the hMT1 transgenic mice showed that they moved much less than their non-transgenic littermates (0.9 +/- 0.3 m for transgenic mice vs. 2.4 +/- 0.7 m for non-transgenic littermates, p < 0.05). This relative reduction in locomotion was also observed following amphetamine administration (48.5 +/- 6.7 m for transgenics vs. 73.7 +/- 9.8 m for non-transgenics, p < 0.05). These results support the finding that there are altered dynamics of DA release and reuptake in hMT1 transgenic mice in vivo, with DAT activity is reduced in the presence of mutant torsinA, which is consistent with behavioral consequences such as reduced locomotion and (previously described) abnormal motor phenotypes such as increased hind-base width and impaired performance on the raised-beam task. These data implies that altered DAT function may contribute to impaired DA neurotransmission and clinical symptoms in human DYT1 dystonia. PMID:20132487

Hewett, Jeff; Johanson, Peter; Sharma, Nutan; Standaert, David; Balcioglu, Aygul

2010-04-01

132

Characterization of Functional Domains of the SMN Protein in Vivo* Received for publication, June 1, 2001, and in revised form, September 20, 2001  

E-print Network

Characterization of Functional Domains of the SMN Protein in Vivo* Received for publication, June 1 previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein

Dreyfuss, Gideon

133

Effect of duration of the GnRH agonists in the luteal phase in the outcome of assisted reproduction cycles.  

PubMed

The effect of long-acting GnRHa, in the luteal phase, during ART cycles varies from one patient to another. The aim of this study was to evaluate whether the effect of long-acting GnRHa in the luteal phase, in ART cycles, affects pregnancy rates according to the duration of its action in such phase. This is a retrospective study of 367 patients submitted to ovulation induction for in vitro fertilization/intracytoplasmic sperm injection procedures that used long-acting depot GnRHa for pituitary suppression. Patients were stratified according to the period of action of the agonist in the luteal phase: group 1, ? 6 days; group 2, 7 to 12 days; and group 3, >12 days. The following variables were analyzed: ovarian response, age, infertility causes and pregnancy rates. Group 1 (n = 53) had a mean age of 33.8 ± 4.55 years (23-44 years) and a pregnancy rate of 45.2%. In group 2 (n = 118), mean age was 33.7 ± 4.5 years (24-44 years) and the pregnancy rate was 38.9%. In group 3 (n = 196), mean age was 33.7 ± 4.4 years (23-43 years) and the pregnancy rate was 47.4%. Regardless of the duration of depot GnRHa action in the luteal phase, no significant association with pregnancy rates was found. PMID:23656392

Geber, Selmo; Sampaio, Marcos

2013-06-01

134

The impact of luteal phase support on endometrial estrogen and progesterone receptor expression: a randomized control trial  

PubMed Central

Background To assess the impact of luteal phase support on the expression of estrogen receptor (ER) alpha and progesterone receptors B (PR-B) on the endometrium of oocyte donors undergoing controlled ovarian hyperstimulation (COH). Methods A prospective, randomized study was conducted in women undergoing controlled ovarian hyperstimulation for oocyte donation. Participants were randomized to receive no luteal support, vaginal progesterone alone, or vaginal progesterone plus orally administered 17 Beta estradiol. Endometrial biopsies were obtained at 4 time points in the luteal phase and evaluated by tissue microarray for expression of ER alpha and PR-B. Results One-hundred and eight endometrial tissue samples were obtained from 12 patients. No differences were found in expression of ER alpha and PR-B among all the specimens with the exception of one sample value. Conclusions The administration of progesterone during the luteal phase of COH for oocyte donor cycles, either with or without estrogen, does not significantly affect the endometrial expression of ER alpha and PR. PMID:22360924

2012-01-01

135

The effects of heat on skin barrier function and in vivo dermal absorption.  

PubMed

Enhanced delivery of ingredients across the stratum corneum (SC) is of great interest for improving the efficacy of topically applied formulations. Various methods for improving dermal penetration have been reported including galvanic devices and micro-needles. From a safety perspective it is important that such approaches do not compromise SC barrier function. This study investigates the influence of topically applied heat in vivo on the dermal uptake and penetration of a model active, allantoin from gel and lotion formulations. A custom designed device was used to deliver 42°C for 30s daily to human subjects after application of two formulations containing allantoin. The results were compared with sites treated with formulations containing no active and no heat, and a control site. In addition to penetration of allantoin, the integrity of the SC was monitored using trans-epidermal water loss (TEWL) measurements. The results showed that just 30s of 42°C topically applied heat was enough to cause significantly more penetration of allantoin from the lotion formulation compared with no application of heat. TEWL data indicated that the integrity of the skin was not compromised by the treatment. However, the application of heat did not promote enhanced penetration of the active from the gel formulation. Vehicle composition is therefore an important factor when considering thermal enhancement strategies for targeting actives to the skin. PMID:24445121

Oliveira, Gabriela; Leverett, Jesse C; Emamzadeh, Mandana; Lane, Majella E

2014-04-10

136

In vivo effects of Eurycoma longifolia Jack (Tongkat Ali) extract on reproductive functions in the rat.  

PubMed

An aqueous extract of Eurycoma longifolia (Tongkat Ali; TA) roots is traditionally used to enhance male sexuality. Because previous studies are limited to only few sperm parameters or testosterone concentration, this study investigated the in vivo effects of TA on body and organ weight as well as functional sperm parameters in terms of safety and efficacy in the management of male infertility. Forty-two male rats were divided into a control, low-dose (200 mg kg(-1) BW) and high-dose (800 mg kg(-1) BW) group (n = 14). Rats were force-fed for 14 days and then sacrificed. Total body and organ weights of the prostate, testes, epididymides, gastrocnemius muscle and the omentum were recorded. Moreover, testosterone concentration, sperm concentration, motility, velocity, vitality, acrosome reaction and mitochondrial membrane potential (MMP) were assessed. Whilst TA decreased BW by 5.7% (P = 0.0276) and omentum fat by 31.9% (P = 0.0496), no changes in organ weights were found for the prostate, testes and epididymides. Testosterone concentration increased by 30.2% (P = 0.0544). Muscle weight also increased, yet not significantly. Whilst sperm concentration, total and progressive motility and vitality increased significantly, MMP improved markedly (P = 0.0765) by 25.1%. Because no detrimental effect could be observed, TA appears safe for possible treatment of male infertility and ageing male problems. PMID:23464350

Solomon, M C; Erasmus, N; Henkel, R R

2014-05-01

137

Neurotrophic Factor Artemin Promotes Invasiveness and Neurotrophic Function of Pancreatic Adenocarcinoma In Vivo and In Vitro  

PubMed Central

Objectives The aim of this study was to investigate the effect of the neurotrophic factor Artemin on neuroplasticity and perineural invasion of pancreatic adenocarcinoma. Methods Artemin expressions were detected in human pancreatic adenocarcinoma tissues by Western blot and immunohistochemistry. Artemin overexpression and RNA interference in the pancreatic cancer cell lines were performed to evaluate the effects of Artemin on cell proliferation, invasion, and neurotrophic activity in vitro and in nude orthotopic transplantation tumor models. Results Artemin expression in pancreatic cancer tissues was related to the incidence of lymphatic metastasis and perineural invasion as well as the mean density and total area of nerve fibers. Overexpression of Artemin in pancreatic cancer cell lines improved colony formation, cell migration, matrigel invasion, and neurotrophic activity in vitro. This overexpression also increased the volume of nude orthotopic transplantation tumors; promoted cancer cell invasion of the peripheral organs, nerves, vessels, and lymph nodes; and stimulated the proliferation of peritumoral nerve fibers. Artemin depletion by RNA interference had an inhibitory effect mentioned previously. Conclusions Artemin could promote invasiveness and neurotrophic function of pancreatic adenocarcinoma in vivo and in vitro. Therefore, Artemin could be used as a new therapeutic target of pancreatic carcinoma. PMID:25243385

Gao, Li; Bo, Haiji; Wang, Yang; Zhang, Jing; Zhu, Minghua

2015-01-01

138

In vivo activation of neutrophil function in hamsters by recombinant human granulocyte colony-stimulating factor.  

PubMed Central

The in vivo effect of Escherichia coli-derived recombinant human granulocyte colony-stimulating factor on neutrophil function was studied in golden Syrian hamsters. Significant increases in superoxide generation and specific binding of N-formylmethionyl-leucyl-phenylalanine were observed in neutrophils isolated 4 h following a single subcutaneous injection of the factor (30 micrograms/kg). However, phagocytotic activity was not significantly stimulated in hamsters treated with the factor. Recombinant human granulocyte colony-stimulating factor hastened the recovery of peripheral neutrophil counts in animals made leukopenic by prior treatment with cyclophosphamide. Beginning several hours after infection, resistance to lethal infection following intraperitoneal injection of Staphylococcus aureus was increased when neutropenic animals were treated daily with the factor. This protective effect was associated with increased peritoneal neutrophil counts and a decreased incidence of positive peritoneal bacterial cultures at 24 h after the start of treatment. These results suggest that recombinant human granulocyte colony-stimulating factor may be a useful adjunct in the treatment of bacterial infections in neutropenic patients. PMID:2459064

Cohen, A M; Hines, D K; Korach, E S; Ratzkin, B J

1988-01-01

139

Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy  

PubMed Central

Respiratory syncytial virus (RSV) can cause devastating lower respiratory tract infections in preterm infants or when other serious health problems are present. Immunoprophylaxis with palivizumab (Synagis), a humanized IgG1 mAb, is the current standard of care for preventing RSV infection in at-risk neonates. We have explored the contribution of effector function to palivizumab efficacy using a plant-based expression system to produce palivizumab N-glycan structure variants with high homogeneity on different antibody isotypes. We compared these isotype and N-glycoform variants with commercially available palivizumab with respect to both in vitro receptor and C1q binding and in vivo efficacy. Whereas the affinity for antigen and neutralization activity of each variant were indistinguishable from those of palivizumab, their Fc? receptor binding profiles were very different, which was reflected in either a reduced or enhanced ability to influence the RSV lung titer in challenged cotton rats. Enhanced Fc? receptor binding was associated with reduced viral lung titers compared with palivizumab, whereas abrogation of receptor binding led to a drastic reduction in efficacy. The results support the hypotheses that classic antibody neutralization is a minor component of efficacy by palivizumab in the cotton rat and that antibody-dependent cell-mediated cytotoxicity activity can significantly enhance the efficacy of this antiviral mAb. PMID:24711420

Hiatt, Andrew; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Piedra, Pedro A.; Gilbert, Brian E.; Zeitlin, Larry

2014-01-01

140

Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy.  

PubMed

Respiratory syncytial virus (RSV) can cause devastating lower respiratory tract infections in preterm infants or when other serious health problems are present. Immunoprophylaxis with palivizumab (Synagis), a humanized IgG1 mAb, is the current standard of care for preventing RSV infection in at-risk neonates. We have explored the contribution of effector function to palivizumab efficacy using a plant-based expression system to produce palivizumab N-glycan structure variants with high homogeneity on different antibody isotypes. We compared these isotype and N-glycoform variants with commercially available palivizumab with respect to both in vitro receptor and C1q binding and in vivo efficacy. Whereas the affinity for antigen and neutralization activity of each variant were indistinguishable from those of palivizumab, their Fc? receptor binding profiles were very different, which was reflected in either a reduced or enhanced ability to influence the RSV lung titer in challenged cotton rats. Enhanced Fc? receptor binding was associated with reduced viral lung titers compared with palivizumab, whereas abrogation of receptor binding led to a drastic reduction in efficacy. The results support the hypotheses that classic antibody neutralization is a minor component of efficacy by palivizumab in the cotton rat and that antibody-dependent cell-mediated cytotoxicity activity can significantly enhance the efficacy of this antiviral mAb. PMID:24711420

Hiatt, Andrew; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Piedra, Pedro A; Gilbert, Brian E; Zeitlin, Larry

2014-04-22

141

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells  

PubMed Central

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4? and HNF-1? and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4? and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases. PMID:24730442

Patil, Pradeep B.; Begum, Setara; Joshi, Meghnad; Kleman, Marika I; Olausson, Michael

2014-01-01

142

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.  

PubMed

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates. PMID:23204504

Rudolf, Mareike; Machettira, Anu B; Groß, Lucia E; Weber, Katrin L; Bolte, Kathrin; Bionda, Tihana; Sommer, Maik S; Maier, Uwe G; Weber, Andreas P M; Schleiff, Enrico; Tripp, Joanna

2013-05-01

143

In vivo functional and myeloarchitectonic mapping of human primary auditory areas  

PubMed Central

In contrast to vision, where retinotopic mapping alone can define areal borders, primary auditory areas such as A1 are best delineated by combining in vivo tonotopic mapping with post mortem cyto- or myelo-architectonics from the same individual. We combined high-resolution (800 ?m) quantitative T1 mapping with phase-encoded tonotopic methods to map primary auditory areas (A1 and R) within the ‘auditory core’ of human volunteers. We first quantitatively characterize the highly myelinated auditory core in terms of shape, area, cortical depth profile, and position, with our data showing considerable correspondence to post-mortem myeloarchitectonic studies, both in cross-participant averages and in individuals. The core region contains two ‘mirror-image‘ tonotopic maps oriented along the same axis as observed in macaque and owl monkey. We suggest that thee two maps within the core are the human analogues of primate auditory areas A1 and R. The core occupies a much smaller portion of tonotopically organized cortex on the superior temporal plane and gyrus than is generally supposed. The multi-modal approach to defining the auditory core will facilitate investigations of structure-function relationships, comparative neuroanatomical studies, and promises new biomarkers for diagnosis and clinical studies. PMID:23152594

Dick, Frederic; Tierney, Adam Taylor; Lutti, Antoine; Josephs, Oliver; Sereno, Martin I.; Weiskopf, Nikolaus

2012-01-01

144

Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo  

SciTech Connect

The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

Girard, C.; Brodeur, J.C.; Hontela, A. [Univ. du Quebec, Montreal, Quebec (Canada)

1995-12-31

145

The effect of glycerol-containing peritoneal dialysis fluid on peritoneal macrophage function in vivo.  

PubMed

Though glucose is universally applied as osmotic agent in CAPD, there is great interest in the use of alternative osmotic agents. Glycerol-containing peritoneal dialysis fluids (G-PDF) have been used in an attempt to minimize the metabolic effects of long-term exposure to glucose, especially in patients with diabetes. Since data were lacking, we studied the effect of G-PDF on peritoneal macrophage (PMO) function. In a randomized cross-over setting eight stable diabetic CAPD patients performed the third and fourth exchange of the day with either G-PDF or with glucose-containing PDF (D-PDF) of comparable osmolality. The next day the patients who had used G-PDF were switched to D-PDF and vice versa. PMO were isolated from the effluents and tested for their phagocytic capacity and chemiluminescence response. No differences were encountered in total and differential white cell counts between G-PDF and D-PDF effluents. PMO phagocytic capacity for both S. epidermidis (SE) and E. coli (EC) was significantly depressed after the instillation of G-PDF as compared to D-PDF (SE: 52 +/- 2.7 vs 69 +/- 5.0%, p less than 0.02, and EC: 44 +/- 5.7 vs 63 +/- 6.7%, p less than 0.02). The same held true for peak chemiluminescence response (5.3 +/- 1.36 vs 7.2 +/- 1.43% of control cells, p less than 0.005). Thus, G-PDF may compromise PMO function in vivo more than D-PDF despite its more favourable metabolic profile as compared to D-PDF for diabetic patients. PMID:1680414

de Fijter, C W; Verbrugh, H A; Oe, P L; Peters, E D; van der Meulen, J; Donker, A J; Verhoef, J; Lameire, N

1991-01-01

146

Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy  

NASA Technical Reports Server (NTRS)

BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

2000-01-01

147

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.  

PubMed

Abstract The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles®). Fluorescence of the Bioparticles® is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles® phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS® Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles®. The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles®. In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development. PMID:25027674

Tartaro, Karrie; VanVolkenburg, Maria; Wilkie, Dean; Coskran, Timothy M; Kreeger, John M; Kawabata, Thomas T; Casinghino, Sandra

2014-07-16

148

SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition  

PubMed Central

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology. PMID:23922875

Hanson, Jesse E.; La, Hank; Plise, Emile; Chen, Yung-Hsiang; Ding, Xiao; Hanania, Taleen; Sabath, Emily V.; Alexandrov, Vadim; Brunner, Dani; Leahy, Emer; Steiner, Pascal; Liu, Lichuan; Scearce-Levie, Kimberly; Zhou, Qiang

2013-01-01

149

In vivo skin biophysical behaviour and surface topography as a function of ageing.  

PubMed

Normal skin ageing is characterised by an alteration of the underlying connective tissue with measurable consequences on global skin biophysical properties. The cutis laxa syndrome, a rare genetic disorder, is considered as an accelerated ageing process since patients appear prematurely aged due to alterations of dermal elastic fibres. In the present study, we compared the topography and the biomechanical parameters of normal aged skin with an 17 year old cutis laxa patient. Skin topography analyses were conducted on normal skin at different ages. The results indicate that the skin relief highly changes as a function of ageing. The cutaneous lines change from a relatively isotropic orientation to a highly anisotropic orientation. This reorganisation of the skin relief during the ageing process might be due to a modification of the skin mechanical properties, and particularly to a modification of the dermis mechanical properties. A specific bio-tribometer, based on the indentationtechnique under light load, has been developed to study the biophysical properties of the human skin in vivo through two main parameters: the physico-chemical properties of the skin surface, by measuring the maximum adhesion force between the skin and the bio-tribometer; and the bulk mechanical properties. Our results show that the pull-off force between the skin and the biotribometer as well as the skin Young's modulus decrease with age. In the case of the young cutis laxa patient, the results obtained were similar to those observed for aged individuals. These results are very interesting and encouraging since they would allow the monitoring of the cutis laxa skin in a standardised and non-invasive way to better characterize either the evolution of the disease or the benefit of a treatment. PMID:23664827

Pailler-Mattei, C; Debret, R; Vargiolu, R; Sommer, P; Zahouani, H

2013-12-01

150

Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo  

PubMed Central

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor. PMID:20582266

Olaghere da Silva, Uade B.; Morabito, Michael V.; Canal, Clinton E.; Airey, David C.; Emeson, Ronald B.; Sanders-Bush, Elaine

2009-01-01

151

Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor  

PubMed Central

Inhibitors of HIV protease have been shown to have antiapoptotic effects in vitro, yet whether these effects are seen in vivo remains controversial. In this study, we have evaluated the impact of the HIV protease inhibitor (PI) nelfinavir, boosted with ritonavir, in models of nonviral disease associated with excessive apoptosis. In mice with Fas-induced fatal hepatitis, Staphylococcal enterotoxin B–induced shock, and middle cerebral artery occlusion–induced stroke, we demonstrate that PIs significantly reduce apoptosis and improve histology, function, and/or behavioral recovery in each of these models. Further, we demonstrate that both in vitro and in vivo, PIs block apoptosis through the preservation of mitochondrial integrity and that in vitro PIs act to prevent pore function of the adenine nucleotide translocator (ANT) subunit of the mitochondrial permeability transition pore complex. PMID:15937550

Weaver, Joel G.R.; Tarze, Agathe; Moffat, Tia C.; LeBras, Morgane; Deniaud, Aurelien; Brenner, Catherine; Bren, Gary D.; Morin, Mario Y.; Phenix, Barbara N.; Dong, Li; Jiang, Susan X.; Sim, Valerie L.; Zurakowski, Bogdan; Lallier, Jessica; Hardin, Heather; Wettstein, Peter; van Heeswijk, Rolf P.G.; Douen, Andre; Kroemer, Romano T.; Hou, Sheng T.; Bennett, Steffany A.L.; Lynch, David H.; Kroemer, Guido; Badley, Andrew D.

2005-01-01

152

FALL STrUCTUrE, dYNAMiCS & FUNCTiON Mouse in-vivo MRI probe and proton RF coil for the UWB 900 MRI scanner.  

E-print Network

FALL STrUCTUrE, dYNAMiCS & FUNCTiON Figure 1. Mouse in-vivo MRI probe and proton RF coil for the UWB 900 MRI scanner. In vivo Mr imaging at 21.1 T Victor D. Schepkin, Samuel C. Grant and Timothy A imaging experiments using the Magnet lab world-record 900 uWB magnet. ExpEriMENTAL Testing the in vivo Mri

Weston, Ken

153

Fucoidan Can Function as an Adjuvant In Vivo to Enhance Dendritic Cell Maturation and Function and Promote Antigen-Specific T Cell Immune Responses  

PubMed Central

Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, including promoting antigen uptake and enhancing anti-viral and anti-tumor effects. However, the effect of fucoidan in vivo, especially its adjuvant effect on in vivo anti-tumor immune responses, was not fully investigated. In this study, we investigated the effect of fucoidan on the function of spleen dendritic cells (DCs) and its adjuvant effect in vivo. Systemic administration of fucoidan induced up-regulation of CD40, CD80 and CD86 expression and production of IL-6, IL-12 and TNF-? in spleen cDCs. Fucoidan also promoted the generation of IFN-?-producing Th1 and Tc1 cells in an IL-12-dependent manner. When used as an adjuvant in vivo with ovalbumin (OVA) antigen, fucoidan promoted OVA-specific antibody production and primed IFN-? production in OVA-specific T cells. Moreover, fucoidan enhanced OVA-induced up-regulation of MHC class I and II on spleen cDCs and strongly prompted the proliferation of OVA-specific CD4 and CD8 T cells. Finally, OVA immunization with fucoidan as adjuvant protected mice from the challenge with B16-OVA tumor cells. Taken together, these results suggest that fucoidan can function as an adjuvant to induce Th1 immune response and CTL activation, which may be useful in tumor vaccine development. PMID:24911024

Jin, Jun-O; Zhang, Wei; Du, Jiang-Yuan; Wong, Ka-Wing; Oda, Tatsuya; Yu, Qing

2014-01-01

154

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo  

PubMed Central

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer. PMID:22754528

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

2012-01-01

155

Increased in vivo stability and functional lifetime of an implantable glucose sensor through platinum catalysis.  

PubMed

Understanding and improving in vivo materials related to signal stability and preservation for active chemical sensor and biosensor transduction systems is critical in achieving implantable medical sensors for long-term in vivo applications. During human in vivo clinical testing of an implantable glucose sensor based on a glucose sensitive hydrogel, post-explant analysis showed that the boronate recognition element had been oxidized from the fluorescent indicator, causing a rapid loss of signal within hours after implant. Additional wet-bench analytical evidence and reproduction in vitro suggests reactive oxygen species, particularly hydrogen peroxide (H2O2), stemming from natural inflammatory response to the material, to be the cause of the observed oxidative de-boronation. A 3-nm thick deposition of metallic platinum (Pt) placed by plasma sputtering onto the porous surface of the hydrogel, showed immediate protection from sensor signal loss due to oxidation both in vitro and in vivo, greatly extending the useful lifetime of the implantable glucose sensor from 1 day to an expected ?6 months. This finding may represent a new strategy to protect an implanted material and/or device from in vivo oxidative damage, leading to much improved overall stability and reliability for long-term applications. PMID:23071075

Colvin, Arthur E; Jiang, Hui

2013-05-01

156

HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility.  

PubMed

Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community. PMID:23416135

Langer, Dominik; van 't Hoff, Marcel; Keller, Andreas J; Nagaraja, Chetan; Pfäffli, Oliver A; Göldi, Maurice; Kasper, Hansjörg; Helmchen, Fritjof

2013-04-30

157

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.  

PubMed

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin > soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically. PMID:19190233

Coote, K; Atherton-Watson, H C; Sugar, R; Young, A; MacKenzie-Beevor, A; Gosling, M; Bhalay, G; Bloomfield, G; Dunstan, A; Bridges, R J; Sabater, J R; Abraham, W M; Tully, D; Pacoma, R; Schumacher, A; Harris, J; Danahay, H

2009-05-01

158

L-type CaV1.2 calcium channels: from in vitro findings to in vivo function.  

PubMed

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that ?-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaV?2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2?1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results. PMID:24382889

Hofmann, Franz; Flockerzi, Veit; Kahl, Sabine; Wegener, Jörg W

2014-01-01

159

Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells  

PubMed Central

The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, expanded safely ex vivo and produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by co-implantation of human endothelial and mesenchymal progenitor cells (EPCs and MPCs) isolated from blood and bone marrow. Evaluation of implants after one week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted EPCs were restricted to the luminal aspect of the vessels; MPCs were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from non-invasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization. PMID:18556575

Melero-Martin, Juan M.; De Obaldia, Maria E.; Kang, Soo-Young; Khan, Zia A.; Yuan, Lei; Oettgen, Peter; Bischoff, Joyce

2009-01-01

160

Slam haplotypes modulate the response to LPS in vivo through control of NKT cell number and function1  

PubMed Central

CD1d-restricted NKT cells comprise an innate-like T cell subset that hasbeen demonstrated to play a role in amplifying the response of innate immune leukocytesto TLR ligands. The Slam locus contains genes that have been implicated in both innate and adaptive immune responses. Here, we demonstrate that divergent Slam locus haplotypesmodulate the response of macrophages to TLR ligands such as LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2-associated 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison to macrophage TNF production in Slam haplotype -1-positive C57BL/6J mice. Although no cell-intrinsic differences in macrophage responses to LPS were observed between strains, 129 mice were found to be deficient in liver NKT cell number, in NKT cell cytokine production in response to the CD1d ligand ?-galactosylceramide, and in NKT cell IFN-? production after LPS challenge in vivo. Using B6.129 c1congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled both the response to LPS in vivo as well as the diminished NKT cell number and function, and that these phenotypes were associated with differential expression of SLAM family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivothrough their effect on NKT cell s. PMID:20530260

Aktan, Idil; Chant, Alan; Borg, Zachary D.; Damby, David E.; Leenstra, Paige; Lilley, Graham; Petty, Joseph; Suratt, Benjamin T.; Teuscher, Cory; Wakeland, Edward K.; Poynter, Matthew E.; Boyson, Jonathan E.

2011-01-01

161

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.  

PubMed Central

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo. Images Fig. 2 Fig. 5 PMID:8876231

Michelson, A D; Barnard, M R; Hechtman, H B; MacGregor, H; Connolly, R J; Loscalzo, J; Valeri, C R

1996-01-01

162

Association Between Energy Balance and Luteal Function in Lactating Dairy Cows  

Microsoft Academic Search

ABSTRACT Our,objective,was,to determine,the relationship,between,energy,balance,and secretion,of progesterone,in,lactating dairy,cows.,Eight primiparous,and,24 muhiparous,lactating Holstein cows,were studied,from,parturition,to 100 d post- partum,or,conception.,Cows,calved normally,and,remained,healthy,through- out,the,study. All cows,were,fed,ad libitum,a total mixed,diet formulated,to satisfy requirements,for maintenance,and lactation. Intake of feed and,production of,milk,per,cow,were,measured,twice daily. Body,weight,was,determined weekly.,Daily energy,balance,was,de- termined,by,subtracting,energy,required for,maintenance,and,lactation,from intake,of,energy.,Concentrations,of progesterone,were,determined,in milk sampled,every,3rd d. For at,least 4 successive d postpartum, 81% of cows were,in negative,energy,balance. Varia- tion,in,energy,balance,was,explained largely by intake,of

A. Villa-Godoy; T. L. Hughes; R. S. Emery; L. T. Chapin; R. L. Fogwell

1988-01-01

163

The S. cerevisiae SAGA complex functions in vivo as a coactivator  

E-print Network

, Massachusetts 02115, USA Previous studies demonstrated that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex, but not the SAGA component Gcn5. We have now examined whether SAGA is directly required as a coactivator in vivo. The SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex of S. cerevisiae is a large multiprotein complex

Winston, Fred

164

Functionalized near-infrared quantum dots for in vivo tumor vasculature imaging  

NASA Astrophysics Data System (ADS)

In this paper, we report the use of near-infrared (NIR)-emitting alloyed quantum dots (QDs) as efficient optical probes for high contrast in vivo imaging of tumors. Alloyed CdTe1 - xSex/CdS QDs were prepared in the non-aqueous phase using the hot colloidal synthesis approach. Water dispersion of the QDs were accomplished by their encapsulation within polyethyleneglycol (PEG)-grafted phospholipid micelles. For tumor-specific delivery in vivo, the micelle-encapsulated QDs were conjugated with the cyclic arginine-glycine-aspartic acid (cRGD) peptide, which targets the ?v?3 integrins overexpressed in the angiogenic tumor vasculatures. Using in vivo NIR optical imaging of mice bearing pancreatic cancer xenografts, implanted both subcutaneously and orthotopically, we have demonstrated that systemically delivered cRGD-conjugated QDs, but not the unconjugated ones, can efficiently target and label the tumors with high signal-to-noise ratio. Histopathological analysis of major organs of the treated mice showed no evidence of systemic toxicity associated with these QDs. These experiments suggest that cRGD-conjugated NIR QDs can serve as safe and efficient probes for optical bioimaging of tumors in vivo. Furthermore, by co-encapsulating these QDs and anticancer drugs within these micelles, we have demonstrated a promising theranostic, nanosized platform for both cancer imaging and therapy.

Hu, Rui; Yong, Ken-Tye; Roy, Indrajit; Ding, Hong; Law, Wing-Cheung; Cai, Hongxing; Zhang, Xihe; Vathy, Lisa A.; Bergey, Earl J.; Prasad, Paras N.

2010-04-01

165

Psychological Stress Exerts an Adjuvant Effect on Skin Dendritic Cell Functions In Vivo1  

Microsoft Academic Search

Psychological stress affects the pathophysiology of infectious, inflammatory, and autoimmune diseases. However, the mechanisms by which stress could modulate immune responses in vivo are poorly understood. In this study, we report that application of a psychological stress before immunization exerts an adjuvant effect on dendritic cell (DC), resulting in increased primary and memory Ag-specific T cell immune responses. Acute stress

Pierre Saint-Mezard; Cyril Chavagnac; Sophie Bosset; Marius Ionescu; Eric Peyron; Dominique Kaiserlian; Jean-Francois Nicolas; Frederic Berard

2003-01-01

166

In-Vivo functional optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source.  

PubMed

In this paper a multi-wavelength optical-resolution photoacoustic microscopy (OR-PAM) system using stimulated Raman scattering is demonstrated for both phantom and in vivo imaging. A 1-ns pulse width ytterbium-doped fiber laser is coupled into a single-mode polarization maintaining fiber. Discrete Raman-shifted wavelength peaks extending to nearly 800 nm are generated with pulse energies sufficient for OR-PAM imaging. Bandpass filters are used to select imaging wavelengths. A dual-mirror galvanometer system was used to scan the focused outputs across samples of carbon fiber networks, 200?m dye-filled tubes, and Swiss Webster mouse ears. Photoacoustic signals were collected in transmission mode and used to create maximum amplitude projection C-scan images. Double dye experiments and in vivo oxygen saturation estimation confirmed functional imaging potential. PMID:24575346

Hajireza, Parsin; Forbrich, Alexander; Zemp, Roger

2014-02-01

167

In-Vivo functional optical-resolution photoacoustic microscopy with stimulated Raman scattering fiber-laser source  

PubMed Central

In this paper a multi-wavelength optical-resolution photoacoustic microscopy (OR-PAM) system using stimulated Raman scattering is demonstrated for both phantom and in vivo imaging. A 1-ns pulse width ytterbium-doped fiber laser is coupled into a single-mode polarization maintaining fiber. Discrete Raman-shifted wavelength peaks extending to nearly 800 nm are generated with pulse energies sufficient for OR-PAM imaging. Bandpass filters are used to select imaging wavelengths. A dual-mirror galvanometer system was used to scan the focused outputs across samples of carbon fiber networks, 200?m dye-filled tubes, and Swiss Webster mouse ears. Photoacoustic signals were collected in transmission mode and used to create maximum amplitude projection C-scan images. Double dye experiments and in vivo oxygen saturation estimation confirmed functional imaging potential. PMID:24575346

Hajireza, Parsin; Forbrich, Alexander; Zemp, Roger

2014-01-01

168

Plasma Progesterone Levels and Luteal Activity during Gestation and Prolonged Oviductal Egg Retention in a Tropical Lizard, Calotes versicolor  

Microsoft Academic Search

Plasma progesterone (P) levels and luteal and adrenal activities were studied during normal gestation and unusual prolonged period of oviductal egg retention in a polyautochronic, multiclutched lizard, Calotes versicolor. The normal gestation period (?15 days) was categorized into four stages: stage I—a few hours following ovulation, stage II—eggs with shell and embryo at primitive streak, stage III—embryonic stages 16–20, and

B. A. Shanbhag; R. S. Radder; S. K. Saidapur

2001-01-01

169

High-mobility group box 1 is dispensable for autophagy, mitochondrial quality control, and organ function in vivo.  

PubMed

In vitro studies have demonstrated a critical role for high-mobility group box 1 (HMGB1) in autophagy and the autophagic clearance of dysfunctional mitochondria, resulting in severe mitochondrial fragmentation and profound disturbances of mitochondrial respiration in HMGB1-deficient cells. Here, we investigated the effects of HMGB1 deficiency on autophagy and mitochondrial function in vivo, using conditional Hmgb1 ablation in the liver and heart. Unexpectedly, deletion of Hmgb1 in hepatocytes or cardiomyocytes, two cell types with abundant mitochondria, did not alter mitochondrial structure or function, organ function, or long-term survival. Moreover, hepatic autophagy and mitophagy occurred normally in the absence of Hmgb1, and absence of Hmgb1 did not significantly affect baseline and glucocorticoid-induced hepatic gene expression. Collectively, our findings suggest that HMGB1 is dispensable for autophagy, mitochondrial quality control, the regulation of gene expression, and organ function in the adult organism. PMID:24606906

Huebener, Peter; Gwak, Geum-Youn; Pradere, Jean-Philippe; Quinzii, Catarina M; Friedman, Richard; Lin, Chyuan-Sheng; Trent, Chad M; Mederacke, Ingmar; Zhao, Enpeng; Dapito, Dianne H; Lin, Yuxi; Goldberg, Ira J; Czaja, Mark J; Schwabe, Robert F

2014-03-01

170

Sweet taste threshold for sucrose inversely correlates with depression symptoms in female college students in the luteal phase.  

PubMed

Influences of depression symptoms on the sweet taste threshold were investigated in healthy college students (30 males and 40 females). Depression symptoms were scored by SDS (Self-Rating Depression Scale), and anxiety levels by STAI (State- and Trait-Anxiety Inventory). Recognition thresholds for sucrose were determined. In female students, the menstrual phase on the day of the experiment was self-reported. Depression symptoms, anxiety levels, and the recognition threshold for sucrose were not different among the 3 groups, i.e. males, females in the follicular phase, and females in the luteal phase. Depression symptoms were positively correlated with state and trait anxiety in all groups. The sweet taste threshold was inversely correlated with depression symptoms (r=-0.472, p=0.031) and trait anxiety (r=-0.506, p=0.019) in females in the luteal phase. In males as well as females in the follicular phase, however, no correlation between sweet taste threshold and depression was found. The results show that the recognition threshold for sucrose reduces with increased depression in females with a higher anxiety trait, but only in the luteal phase. It is hypothesized that brain regions, which spatially overlap and are responsible for both aversive emotions and gustatory processing, are susceptible to periodic changes in gonadal hormones due to the menstrual cycle. PMID:25576640

Nagai, Masanori; Matsumoto, Sayaka; Endo, Junko; Sakamoto, Reiko; Wada, Maki

2015-03-15

171

HIV1 viral protein R compromises cellular immune function in vivo  

Microsoft Academic Search

HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-?B-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect

Velpandi Ayyavoo; Karuppiah Muthumani; Sagar Kudchodkar; Donghui Zhang; P. Ramanathan; Nathanael S. Dayes; J. J. Kim; Jeong-Im Sin; Luis J. Montaner; David B. Weiner

2002-01-01

172

Simultaneous estimation of physiological parameters and the input function - in vivo PET data  

Microsoft Academic Search

Dynamic imaging with positron emission tomography (PET) is widely used for the in-vivo measurement of the regional cerebral metabolic rate for glucose (rCMRGlc) with [ 18F]fluorodeoxy-D-glucose (FDG), and is used for the clinical evaluation of neurological diseases. However, in addition to the acquisition of dynamic images, continuous arterial blood sampling is the conventional method of obtaining the tracer time-activity curve

Koon-pong Wong; David Dagan Feng; Steven R. Meikle; Michael J. Fulham

2001-01-01

173

Noninvasive Assessment of Gene Transfer and Expression by In Vivo Functional and Morphologic Imaging in a Rabbit Tumor Model  

PubMed Central

Purpose To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model. Materials and Methods Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, 111In-octreotide was administered intravenously after CT imaging using a clinical scanner. 111In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine 111In-octreotide biodistribution ex vivo. Results VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater 111In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r?=?0.72, P<0.01, Coefficient of the x-variable?=?.72) at 2 weeks than without excluding necrosis (P<0.01). Conclusion Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo. PMID:23762226

Ravoori, Murali K.; Han, Lin; Singh, Sheela P.; Dixon, Katherine; Duggal, Jyoti; Liu, Ping; Uthamanthil, Rajesh; Gupta, Sanjay; Wright, Kenneth C.; Kundra, Vikas

2013-01-01

174

Luteal Phase Support in assisted reproductive technology treatment: focus on Endometrin® (progesterone) vaginal insert  

PubMed Central

Supplementation of progesterone in the luteal phase and continuance of progesterone therapy during the first trimester has been found in several studies to have benefits in promoting fertility, preventing miscarriages and even preventing pre-term labor. Though it can be administered orally, intramuscularly or even sublingually, a very effective route with fewer side effects can be achieved by an intravaginal route. The first vaginal preparations were not made commercially but were compounded by pharmacies. This had the disadvantage of lack of control by the Food and Drug Administration (FDA) ensuring efficacy of the preparations. Furthermore there was a lack of precise dosing leading to batch to batch variation. The first commercially approved vaginal progesterone preparation in the United States was a vaginal gel which has proven very effective. The main side effect was accumulation of a buildup of the vaginal gel sometimes leading to irritation. Natural micronized progesterone for vaginal administration with the brand name of Utrogestan A® had been approved even before the gel in certain European countries. Endometrin® vaginal tablets are the newest natural progesterone approved by the FDA. Comparisons to the vaginal gel and to intramuscular progesterone have shown similar efficacy especially in studies following controlled ovarian hyperstimulation and oocyte egg retrieval and embryo transfer. Larger studies are needed to compare side effects. PMID:19753133

Check, Jerome H

2009-01-01

175

Interference with Ca2+ release activated Ca2+ (CRAC) channel function delays T-cell arrest in vivo  

PubMed Central

Summary Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca2+]i) elevation. TCR activation triggers increased [Ca2+]i and can arrest T-cell motility in vitro. However the requirement for [Ca2+]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca2+ release-activated Ca2+ (CRAC) channel pathway required for [Ca2+]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4+ T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca2+]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen specific ORAI1-DN T cells had a two-fold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest. PMID:23939929

Waite, Janelle C.; Vardhana, Santosh; Shaw, Patrick J.; Jang, Jung-Eun; McCarl, Christie-Ann; Cameron, Thomas O.; Feske, Stefan; Dustin, Michael L.

2014-01-01

176

Role of vascular networks in extending glucose sensor function: Impact of angiogenesis and lymphangiogenesis on continuous glucose monitoring in vivo.  

PubMed

The concept of increased blood vessel (BV) density proximal to glucose sensors implanted in the interstitial tissue increases the accuracy and lifespan of sensors is accepted, despite limited existing experimental data. Interestingly, there is no previous data or even conjecture in the literature on the role of lymphatic vessels (LV) alone, or in combination with BV, in enhancing continuous glucose monitoring (CGM) in vivo. To investigate the impact of inducing vascular networks (BV and LV) at sites of glucose sensor implantation, we utilized adenovirus based local gene therapy of vascular endothelial cell growth factor-A (VEGF-A) to induce vessels at sensor implantation sites. The results of these studies demonstrated that (1) VEGF-A based local gene therapy increases vascular networks (blood vessels and lymphatic vessels) at sites of glucose sensor implantation; and (2) this local increase of vascular networks enhances glucose sensor function in vivo from 7 days to greater than 28 days postsensor implantation. This data provides "proof of concept" for the effective usage of local angiogenic factor (AF) gene therapy in mammalian models in an effort to extend CGM in vivo. It also supports the practice of a variety of viral and nonviral vectors as well as gene products (e.g. anti-inflammatory and anti-fibrosis genes) to engineer "implant friendly tissues" for the usage with implantable glucose sensors as well as other implantable devices. PMID:24243850

Klueh, Ulrike; Antar, Omar; Qiao, Yi; Kreutzer, Donald L

2014-10-01

177

Long-Term Persistence of Functional Thymic Epithelial Progenitor Cells In Vivo under Conditions of Low FOXN1 Expression  

PubMed Central

Normal thymus function reflects interactions between developing T-cells and several thymic stroma cell types. Within the stroma, key functions reside in the distinct cortical and medullary thymic epithelial cell (TEC) types. It has been demonstrated that, during organogenesis, all TECs can be derived from a common thymic epithelial progenitor cell (TEPC). The properties of this common progenitor are thus of interest. Differentiation of both cTEC and mTEC depends on the epithelial-specific transcription factor FOXN1, although formation of the common TEPC from which the TEC lineage originates does not require FOXN1. Here, we have used a revertible severely hypomorphic allele of Foxn1, Foxn1R, to test the stability of the common TEPC in vivo. By reactivating Foxn1 expression postnatally in Foxn1R/? mice we demonstrate that functional TEPCs can persist in the thymic rudiment until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression, suggesting that manipulation of FOXN1 activity may prove a valuable method for long term maintenance of TEPC in vitro. PMID:25531271

Jin, Xin; Nowell, Craig S.; Ulyanchenko, Svetlana; Stenhouse, Frances H.; Blackburn, C. Clare

2014-01-01

178

Methods: implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing.  

PubMed

Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies. PMID:23173903

Freebern, Wendy J; Bigwarfe, Tammy J; Price, Karen D; Haggerty, Helen G

2013-01-01

179

In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model.  

PubMed

Loss of neurons after brain injury and in neurodegenerative disease is often accompanied by reactive gliosis and scarring, which are difficult to reverse with existing treatment approaches. Here, we show that reactive glial cells in the cortex of stab-injured or Alzheimer's disease (AD) model mice can be directly reprogrammed into functional neurons in vivo using retroviral expression of a single neural transcription factor, NeuroD1. Following expression of NeuroD1, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and GABAergic neurons. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting that they integrated into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. Our studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide an alternative approach for repair of injured or diseased brain. PMID:24360883

Guo, Ziyuan; Zhang, Lei; Wu, Zheng; Chen, Yuchen; Wang, Fan; Chen, Gong

2014-02-01

180

Encapsulation of human islets in novel inhomogeneous alginate-Ca2+/Ba2+ microbeads: in vitro and in vivo function  

PubMed Central

Microencapsulation may allow for immunosuppression free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3±2.5 and 87.5±2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4±0.5 and 6.3±0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30–55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca2+/Ba2+ microbeads allows for long-term in vivo human islet graft function, despite long-distance shipment. PMID:18925451

Qi, Meirigeng; Strand, Berit Løkensgard; Mørch, Yrr; Lacík, Igor; Wang, Yong; Salehi, Payam; Barbaro, Barbara; Gangemi, Antonio; Kuechle, Joseph; Romagnoli, Travis; Hansen, Michael A.; Rodriguez, Lisette A.; Benedetti, Enrico; Hunkeler, David; Skjåk-Bræk, Gudmund; Oberholzer, José

2013-01-01

181

Functional diversity for REST (NRSF) is defined by in vivo binding affinity hierarchies at the DNA sequence level  

PubMed Central

The molecular events that contribute to, and result from, the in vivo binding of transcription factors to their cognate DNA sequence motifs in mammalian genomes are poorly understood. We demonstrate that variations within the DNA sequence motifs that bind the transcriptional repressor REST (NRSF) encode in vivo DNA binding affinity hierarchies that contribute to regulatory function during lineage-specific and developmental programs in fundamental ways. First, canonical sequence motifs for REST facilitate strong REST binding and control functional classes of REST targets that are common to all cell types, whilst atypical motifs participate in weak interactions and control those targets, which are cell- or tissue-specific. Second, variations in REST binding relate directly to variations in expression and chromatin configurations of REST's target genes. Third, REST clearance from its binding sites is also associated with variations in the RE1 motif. Finally, and most surprisingly, weak REST binding sites reside in DNA sequences that show the highest levels of constraint through evolution, thus facilitating their roles in maintaining tissue-specific functions. These relationships have never been reported in mammalian systems for any transcription factor. PMID:19401398

Bruce, Alexander W.; López-Contreras, Andrés J.; Flicek, Paul; Down, Thomas A.; Dhami, Pawandeep; Dillon, Shane C.; Koch, Christoph M.; Langford, Cordelia F.; Dunham, Ian; Andrews, Robert M.; Vetrie, David

2009-01-01

182

The Ras/Rap GTPase activating protein RASA3: From gene structure to in vivo functions.  

PubMed

RASA3 (or GTPase Activating Protein III, R-Ras GTPase-activating protein, GAP1(IP4BP)) is a GTPase activating protein of the GAP1 subfamily which targets Ras and Rap1. RASA3 was originally purified from pig platelet membranes through its intrinsic ability to bind inositol 1,3,4,5-tetrakisphosphate (I(1,3,4,5)P4) with high affinity, hence its first name GAP1(IP4BP) (for GAP1 subfamily member which binds I(1,3,4,5)P4). RASA3 was thus the first I(1,3,4,5)P4 receptor identified and cloned. The in vitro and in vivo functions of RASA3 remained somewhat elusive for a long time. However, recently, using genetically-modified mice and cells derived from these mice, the function of RASA3 during megakaryopoiesis, megakaryocyte adhesion and migration as well as integrin signaling has been reported. The goal of this review is thus to summarize and comment recent and less recent data in the literature on RASA3, in particular on the in vivo function of this specific GAP1 subfamily member. PMID:25294679

Schurmans, Stéphane; Polizzi, Séléna; Scoumanne, Ariane; Sayyed, Sufyan; Molina-Ortiz, Patricia

2015-01-01

183

Selection of Antibodies for Intracellular Function Using a Two-Hybrid in vivo System  

Microsoft Academic Search

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in

Michela Visintin; Eric Tse; Hakan Axelson; Terence H. Rabbitts; Antonino Cattaneo

1999-01-01

184

Disruption of Tacc3 function leads to in vivo tumor regression.  

PubMed

The formation of the bipolar spindle is responsible for accurate chromosomal segregation during mitosis. The dynamic instability of microtubules has an important role in this process, and has been shown to be an effective target for cancer chemotherapy. Several agents that target non-microtubule mitotic proteins, including the motor protein Eg5, Aurora kinases and Polo-like kinases, are currently being developed as chemotherapeutic drugs. However, because the efficacies of these drugs remain elusive, new molecular targets that have essential roles in tumor cells are desired. Here, we provide in vivo evidence that transforming acidic coiled-coil-3 (Tacc3) is a potential target for cancer chemotherapy. Using MRI, we showed that Tacc3 loss led to the regression of mouse thymic lymphoma in vivo, which was accompanied by massive apoptosis. By contrast, normal tissues, including the thymus, showed no overt abnormalities, despite high Tacc3 expression. in vitro analysis indicated that Tacc3 depletion induced multi-polar spindle formation, which led to mitotic arrest, followed by apoptosis. Similar responses have been observed in Burkitt's lymphoma and T-ALL. These results show that Tacc3 is a vulnerable component of the spindle assembly in lymphoma cells and is a promising cancer chemotherapy target. PMID:21685933

Yao, R; Natsume, Y; Saiki, Y; Shioya, H; Takeuchi, K; Yamori, T; Toki, H; Aoki, I; Saga, T; Noda, T

2012-01-12

185

The Intramembrane Proteases Signal Peptide Peptidase-Like 2a and 2b Have Distinct Functions In Vivo  

PubMed Central

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system. PMID:24492962

Schneppenheim, Janna; Hüttl, Susann; Mentrup, Torben; Lüllmann-Rauch, Renate; Rothaug, Michelle; Engelke, Michael; Dittmann, Kai; Dressel, Ralf; Araki, Masatake; Araki, Kimi; Wienands, Jürgen; Fluhrer, Regina; Saftig, Paul

2014-01-01

186

Goserelin can inhibit ovarian cancer proliferation and simultaneously protect ovarian function from cisplatin: an in vitro and in vivo study.  

PubMed

This study investigates whether goserelin can inhibit ovarian cancer proliferation and protect ovarian function from cisplatin (CDDP). We evaluated proliferation and AKT phosphorylation in goserelin-treated ES-2 and SKOV3-ip ovarian cancer cells. Anti-Müllerian hormone (AMH) in human granulosa cells (hGCs) cotreated with goserelin and CDDP was measured by ELISA. Tumour volumes, Ki-67 expression, estrus, follicles, ovarian volumes, and serum AMH were compared in nude mice bearing transplanted tumours treated with goserelin and/or CDDP. Our results showed that goserelin inhibited cellular proliferation and AKT phosphorylation in vitro, and inhibited tumour growth and Ki-67 expression in vivo. Goserelin and CDDP cotreatment decreased the estrus cycles of the nude mice and prolonged estrus duration. Goserelin abrogated the CDDP-induced down-regulation of primary and preantral follicle percentage and ovarian volume. Goserelin increased AMH secretion in vitro and in vivo. In conclusion, goserelin inhibited ovarian cancer proliferation and simultaneously protected ovarian function from CDDP. PMID:23684357

Zhang, Ying; Ding, Jing Xin; Tao, Xiang; Lu, Zhi Ying; Wang, Jia Jia; Feng, Wei Wei; Hua, Ke Qin

2013-04-01

187

Delta-catenin is required for the maintenance of neural structure and function in mature cortex in vivo.  

PubMed

Delta-catenin is a brain-specific member of the adherens junction complex that localizes to the postsynaptic and dendritic compartments. This protein is likely critical for normal cognitive function; its hemizygous loss is linked to the severe mental retardation syndrome Cri-du-Chat and it directly interacts with presenilin-1 (PS1), the protein most frequently mutated in familial Alzheimer's disease. Here we examine dendritic structure and cortical function in vivo in mice lacking delta-catenin. We find that in cerebral cortex of 5-week-old mice, dendritic complexity, spine density, and cortical responsiveness are similar between mutant and littermate controls; thereafter, mutant mice experience progressive dendritic retraction, a reduction in spine density and stability, and concomitant reductions in cortical responsiveness. Our results indicate that delta-catenin regulates the maintenance of dendrites and dendritic spines in mature cortex but does not appear to be necessary for the initial establishment of these structures during development. PMID:19914181

Matter, Cheryl; Pribadi, Mochtar; Liu, Xin; Trachtenberg, Joshua T

2009-11-12

188

Lipopolysaccharide enhances Fc?R-dependent functions in vivo through CD11b/CD18 up-regulation  

PubMed Central

Fc receptors for immunoglobulin G (IgG) (Fc?R) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases Fc?R-dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-? (TNF-?) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fc?-dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fc?-receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting Fc?R expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of Fc?R functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and Fc?R could be critical for the modulation of effector functions during an inflammatory response. PMID:10447764

Rubel, C; Miliani De Marval, P; Vermeulen, M; Isturiz, M A; Palermo, M S

1999-01-01

189

In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands  

PubMed Central

To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (?50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a ?-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ?0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with ‘CFTR-related’ conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics. PMID:24204751

Wine, Jeffrey J.; Char, Jessica E.; Chen, Jonathan; Cho, Hyung-ju; Dunn, Colleen; Frisbee, Eric; Joo, Nam Soo; Milla, Carlos; Modlin, Sara E.; Park, Il-Ho; Thomas, Ewart A. C.; Tran, Kim V.; Verma, Rohan; Wolfe, Marlene H.

2013-01-01

190

Development of an Ex Vivo Model for the Study of Cerebrovascular Function Utilizing Isolated Mouse Olfactory Artery  

PubMed Central

Objective Cerebral vessels, such as intracerebral perforating arterioles isolated from rat brain, have been widely used as an ex vivo model to study the cerebrovascular function associated with cerebrovascular disorders and the therapeutic effects of various pharmacological agents. These perforating arterioles, however, have demonstrated differences in the vascular architecture and reactivity compared with a larger leptomeningeal artery which has been commonly implicated in cerebrovascular disease. In this study, therefore, we developed the method for studying cerebrovascular function utilizing the olfactory artery isolated from the mouse brain. Methods The olfactory artery (OA) was isolated from the C57/BL6 wild-type mouse brain. After removing connective tissues, one side of the isolated vessel segment (approximately -500 µm in length) was cannulated and the opposite end of the vessel was completely sealed while being viewed with an inverted microscope. After verifying the absence of pressure leakage, we examined the vascular reactivity to various vasoactive agents under the fixed intravascular pressure (60 mm Hg). Results We found that the isolated mouse OAs were able to constrict in response to vasoconstrictors, including KCl, phenylephrine, endothelin-1, and prostaglandin PGH2. Moreover, this isolated vessel demonstrated vasodilation in a dose-dependent manner when vasodilatory agents, acetylcholine and bradykinin, were applied. Conclusion Our findings suggest that the isolated olfactory artery would provide as a useful ex vivo model to study the molecular and cellular mechanisms of vascular function underlying cerebrovascular disorders and the direct effects of such disease-modifying pathways on cerebrovascular function utilizing pharmacological agents and genetically modified mouse models.

Dietrich, Hans H.; Han, Byung Hee; Zipfel, Gregory J.

2015-01-01

191

Circumferential and functional re-entry of in-vivo slow wave activity in the porcine small intestine  

PubMed Central

Background Slow waves modulate the pattern of small intestine contractions. However, the large-scale spatial organization of intestinal slow wave pacesetting remains uncertain because most previous studies have had limited resolution. This study applied high-resolution (HR) mapping to evaluate intestinal pacesetting mechanisms and propagation patterns in-vivo. Methods HR serosal mapping was performed in anesthetized pigs using flexible arrays (256 electrodes; 32×8; 4 mm spacing), applied along the jejunum. Slow wave propagation patterns, frequencies, and velocities were calculated. Slow wave initiation sources were identified and analyzed by animation and isochronal activation mapping. Key Results Analysis comprised 32 recordings from nine pigs (mean duration 5.1±3.9 min). Slow wave propagation was analyzed, and a total of 26 sources of slow wave initiation were observed and classified as focal pacemakers (31%), sites of functional re-entry (23%) and circumferential re-entry (35%), or indeterminate sources (11%). The mean frequencies of circumferential and functional re-entry were similar (17.0±0.3 vs 17.2±0.4 cycle min?1; p=0.5), and greater than that of focal pacemakers (12.7±0.8 cycle min?1; p<0.001). Velocity was anisotropic (12.9±0.7 mm s?1 circumferential vs 9.0±0.7 mm s?1 longitudinal; p<0.05), contributing to the onset and maintenance of re-entry. Conclusions & Inferences This study has shown multiple patterns of slow wave initiation in the jejunum of anesthetized pigs. These results constitute the first description and analysis of circumferential re-entry in the gastrointestinal tract and functional re-entry in the in-vivo small intestine. Re-entry can control the direction, pattern, and frequency of slow wave propagation, and its occurrence and functional significance merit further investigation. PMID:23489929

Angeli, Timothy R.; O’Grady, Gregory; Du, Peng; Paskaranandavadivel, Niranchan; Bissett, Ian P; Cheng, Leo K

2013-01-01

192

Pregnancy Rate Following Luteal Phase Support in Iranian Women with Polycystic Ovarian Syndrome  

PubMed Central

Background To assess the efficacy of luteal phase support (LPS) using intravaginal progesterone (P) on pregnancy rate in Iranian women with polycystic ovarian syndrome (PCOS) who used a combination for ovulation induction consisting of letrozole or clomi- phene citrate (CC) and human menopausal gonadotropin (HMG). Materials and Methods This was a randomized clinical trial undertaken in a fertility clinic in Kashan, Isfahan Province, Iran. A total of 198 patients completed treatment and follow up. Base on chosen ovulation induction programs, they were divided into two following group: i. CC group (n=98) used a combination consisting of CC (100 mg×5 day) and HMG (150 IU×5 day) and ii. letrozole group (n=100) used a combination consisting of letrozole (5 mg×5 day) and HMG (150 IU×5 day). After human chorionic gonadotropin (hCG) administration (5000 IU), the patients (n=122) who randomly re- ceived intravaginal P (Cyclogest, 400 mg daily) were included in LPS group, while the rest (n=123) were included in non-P cycles group. The outcome was the comparison of chemical pregnancy rate between the groups. Results Our findings showed that LPS was associated with a 10% higher pregnancy rate than in non-P cycles, although this difference did not reach statistical significant (p=0.08). LPS improved pregnancy rate in both CC (4%) and letrozole (6%) groups. In addition, patients who used letrozole for ovulation induction along with intravaginal P showed higher pregnancy rates than CC group. Conclusion Administration of vaginal P for LPS may improve the pregnancy rate in women with PCOS using letrozole or CC in combination with HMG for ovulation induc- tion (Registration Number: IRCT201206072967N4). PMID:25379150

Foroozanfard, Fatemeh; Saberi, Hamidreza; Moraveji, Seyed Alireza; Bazarganipour, Fatemeh

2014-01-01

193

Premenstrual Dysphoria and Luteal Stress in Dominant-Social-Status Female Macaques  

PubMed Central

The current study aims to extend our previous work to develop nonhuman primate model for prospectively studying the mechanism underlying premenstrual dysphoric disorder (PMDD). Thirty young dominant-status female monkeys were randomly divided into the control group, the model group, and JQP group. For two consecutive menstrual cycles, from day 18 to 22, monkeys in the model and JQP groups were housed and immobilized singly in specially designed isolation cages for 5-6 hours per day. At the same time, the pharmaceutical interference effect of jingqianping (JQP) granule, a traditional Chinese medicine specifically used to cure PMDD patients, was tested using monkeys in the JQP group. The behavior and facial expressions of monkeys were photographed with an automatic vidicon and were quantitatively analyzed by “the emotion evaluation scale of female experimental macaque.” Changes in serum level of progesterone and estradiol were measured with RIA, and serum level of 5-HT, noradrenaline, and dopamine were measured with HPLC. After experiencing mentioned above stress, 70% of monkeys of model group showed PMDD symptoms during three consecutive menstrual cycles. Estradiol and progesterone serum level decreased (P < 0.01). Moreover, the peak value of secreted hormones in their follicular phase did not occur. Serum level of 5-HT and dopamine were significantly lower (P < 0.01), but the serum noradrenaline level was higher (P < 0.01). Moreover, in monkeys administered by JQP granule, both PMDD symptoms and the anormal serum level of neurotransmitters could be obviously reversed. This special luteal-phase treatment on dominant-social-status monkeys might be a feasible way to create models mimicking PMDD. PMID:24371458

Qiao, Mingqi; Zhao, Qitao; Wei, Sheng; Zhang, Huiyun; Wang, Haijun

2013-01-01

194

Relationships between LH and estradiol-17 beta after removal of luteal progesterone in the ewe.  

PubMed

Three experiments were conducted to examine the relationship between systemic concentrations of luteinizing hormone (LH) and estradiol-17 beta (E2) after withdrawal of progesterone in cycling ewes. In Exp. 1, ewes were assigned randomly to one of three treatments: laparotomy (C), removal of the luteal ovary (ULO), or ULO plus anesthesia with sodium pentobarbital for 6 h beginning 4 h after surgery. Anesthesia was used in an attempt to block the expected increase in tonic secretion of LH. Patterns of LH and E2 in these three groups did not differ during the 24-h experimental period. In Exp. 2, a longer period of anesthesia was utilized. Forty-eight ewes were assigned at random to one of four treatments: C, ULO, lutectomy or an intrafollicular injection of prostaglandin F2 alpha (PGF2 alpha). One-half of the ewes in each group were anesthetized with sodium pentobarbital from initiation of treatment (0 h) until 10 h after surgery. Sodium pentobarbital did not suppress the increases in LH and E2 after progesterone withdrawal. The regression of concentrations of E2 on concentration of LH was not significant. In Exp. 3, ewes were infused with either saline or dopamine after receiving an im injection of PGF2 alpha. Tonic secretion of LH increased after 4 h in ewes infused with saline, but not in ewes infused with dopamine. Despite the suppression of LH, concentrations of E2 increased in dopamine-treated ewes as in control ewes. Therefore, the initial increase in E2 after a decline of progesterone in cycling ewes is independent of increases in LH. PMID:6584418

Gust, C M; Deaver, D R; Dailey, R A; Inskeep, E K

1984-02-01

195

Functional remodeling of benign human prostatic tissues in vivo by spontaneously immortalized progenitor and intermediate cells.  

PubMed

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133(high)/CD44(high)/OCT4(high)/PTEN(high)) was characterized as a putative progenitor cell, and BHPrE1 (p63(high)/p53(high)/p21(WAF1)(high)/RB(high)) was characterized as a putative epithelial intermediate cell. Genomic analysis demonstrated an abnormal karyotype with genomic rearrangements including PTEN amplification in NHPrE1 and CTNNB1 (beta-catenin) amplification in BHPrE1 cells. Embedded three-dimensional culture of NHPrE1 showed greater branching than BHPrE1. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme, were grafted to the renal capsule of severe combined immunodeficient mice. Both cell lines were able to regenerate benign secretory ductal-acinar architecture in vivo, containing intact basal and luminal epithelial layers confirmed by the expression of appropriate CK profiles. Prostate-specific antigen, 15-lipoxygenase-2, androgen receptor, and NKX3.1 proteins were appropriately expressed in the regenerated epithelia. Regeneration of benign prostatic glandular structures could be achieved using as few as 10 NHPrE1 cells, whereas 200,000 BHPrE1 cells were required to achieve prostatic architecture. This suggests a greater proportion of progenitor/stem cells in NHPrE1 than in BHPrE1. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis, and carcinogenesis. PMID:20020426

Jiang, Ming; Strand, Douglas W; Fernandez, Suzanne; He, Yue; Yi, Yajun; Birbach, Andreas; Qiu, Qingchao; Schmid, Johannes; Tang, Dean G; Hayward, Simon W

2010-02-01

196

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo  

PubMed Central

To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3?324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo. PMID:20185759

Liu, Minmin; Bandaru, Viswanath; Bond, Jeffrey P.; Jaruga, Pawel; Zhao, Xiaobei; Christov, Plamen P.; Burrows, Cynthia J.; Rizzo, Carmelo J.; Dizdaroglu, Miral; Wallace, Susan S.

2010-01-01

197

TIMELESS Is an Important Mediator of CK2 Effects on Circadian Clock Function In Vivo  

PubMed Central

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of PERIOD (PER) and TIMELESS (TIM) proteins govern period length. Yet little is known about the kinases that phosphorylate TIM in vivo. It has been shown previously that the protein kinase CK2 phosphorylates TIM in vitro. Here, we identify a role for CK2 in TIM regulation in vivo. Induction of a dominant-negative CK2?, CK2?Tik (Tik), increases TIM protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic TIM localization. Exposure to light and subsequent TIM degradation results in an increase in the fraction of the transcriptional repressor PER that is nuclear and suppression of per and tim RNA levels. TIM protein, but not tim transcript, levels are elevated in Tik mutants in a per01 background. In contrast, Tik effects on PER are undetectable in a tim01 background, suggesting that TIM is required for CK2 effects on PER. To identify potential CK2 target sites, we assayed TIM phosphorylation rhythms in a deletion mutant that removes a conserved serine-rich domain and found that TIM protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a timUL mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that TIM is an important mediator of CK2 effects on circadian rhythms. PMID:18815259

Meissner, Rose-Anne; Kilman, Valerie L.; Lin, Jui-Ming; Allada, Ravi

2011-01-01

198

Ageing alters perivascular nerve function of mouse mesenteric arteries in vivo  

PubMed Central

Mesenteric arteries (MAs) are studied widely in vitro but little is known of their reactivity in vivo. Transgenic animals have enabled Ca2+ signalling to be studied in isolated MAs but the reactivity of these vessels in vivo is undefined. We tested the hypothesis that ageing alters MA reactivity to perivascular nerve stimulation (PNS) and adrenoreceptor (AR) activation during blood flow control. First- (1A), second- (2A) and third-order (3A) MAs of pentobarbital-anaesthetized Young (3–6 months) and Old (24–26 months) male and female Cx40BAC-GCaMP2 transgenic mice (C57BL/6 background; positive or negative for the GCaMP2 transgene) were studied with intravital microscopy. A segment of jejunum was exteriorized and an MA network was superfused with physiological salt solution (pH 7.4, 37°C). Resting tone was ? 10% in MAs of Young and Old mice; diameters were ?5% (1A), 20% (2A) and 40% (3A) smaller (P? 0.05) in Old mice. Throughout MA networks, vasoconstriction increased with PNS frequency (1–16 Hz) but was ?20% less in Young vs. Old mice (P? 0.05) and was inhibited by tetrodotoxin (1 ?m). Capsaicin (10 ?m; to inhibit sensory nerves) enhanced MA constriction to PNS (P? 0.05) by ?20% in Young but not Old mice. Phenylephrine (an ?1AR agonist) potency was greater in Young mice (P? 0.05) with similar efficacy (?60% constriction) across ages and MA branches. Constrictions to UK14304 (an ?2AR agonist) were less (?20%; P? 0.05) and were unaffected by ageing. Irrespective of sex or transgene expression, ageing consistently reduced the sensitivity of MAs to ?1AR vasoconstriction while blunting the attenuation of sympathetic vasoconstriction by sensory nerves. These findings imply substantive alterations in splanchnic blood flow control with ageing. PMID:23247111

Westcott, Erika B; Segal, Steven S

2013-01-01

199

Profiling of Luteal Transcriptome during Prostaglandin F2-Alpha Treatment in Buffalo Cows: Analysis of Signaling Pathways Associated with Luteolysis  

PubMed Central

In several species including the buffalo cow, prostaglandin (PG) F2? is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF2? in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF2? treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression changes in several of immediate early genes and transcription factors within 3 h of treatment. Also, changes in expression of genes associated with cell to cell signaling, cytokine signaling, steroidogenesis, PG synthesis and apoptosis were observed. Analysis of various components of LH/CGR signaling in CL tissues indicated decreased LH/CGR protein expression, pCREB levels and PKA activity post PGF2? treatment. The novel finding of this study is the down regulation of CYP19A1 gene expression accompanied by decrease in expression of E2 receptors and circulating and intra luteal E2 post PGF2? treatment. Mining of microarray data revealed several differentially expressed E2 responsive genes. Since CYP19A1 gene expression is low in the bovine CL, mining of microarray data of PGF2?-treated macaques, the species with high luteal CYP19A1 expression, showed good correlation between differentially expressed E2 responsive genes between both the species. Taken together, the results of this study suggest that PGF2? interferes with luteotrophic signaling, impairs intra-luteal E2 levels and regulates various signaling pathways before the effects on structural luteolysis are manifest. PMID:25102061

Suganthi, Hepziba; Rudraiah, Medhamurthy

2014-01-01

200

RANKL expression in normal and malignant breast tissue responds to progesterone and is up-regulated during the luteal phase.  

PubMed

The receptor activator of nuclear factor-?B ligand (RANKL) acts as a paracrine factor in progesterone-induced mammary epithelial proliferation and tumorigenesis. This evidence comes mainly from mouse models. Our aim was to examine whether RANKL expression in human normal and malignant breast is under the control of progesterone throughout the menstrual cycle. Breast epithelial samples were obtained by random fine needle aspiration (rFNA) of the contralateral unaffected breasts (CUB) of 18 breast cancer patients, with simultaneous serum hormone measurements. Genes correlated with serum progesterone levels were identified through Illumina microarray analysis. Validation was performed using qRT-PCR in rFNA samples from CUB of an additional 53 women and using immunohistochemistry in tissue microarrays of 61 breast cancer samples. Expression of RANKL, DIO2, and MYBPC1 was correlated with serum progesterone in CUB, and was significantly higher in luteal phase. RANKL and MYBPC1 mRNA expression were highly correlated between CUB and matched tumor samples. RANKL protein expression was also significantly increased in the luteal phase and highly correlated with serum progesterone levels in cancer samples, especially in hormone receptor positive tumors. The regulatory effects of progesterone on the expression of RANKL, DIO2, and MYBPC1 were confirmed in three-dimensional cultures of normal breast organoids. In normal breast and in breast cancer, RANKL mRNA and protein expression fluctuate with serum progesterone with highest levels in the luteal phase, suggesting that RANKL is a modulator of progesterone signaling in normal and malignant breast tissue and a potential biomarker of progesterone action and blockade. PMID:25007964

Hu, Hong; Wang, Jun; Gupta, Akash; Shidfar, Ali; Branstetter, Daniel; Lee, Oukseub; Ivancic, David; Sullivan, Megan; Chatterton, Robert T; Dougall, William C; Khan, Seema A

2014-08-01

201

Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-d-glucose gold nanoparticles in vivo  

NASA Astrophysics Data System (ADS)

We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-d-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the 18F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.

Roa, Wilson; Xiong, Yeping; Chen, Jie; Yang, Xiaoyan; Song, Kun; Yang, Xiaohong; Kong, Beihua; Wilson, John; Xing, James Z.

2012-09-01

202

Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo.  

PubMed

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties. PMID:23748743

Grelier, Aurore; Cras, Audrey; Balitrand, Nicole; Delmau, Catherine; Lecourt, Séverine; Lepelletier, Yves; Riesterer, Hélène; Freida, Delphine; Lataillade, Jean-Jacques; Lebousse-Kerdiles, Marie-Caroline; Cuccini, Wendy; Peffault de Latour, Regis; Marolleau, Jean-Pierre; Uzan, Georges; Larghero, Jérôme; Vanneaux, Valérie

2013-10-01

203

In vivo assessment of corneal barrier function through non-invasive impedance measurements using a flexible probe  

NASA Astrophysics Data System (ADS)

The cornea is a transparent structure composed of three layers: the epithelium, the stroma and the endothelium. To maintain its ransparency the stroma remains in a constant state of dehydration. Consequently, any ion flow disorder through the covering layers can compromise the barrier function and, therefore the corneal homeostasis. Since ionic permeability has a fundamental impact on the passive electrical properties of living tissues, in this work it is proposed and demonstrated a diagnosis method based on tetrapolar impedance measurements performed by electrodes placed on the corneal surface. The contribution of each cornea layer to the total measured impedance has been analysed over a frequency range. Following the obtained guidelines, a flexible probe with integrated electrodes has been developed and manufactured using SU-8 photoresin. The feasibility of the proposed method has been evaluated in vivo by monitoring corneal epithelium wound healing. Obtained impedance measurements have been compared with measurements of permeability to sodium fluorescein from different excised corneas. Successful results demonstrate the feasibility of this novel flexible sensor and its capability to quantify corneal permeability in vivo in a noninvasive way.

Guimera, A.; Illa, X.; Traver, E.; Marchan, S.; Herrero, C.; Lagunas, C.; Maldonado, M. J.; Ivorra, A.; Villa, R.

2013-04-01

204

Functionalized conducting polymer as an enzyme-immobilizing substrate: an amperometric glutamate microbiosensor for in vivo measurements.  

PubMed

The functionalized conducting polymer (CP) of 5, 2':5', 2' '-terthiophene-3'-carboxylic acid on a platinum microelectrode was prepared through the electropolymerization process using cyclic voltammetry and was used as a substrate for the immobilization of enzymes. The nanoparticles of the CP were obtained at a high scan rate in the cyclic voltammetric experiment. A needle-type amperometric glutamate microbiosensor based on the covalent immobilization of glutamate oxidase (GlOx) onto the CP layer was fabricated for in vivo measurements. The surfaces of the CP/Pt and GlOx/CP/Pt were characterized by QCM, ESCA, and AFM. The biosensor efficiently detected glutamate through the oxidation of enzymatically generated H2O2 at approximately +0.45 V versus Ag/AgCl. Various experimental parameters, such as pH, temperature, and the applied potential in the detection step were optimized. The interference effects from other biological compounds were examined, and ascorbate and dopamine interferences were observed, which were completely minimized by coimmobilizing ascorbate oxidase and by coating the sensor surface with a cationic polymer, polyethyleneimine. A linear calibration plot for glutamate was obtained between 0.2 and 100 microM with a detection limit of 0.1 +/- 0.03 microM. The proposed glutamate microbiosensor was successfully used for in vivo monitoring of the extracellular glutamate released by cocaine stimulation. PMID:16053298

Rahman, Md Aminur; Kwon, Nak-Hyun; Won, Mi-Sook; Choe, Eun Sang; Shim, Yoon-Bo

2005-08-01

205

In Vivo Regulation of NGF-Mediated Functions by Nedd4-2 Ubiquitination of TrkA  

PubMed Central

Trk neurotrophin receptor ubiquitination in response to ligand activation regulates signaling, trafficking, and degradation of the receptors. However, the in vivo consequences of Trk ubiquitination remain to be addressed. We have developed a mouse model with a mutation in the TrkA neurotrophin receptor (P782S) that results in reduced ubiquitination due to a lack of binding to the E3 ubiquitin ligase, Nedd4-2. In vivo analyses of TrkAP782S indicate that defective ubiquitination of the TrkA mutant results in an altered trafficking and degradation of the receptor that affects the survival of sensory neurons. The dorsal root ganglia from the TrkAP782S knock-in mice display an increased number of neurons expressing CGRP and substance P. Moreover, the mutant mice show enhanced sensitivity to thermal and inflammatory pain. Our results indicate that the ubiquitination of the TrkA neurotrophin receptor plays a critical role in NGF-mediated functions, such as neuronal survival and sensitivity to pain. PMID:24760869

Yu, Tao; Calvo, Laura; Anta, Begoña; López-Benito, Saray; López-Bellido, Roger; Vicente-García, Cristina; Tessarollo, Lino; Rodriguez, Raquel E.

2014-01-01

206

Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection  

PubMed Central

Acute lung injury (ALI) is a complex syndrome with many aetiologies, resulting in the upregulation of inflammatory mediators in the host, followed by dyspnoea, hypoxemia and pulmonary oedema. A central mediator is inducible nitric oxide synthase (iNOS) that drives the production of NO and continued inflammation. Thus, it is useful to have diagnostic and therapeutic agents for targeting iNOS expression. One general approach is to target the precursor iNOS mRNA with antisense nucleic acids. Peptide nucleic acids (PNAs) have many advantages that make them an ideal platform for development of antisense theranostic agents. Their membrane impermeability, however, limits biological applications. Here, we report the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabelled antisense PNA-YR9 · oligodeoxynucleotide (ODN) hybrid and a cationic shell-cross-linked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for 123I radiolabelling, whereas the R9 (arginine9) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR9 · ODN hybrid to the cSCK was achieved at an 8 : 1 cSCK amine to ODN phosphate (N/P) ratio by a gel retardation assay. The antisense PNA-YR9 · ODN · cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR9 · ODN alone was not taken up. Low concentrations of 123I-labelled antisense PNA-YR9 · ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide+interferon-?-activated RAW264.7 cells when compared with a mismatched PNA. Moreover, statistically, greater retention of radioactivity from the antisense complex was also observed in vivo in an iNOS-induced mouse lung after intratracheal administration of the nanocomplexes. This study demonstrates the specificity and sensitivity by which the radiolabelled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of ALI. PMID:24427537

Shen, Yuefei; Shrestha, Ritu; Ibricevic, Aida; Gunsten, Sean P.; Welch, Michael J.; Wooley, Karen L.; Brody, Steven L.; Taylor, John-Stephen A.; Liu, Yongjian

2013-01-01

207

In Vitro and In Vivo Evaluation of a Novel Ferrocyanide Functionalized Nanopourous Silica Decorporation Agent for Cesium in Rats  

SciTech Connect

Novel decorporation agents are being developed to protect against radiological terrorist attacks. These sorbents, known as the self-assembled monolayer on mesoporous supports (SAMMS™), are hybrid materials where differing organic moieties are grafted onto mesoporous silica (SiO2). In vitro experiments focused on the evaluation, and optimization of SAMMS for capturing radiocesium (137Cs); based on these studies, a ferrocyanide copper (FC-Cu-EDA)-SAMMS was advanced for in vivo evaluation. In vivo experiments were conducted comparing the performance of the SAMMS vs. insoluble Prussian blue. Groups of jugular cannulated rats (4/treatment) were evaluated. Group I was administered 137Cs (~40 ?geq/kg) by intravenous (iv) injection and oral gavage; Group II was administered pre-bound 137Cs-SAMMS and sequential 137Cs + SAMMS (~61 ngeq/kg) by oral gavage; and Group III evaluated orally administered 137Cs (~0.06 ?geq/kg) followed by 0.1 g of either SAMMS or Prussian blue. Following dosing the rats were maintained in metabolism cages for 72 hour and blood, urine and fecal samples were collected for 137Cs analysis (gamma counting). Rats were then humanely euthanized, and selected tissues analyzed. Orally administered 137Cs was rapidly and well absorbed (~100% relative to iv dose), and the pharmacokinetics (blood, urine, feces & tissues) were very comparable to the iv dose group. For both exposures the urine and feces accounted for 20 and 3% of the dose, respectively. The prebound 137Cs-SAMMS was retained primarily within the feces (72% of the dose), with ~1.4% detected in the urine, suggesting that the 137Cs remained tightly bound to SAMMS. SAMMS & Prussian blue both effectively captured available 137Cs in the gut with feces accounting for 80-88% of the administered dose, while less than 2% was detected in the urine. This study suggests that the functionalized SAMMS out performs Prussian blue in vitro at low pH, but demonstrates comparable in vivo sequestration efficacy at low exposure concentrations. The comparable response may be the result of the low 137Cs dose and high sorbent dosage that was utilized. Future studies are planned to optimize SAMMS in vivo performance over a broader range of doses and conditions.

Timchalk, Charles; Creim, Jeffrey A.; Sukwarotwat, Vichaya; Wiacek, Robert J.; Addleman, Raymond S.; Fryxell, Glen E.; Yantasee, Wassana

2010-09-01

208

Interaction of bovine respiratory syncytial virus with bovine alveolar macrophages in vivo: effects of virus infection upon selected cell functions.  

PubMed Central

The effect of bovine respiratory syncytial virus (BRSV) upon alveolar macrophage (AM) function was investigated using an in vivo calf inoculation model. Alveolar macrophages were collected sequentially from live calves at multiple time points during the 14 day period following viral inoculation. Alveolar macrophages from bronchoalveolar lavage fluids were purified by density gradient centrifugation (> 95% AM) prior to in vitro evaluation of cell functions. There were significant but variable and inconsistent differences in the functions of AM from the BRSV inoculated calves compared to the control calves. Fc-receptor mediated phagocytosis was either increased or unchanged by BRSV inoculation. Nonopsonized phagocytosis was decreased during the early postinoculation period and later increased. There was a variable effect on AM phagosome lysosome fusion with increased fusion activity on postinoculation days 2 through 5, 7 and 12 but reduced activity on days 6 and 10. The AM respiratory burst, as measured by nitroblue tetrazolium dye reduction, was essentially unaffected with a reduction in activity on day 10 only. In this model, BRSV inoculation of calves primarily resulted in an alteration of the membrane associated phagocytic functions of the alveolar macrophages (p < 0.05). PMID:8143252

Olchowy, T W; Ames, T R; Molitor, T W

1994-01-01

209

Functional effects of dopamine transporter gene genotypes on in vivo dopamine transporter functioning: a meta-analysis.  

PubMed

Much psychiatric genetic research has focused on a 40-base pair variable number of tandem repeats (VNTR) polymorphism located in the 3'-untranslated region (3'UTR) of the dopamine active transporter (DAT) gene (SLC6A3). This variant produces two common alleles with 9- and 10-repeats (9R and 10R). Studies associating this variant with in vivo DAT activity in humans have had mixed results. We searched for studies using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to evaluate this association. Random effects meta-analyses assessed the association of the 3'UTR variant with DAT activity. We also evaluated heterogeneity among studies and evidence for publication bias. We found twelve studies comprising 511 subjects, 125 from PET studies and 386 from SPECT studies. The PET studies provided highly significant evidence that the 9R allele was associated with increased DAT activity in human adults. The SPECT studies were highly heterogeneous. As a group, they suggested no association between the 3'UTR polymorphism and DAT activity. When the analysis was limited to the most commonly used ligand, [123I]?-CIT, stratification by affection status dramatically reduced heterogeneity and revealed a significant association of the 9R allele with increased DAT activity for healthy subjects. In humans, the 9R allele of the 3'UTR polymorphism of SLC6A3 regulates dopamine activity in the striatal brain regions independent of the presence of neuropsychiatric illness. Differences in study methodology account for the heterogeneous results across individual studies. PMID:24061496

Faraone, S V; Spencer, T J; Madras, B K; Zhang-James, Y; Biederman, J

2014-08-01

210

Polymer Fiber Probes Enable Optical Control of Spinal Cord and Muscle Function In Vivo  

E-print Network

Restoration of motor and sensory functions in paralyzed patients requires the development of tools for simultaneous recording and stimulation of neural activity in the spinal cord. In addition to its complex neurophysiology, ...

Lu, Chi

211

Comparison of oral dydrogesterone with suppository vaginal progesterone for luteal-phase support in in vitro fertilization (IVF): A randomized clinical trial  

PubMed Central

Background: Luteal phase support is mandatory in assisted reproductive technologies (ART) for optimizing outcome, so the luteal phase is supported with either progesterone, addition of estradiol to progesterone, hCG or gonadotropin releasing hormone (GnRH) agonists. Supplementation of luteal phase with progesterone is prescribed for women undergoing routine IVF treatment. Objective: To compare oral dydrogestrone with vaginal progesterone for luteal-phase support in IVF. Materials and Methods: We performed this prospective, randomized trial in a tertiary infertility care unit in Taleghani Hospital, Tehran, Iran. In total 80 Women with a history of male factor infertility undergoing controlled ovarian stimulation for IVF treatment (fresh cycle) randomly were divided in two groups (group A or oral dydrogesterone group and group B or vaginal progesterone group). The inclusion criteria were the use of GnRH analogue down-regulation and age less than 40 years old with regular menstrual cycles. All women were euthyroid and normoprolactinemic. Group A (n=40) received 10 mg dydrogesterone QID (40mg daily) and group B (n=40) received 400 mg suppository vaginal progesterone (cyclogest) twice per day (800 mg daily). Results: Clinical pregnancy rate in cyclogest group was higher than dydrogesterone group but the difference was not significant (p=0.52), furthermore the miscarriage rate in two group was the same .The difference between two groups regarding antral follicle, embryo number, luteal-phase duration, endometrial thickness, oocyte number and metaphase-II was not significant (p>0.05). Conclusion: The results showed that oral dydrogesterone is as effective as vaginal progesterone for luteal-phase support in women undergoing IVF. PMID:24639716

Salehpour, Saghar; Tamimi, Maryam; Saharkhiz, Nasrin

2013-01-01

212

Docosahexaenoic acid and phosphatidylserine improves the antioxidant activities in vitro and in vivo and cognitive functions of the developing brain.  

PubMed

Fish oil during early postnatal period may modulate the impact of oxidative stress in the developing brain and thus improve memory and cognitive behaviour. This study investigated the impacts of docosahexaenoic acid (DHA, C22:6, n-3) and/or phosphatidylserine (PS) on antioxidant activities in vitro, and the beneficial effects of feeding with DHA and/or PS on antioxidant activities in brain and liver tissues and on the cognitive functions of the developing brain. Results indicated that DHA and/or PS significantly enhanced antioxidant activities and increased cell viabilities in vitro. Feeding with DHA and/or PS supplementation not only significantly improved escape latency of animals, but it also improved the oxidative parameters in the brain, enhanced glutathione peroxidase activity as well as reduced nitric mono-oxide levels in the liver. DHA and PS may serve to protect cells from oxidative stress and further improve learning and memory ability in vivo. PMID:23265497

Chaung, Hso-Chi; Chang, Chin-Dong; Chen, Pi-Hang; Chang, Chia-Jung; Liu, Shyh-Hwa; Chen, Chih-Cheng

2013-05-01

213

HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro  

PubMed Central

Abstract Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-?B, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4+ T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients. PMID:22280204

Hernández, Juan C.; Stevenson, Mario; Latz, Eicke

2012-01-01

214

Enhanced control of in vivo bone formation with surface functionalized alginate microbeads incorporating heparin and human bone morphogenetic protein-2.  

PubMed

In this study, we tested the hypothesis that a surface functionalization delivery platform incorporating heparin onto strontium alginate microbeads surfaces would convert this "naive carriers" into "mini-reservoirs" for localized in vivo delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) that will induce functional bone regeneration. In vitro evaluation confirmed that (1) heparin incorporation could immobilize and prolong rhBMP-2 release for approximately 3 weeks; (2) a significant decrease (p<0.01) in rhBMP-2 burst release is attainable depending on initial protein load; and (3) rhBMP-2 released from surface functionalized microbeads retained bioactivity and stimulated higher alkaline phosphatase activity in cultured C(2)C(12) cells when compared with daily administration of fresh bolus rhBMP-2. Subsequently, surface functionalized microbeads were used for in vivo delivery of rhBMP-2 at local sites of posterolateral spinal fusion surgery in rats. The microbeads were loaded into the pores of medical-grade polyepsilone caprolactone-tricalcium phosphate scaffolds before implantation. Results revealed robust bone formation and a biomechanically solid fusion after 6 weeks. When compared with a control group consisting of an equivalent amount of rhBMP-2 that was directly adsorbed onto bare-surfaced microbeads with no heparin, a 5.3-fold increase in bone volume fraction and a 2.6-fold increase in bending stiffness (flexion/extension) were observed. When compared with collagen sponge carriers of rhBMP-2, a 1.5-fold and a 1.3-fold increase in bone volume fraction and bending stiffness were observed, respectively. More importantly, 3D micro-computed tomography images enabled the visualization of a well-contained newly formed bone at ipsilateral implant sites with surface functionalized rhBMP-2 delivery. This was absent with collagen sponge carriers where newly formed bone tissue was poorly contained and crossed over the posterior midline to contralateral implants. These findings are important because of complications with current rhBMP-2 delivery method, including excessive, uncontrolled bone formation. PMID:22894570

Abbah, Sunny Akogwu; Liu, Jing; Goh, James Cho Hong; Wong, Hee-Kit

2013-02-01

215

In Vivo Characterization of Traumatic Brain Injury Neuropathology with Structural and Functional Neuroimaging  

PubMed Central

Quantitative neuroimaging is increasingly used to study the effects of traumatic brain injury (TBI) on brain structure and function. This paper reviews quantitative structural and functional neuroimaging studies of patients with TBI, with an emphasis on the effects of diffuse axonal injury (DAI), the primary neuropathology in TBI. Quantitative structural neuroimaging has evolved from simple planometric measurements through targeted region-of-interest analyses to whole-brain analysis of quantified tissue compartments. Recent studies converge to indicate widespread volume loss of both gray and white matter in patients with moderate-to-severe TBI. These changes can be documented even when patients with focal lesions are excluded. Broadly speaking, performance on standard neuropsychological tests of speeded information processing are related to these changes, but demonstration of specific brain-behavior relationships requires more refined experimental behavioral measures. The functional consequences of these structural changes can be imaged with activation functional neuroimaging. Although this line of research is at an early stage, results indicate that TBI causes a more widely dispersed activation in frontal and posterior cortices. Further progress in analysis of the consequences of TBI on neural structure and function will require control of variability in neuropathology and behavior. PMID:17020478

LEVINE, BRIAN; FUJIWARA, ESTHER; O’CONNOR, CHARLENE; RICHARD, NADINE; KOVACEVIC, NATASA; MANDIC, MARINA; RESTAGNO, ADRIANA; EASDON, CRAIG; ROBERTSON, IAN H.; GRAHAM, SIMON J.; CHEUNG, GORDON; GAO, FUQIANG; SCHWARTZ, MICHAEL L.; BLACK, SANDRA E.

2007-01-01

216

In vitro granulocyte adherence and in vivo margination: two associated complement-dependent functions  

PubMed Central

To study mechanisms and mediators regulating the distribution of intravascular granulocytes between circulating and marginated pools, a human model with extreme transient margination, the neutropenia of continuous flow filtration leukophoresis, was analyzed. Studies in animals demonstrated the existence of a complement (C)-derived granulocytopenia-inducing factor. Thus, autologous plasma, exposed to nylon fibers (NF) of the filtration system, produced an acute selective decrement of circulating granulocytes and monocytes. This phenomenon was blocked by decomplementing plasma, by pretreatment of plasma with EDTA or hydrazine, and by preheating at 56 degrees C, but did occur after recombination of heat-inactivated and hydrazine-treated plasma before NF exposure. Preheating plasma at 50 degrees C did not inhibit the neutropenic response, suggesting involvement of the classical pathway of C activation. Ultrafiltration studies indicated that the NF-provoked neutropenia-inducing factor has a mol wt in the range of 10,000-30,000, and is heat stable (56 degrees C). To analyze the hypothesis that C- induced neutrophil margination might be consequent to increased cell adhesiveness to endothelial surfaces, the role of C in promoting granulocyte adherence was evaluated in vitro. Measured with a plastic Petridish assay, granulocyte adherence was significantly reduced in heat- inactivated (56 degrees C) and hydrazine-treated plasma, but adherence promoting capacity was restored by mixing the two plasmas, or by adding purified C3 to hydrazine-treated plasma. After exposure to activated C, neutrophils showed significantly increased adhesiveness which was maintained when cells were resuspended in heat-inactivated plasma, but progressively lost when resuspended in fresh plasma. On the basis of these results we conclude that granulocyte adhesiveness in vitro and margination in vivo are closely associated, C-dependent phenomena. PMID:894188

Fehr, J; Jacob, HS

1977-01-01

217

In vivo mitochondrial inhibition alters corticostriatal synaptic function and the modulatory effects of neurotrophins.  

PubMed

Experimental evidence has revealed the role of mitochondria in various aspects of neuronal physiology. Mitochondrial failure results in alterations that underlie the pathogeneses of many neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease (HD) and amyotrophic lateral sclerosis. The mitochondrial toxin 3-nitropropionic acid (3-NP) has been used to model failure; for example, systemic administration of 3-NP imitates the striatal degeneration that is exhibited in the postmortem tissue of patients afflicted with HD. We have demonstrated that low, sub-chronic doses of 3-NP are sufficient to initiate the damage to striatal neurons that is associated with changes in neurotrophin expression levels. However, the mechanisms underlying the alterations in neuronal activity and neurotransmission due to 3-NP-induced mitochondrial dysfunction remain to be elucidated. In this paper, we focus on how corticostriatal transmission and its modulation by neurotrophins are altered in vivo after 5 days of mitochondrial inhibition with 3-NP. Recordings of population spikes and a paired pulse (PP) stimulation protocol were used to document changes in corticostriatal synapses in 3-NP-treated brain slices. The corticostriatal synapses were modulated by neurotrophins but displayed differential amplitude increases in the presence of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) under control conditions. Neurotrophin-mediated synaptic modulation was decreased in slices from 3-NP-treated mice. The protein and mRNA levels of neurotrophins and their receptors were also modified in the 3-NP-treated tissue. Neuronal structural evaluation demonstrated that synaptic length and density were reduced in the 3-NP-treated mice, which partially explained the changes in the amplitudes of the synaptic field responses. Our results demonstrate that corticostriatal synapses are differentially modulated by neurotrophins and that this modulation is altered by mitochondrial failure. Mitochondrial dysfunction also affects neurotransmitter release in corticostriatal synapses, neurotrophin availability, dendritic arborization and the lengths of the striatal medium spiny neurons (MSNs). PMID:25241069

Mendoza, E; Miranda-Barrientos, J A; Vázquez-Roque, R A; Morales-Herrera, E; Ruelas, A; De la Rosa, G; Flores, G; Hernández-Echeagaray, E

2014-11-01

218

Follicular characteristics and luteal development after follicle-stimulating hormone induced multiple ovulations in heifers.  

PubMed

A protocol based on small doses of FSH was examined for the induction of double or triple (multiple) ovulations in cattle. Ovulation rate, follicular characteristics, and luteal responses were determined. In Exp. 1, three groups of estrous-synchronized, cyclic Holstein heifers were treated once daily, on d 3 to 6 of the cycle, with a FSH product (Folltropin-V): large FSH dose (total of 150 mg; n=18), medium FSH dose (total of 130 mg, n=12), and small FSH dose (total of 80 mg; n=7). Controls received saline (n=6). Prostaglandin F(2?) was injected on d 6, ultrasound-guided aspiration of surplus follicles (if needed) was performed on d 7, and GnRH was injected on d 8 to induce ovulation. The large FSH dose induced growth of more (2.6±0.3, P<0.05) large follicles than controls on d 8; medium and small FSH doses insufficiently stimulated growth of <2 large follicles. Ovulation rates were determined in subgroups of heifers (n=10, 13, 4, and 6, respectively). The large FSH dose induced greater rates (P<0.01) of mostly double and triple ovulations (90% multiple ovulations, 70% double ovulations), most of which (89%) were bilateral, with only 2 out of 10 heifers requiring aspiration of surplus follicles. Medium and small FSH doses induced fewer multiple ovulations (38% and 25%, respectively). Estradiol concentrations on d 8 did not differ among treatments, but the concentration per large follicle in controls was greater (P<0.05) than in FSH treatments. Mean corpus luteum (CL) volume in single-ovulation controls was greater (P<0.05) than that of multiple ovulations in the large FSH group and total CL volume and progesterone concentrations were numerically greater in multiple ovulations. In Exp. 2, the characteristics of follicles aspirated on d 7 from large FSH (n=11) and control heifers (n=10) were compared. Based on estradiol-to-progesterone ratio, 57% of the large FSH-treated follicles were classified as codominant/healthy follicles and 43% as subordinate/early atretic. Although concentrations of estradiol and androstenedione in FSH-treated codominant follicles were less (P<0.05) than in controls, estradiol-to-progesterone ratio indicated that those follicles were steroidogenically active. Finely tuned small doses of FSH administered during the first follicular wave can induce a large incidence of double/triple, mainly bilateral, ovulations in cattle, which may serve as a basis for treatment aimed at promoting twinning in beef cattle. PMID:23097398

Glick, G; Hogeg, M; Moallem, U; Lavon, Y; Wolfenson, D

2013-01-01

219

Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages  

PubMed Central

Class II major histocompatibility complex (Ia)-bearing dendritic cells (DC) from airway epithelium and lung parenchyma express low-moderate antigen presenting cell (APC) activity when freshly isolated. However, this function is markedly upregulated during overnight culture in a manner analogous to epidermal Langerhans cells. The in vitro "maturation" process is inhibited by coculture with pulmonary alveolar macrophages (PAM) across a semipermeable membrane, and the degree of inhibition achieved can be markedly increased by the presence of tumor necrosis factor alpha. In addition, PAM-mediated suppression of DC function is abrogated via inhibition of the nitric oxide synthetase pathway. Functional maturation of the DC is accompanied by increased expression of surface Ia, which is also inhibited in the presence of PAM. Prior elimination of PAM from DC donors via intratracheal administration of the cytotoxic drug dichloromethylene diphosphonate in liposomes, 24-72 h before lung DC preparation, achieves a comparable upregulation of APC activity, suggesting that (consistent with the in vitro data) the resident PAM population actively suppresses the APC function of lung DC in situ. In support of the feasibility of such a regulatory mechanism, electron microscopic examination of normal lung fixed by intravascular perfusion in the inflated state (which optimally preserves PAM in situ), revealed that the majority are preferentially localized in recesses at the alveolar septal junctions. In this position, the PAM are in intimate association with the alveolar epithelial surface, and are effectively separated by as little as 0.2 microns from underlying interstitial spaces which contain the peripheral lung DC population. A similar juxtaposition of airway intraepithelial DC is demonstrated with underlying submucosal tissue macrophages, where the separation between the two cell populations is effectively the width of the basal lamina. PMID:8426110

1993-01-01

220

Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo  

PubMed Central

Selective targeting of cancer cells is a critical step in cancer diagnosis and therapy. To address this need, DNA aptamers have attracted significant attention as possible targeting ligands. However, while their use in targeting cancer cells in vitro has been reported, their effectiveness has rarely been established in vivo. Here we report the development of a liposomal drug delivery system for targeted anticancer chemotherapy. Liposomes were prepared containing doxorubicin as a payload, and functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin. AS1411 aptamer-functionalized liposomes increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as compared to non-targeting liposomes. Furthermore, targeted liposomal doxorubicin improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice, attributable to their enhanced tumor tissue penetration. This study suggests that AS1411 aptamer-functionalized liposomes can recognize nucleolin overexpressed on MCF-7 cell surface, and therefore enable drug delivery with high specificity and selectivity. PMID:24159374

Xing, Hang; Tang, Li; Yang, Xujuan; Hwang, Kevin; Wang, Wendan; Yin, Qian; Wong, Ngo Yin; Dobrucki, Lawrence W.; Yasui, Norio; Katzenellenbogen, John A.; Helferich, William G.; Cheng, Jianjun; Lu, Yi

2013-01-01

221

Body Size in Relation to Urinary Estrogens and Estrogen Metabolites (EM) among Premenopausal Women during the Luteal Phase  

PubMed Central

Estrogen metabolism profiles may play an important role in the relationship between body size and breast carcinogenesis. Previously, we observed inverse associations between current body mass index (BMI) and plasma levels of parent estrogens (estrone and estradiol) among premenopausal women during both follicular and luteal phases. Using data from the Nurses’ Health Study II (NHS II), we assessed whether height, current BMI, and BMI at age 18 were associated with the urinary concentrations of 15 estrogens and estrogen metabolites (jointly referred to as EM) measured during the luteal phase among 603 premenopausal women. We observed inverse associations with total EM for height (Ptrend=0.01) and current BMI (Ptrend=0.01), but not BMI at age 18 (Ptrend=0.26). Six EMs were 18–27% lower in women with a height 68+ inches versus ?62 inches, primarily in the methylated catechol pathway (Ptrend=0.04). Eight EMs were 18–50% lower in women with a BMI of 30+ versus <20, primarily in the 2-catechol and methylated catechol pathways (Ptrend<0.001 for both). Our results suggest that height and current BMI are associated with estrogen metabolism profiles in premenopausal women. Further studies with timed urine and blood collections are required to confirm and extend our findings. PMID:23011724

Xie, Jing; Eliassen, A. Heather; Xu, Xia; Matthews, Charles E.; Hankinson, Susan E.; Ziegler, Regina G.; Tworoger, Shelley S.

2012-01-01

222

GnRH agonist plus vaginal progesterone for luteal phase support in ICSI cycles: a randomized study.  

PubMed

In this prospective randomized study, the effect of daily gonadotrophin-releasing hormone agonist (GnRHa) in the luteal phase on IVF and intracytoplasmic sperm injection (ICSI) outcomes was assessed. Women (n = 446) were counselled for IVF-ICSI, and randomized on the day of embryo transfer to group 1 (daily 0.1?mg subcutaneous GnRHa until day of beta-HCG) (n = 224) and group 2 (stopped GnRHa on day of HCG injection) (n = 222). Both groups received daily vaginal progesterone suppositories. Primary outcome was clinical pregnancy rate. Secondary outcome was ongoing pregnancy rate beyond 20 weeks. Mean age, oestradiol on day of HCG, number of oocytes retrieved, number of embryos transferred, and clinical and ongoing pregnancy rates were 28.9 ± 4.5 years, 2401 ± 746?pg/mL; 13.5 ± 6.0 oocytes; 2.6 ± 0.6 embryos, and 36.2% and 30.4% consecutively in group 1 compared with 29.7 ± 4.7 years, 2483 ± 867?pg/mL, 13.7 ± 5.5 oocytes, 2.7 ± 0.6 embryos, 30.6% pregnancy rate, and 25.7% ongoing pregnancy rate in group 2. No significant difference was found between the groups. Subcutaneous GnRHa during the luteal phase of long GnRHa protocol cycles does not increase clinical or ongoing pregnancy rates after IVF-ICSI. PMID:25456166

Aboulghar, Mohamed A; Marie, Heba; Amin, Yahia M; Aboulghar, Mona M; Nasr, Ahmed; Serour, Gamal I; Mansour, Ragaa T

2015-01-01

223

An approach to the functional anatomy of the sacroiliac joints in vivo  

Microsoft Academic Search

Summary This first part of this paper is a review of the literature on the functional anatomy of the sacroiliac joint followed by a preliminary biomechanical study of the fresh post mortem pelvis. The latter was done in order to determine the coefficients of the screw matrix and the position of the instantaneous centers of rotation during the symmetrical movements

B. Lavignolle; J. M. Vital; J. Senegas; J. Destandau; B. Toson; P. Bouyx; P. Morlier; G. Delorme; A. Calabet

1983-01-01

224

The human protein Hugl-1 substitutes for Drosophila Lethal giant larvae tumour suppressor function in vivo  

Microsoft Academic Search

Drosophila lethal giant larvae (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and

Daniela Grifoni; Flavio Garoia; Christoph C Schimanski; Gösta Schmitz; Elisa Laurenti; Peter R Galle; Annalisa Pession; Sandro Cavicchi; Dennis Strand

2004-01-01

225

In vivo P-glycoprotein function before and after epilepsy surgery  

PubMed Central

Objectives: To study the functional activity of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier of patients with temporal lobe epilepsy using (R)-[11C]verapamil (VPM)-PET before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in Pgp function. Methods: Seven patients with drug-resistant temporal lobe epilepsy underwent VPM-PET scans pre- and postsurgery. Patients were followed up for a median of 6 years (range 4–7) after surgery. Pgp immunoreactivity in surgically resected hippocampal specimens was determined with immunohistochemistry. Results: Optimal surgical outcome, defined as seizure freedom and withdrawal of antiepileptic drugs, was associated with higher temporal lobe Pgp function before surgery, higher Pgp-positive staining in surgically resected hippocampal specimens, and reduction in global Pgp function postoperatively, compared with nonoptimal surgery outcome. Conclusions: The data from our pilot study suggest that Pgp overactivity in epilepsy is dynamic, and complete seizure control and elimination of antiepileptic medication is associated with reversal of overactivity, although these findings will require confirmation in a larger patient cohort. PMID:25186858

Bauer, Martin; Karch, Rudolf; Zeitlinger, Markus; Liu, Joan; Koepp, Matthias J.; Asselin, Marie-Claude; Sisodiya, Sanjay M.; Hainfellner, Johannes A.; Wadsak, Wolfgang; Mitterhauser, Markus; Müller, Markus; Pataraia, Ekaterina

2014-01-01

226

Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups.  

PubMed

We have developed a generalized profile that identifies members of the root-nodulation-cell-division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z-score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild-type to oxidative stress-inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family. PMID:21255364

Godoy, Patricia; Molina-Henares, Antonio J; de la Torre, Jesús; Duque, Estrella; Ramos, Juan L

2010-11-01

227

Lack of Functionally-Active Sweet Taste Receptors in the Jejunum in vivo in the Rat  

PubMed Central

BACKGROUND When studied in enterocyte-like cell lines (Caco-2 and RIE cells), agonists and antagonists of the sweet taste receptor (STR) augment and decrease glucose uptake, respectively. We hypothesize that exposure to STR agonists and antagonists in vivo will augment glucose absorption in the rat. MATERIAL/METHODS 30-cm segments of jejunum in anesthetized rats were perfused with iso-osmolar solutions containing 10, 35, and 100 mM glucose solutions (n=6 rats, each group) with and without the STR agonist 2 mM acesulfame potassium (AceK) and the STR inhibitor 10 ?M U-73122 (inhibitor of the PLC pathway). Carrier-mediated absorption of glucose was calculated by using stereospecific and non-stereospecific 14C-D-glucose and 3H-L-glucose, respectively. RESULTS Addition of the STR agonist AceK to the 10, 35, and 100 mM glucose solutions had no substantive effects on glucose absorption from 2.1±0.2 to 2.0±0.3, 5.8±0.2 to 4.8±0.2, and 15.5±2.3 to 15.7±2.7 ?mol/min/30-cm intestinal segment (p>0.05), respectively. Addition of the STR inhibitor (U-73122) also had no effect on absorption in the 10, 35, and 100 mM solutions from 2.3±0.1 to 2.1±0.2, 7.7±0.5 to 7.2±0.5, and 15.7±0.9 to 15.2±1.1 ?mol/min/30-cm intestinal segment, respectively. CONCLUSION Provision of glucose directly into rat jejunum does not augment glucose absorption via STR-mediated mechanisms within the jejunum in the rat. Our experiments show either no major role of STRs in mediating postprandial augmentation of glucose absorption or that proximal gastrointestinal tract stimulation of STR or other luminal factors may be required for absorption of glucose to be augmented by STR. PMID:23531453

Chaudhry, Rizwan M.; Garg, Alok; Abdelfatah, Mohamed M.; Duenes, Judith A.; Sarr, Michael G.

2013-01-01

228

Effects of systemic glucocorticosteroids on peripheral neutrophil functions in asthmatic subjects: an ex vivo study  

PubMed Central

In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10% E. coli; superoxide anion generation after PMA; leukotriene B4 (LTB4) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 – 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects (39.2 ± 14.1 vs. 25.2 ± 7.3 nmol, p < 0.05); there was a significant correlation between FEV1 (% of predicted) and neutrophil chemotaxis (r = ?0.52, p = 0.04). After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV1 increase > 20% after GCS treatment (from 131 ± 18 to 117 ± 21 ?m, p = 0.005). Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS (r = 0.57, p < 0.05). These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease. PMID:18475647

Bancalari, L.; Giannessi, D.; Bernini, W.; Lazzerini, G.; Sicari, R.; Bacci, E.; Dente, F. L.; Vagaggini, B.; Caterina, R. De

1995-01-01

229

Comparison of polyethylene tibial insert damage from in vivo function and in vitro wear simulation.  

PubMed

Function and wear of total knee arthroplasties were compared by analysis of damage patterns on polyethylene tibial inserts retrieved from patients (Group R) with inserts obtained after in vitro force-controlled knee joint wear simulation. Two simulator input profiles were evaluated, including standard walking (Group W), and combined walking and stair descent (Group W + S), simulating varied activities and a more severe physiological environment. Damage regions on all inserts were quantitatively assessed. On average, inserts in all groups had internally rotated damage patterns and the greatest articular deformation in the lateral compartment. These patterns were more pronounced in Group W + S compared to Group W. Deformation rates of simulated inserts were analogous to about six years of physiologic function. However, both groups of simulated inserts generally underestimated the magnitude of damage area and extent observed on retrieved inserts, consistent with differences in the simulator's tibiofemoral contact mechanics and those known to occur in patients during functional activities. Modification of simulator inputs, such as the increased anteroposterior excursion and more severe loading conditions in Group W + S, can generate greater wear volume, larger damage areas, and increased surface deformation rates compared to standard inputs. PMID:18932244

Harman, Melinda K; DesJardins, John; Benson, Lisa; Banks, Scott A; LaBerge, Martine; Hodge, W Andrew

2009-04-01

230

Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays  

PubMed Central

Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling. PMID:25232310

Ueno, Taro; Kume, Kazuhiko

2014-01-01

231

FAM20C functions intracellularly within both ameloblasts and odontoblasts in vivo  

PubMed Central

FAM20C, also known as Golgi Casein Kinase (G-CK), is proposed to be the archetype for a family of secreted kinases that phosphorylate target proteins in the Golgi and in extracellular matrices, but FAM20C serving an extracellular function is controversial. FAM20C phosphorylates secretory calcium-binding phosphoproteins (SCPPs), which are associated with the evolution of biomineralization in vertebrates. Current models of biomineralization assume SCPP proteins are secreted as phosphoproteins and their phosphates are essential for protein conformation and function. It would be a radical departure from current theories if proteins in mineralizing matrices were dephosphorylated as part of the mineralization mechanism and rephosphorylated in the extracellular milieu by FAM20C using ATP. To see if such mechanisms are possible in the formation of dental enamel, we tested the hypothesis that FAM20C is secreted by ameloblasts and accumulates in the enamel extracellular matrix during tooth development. FAM20C localization was determined by immunohistochemistry in Day 5 mouse incisors and molars and by Western blot analyses of proteins extracted from pig enamel organ epithelia (EOE) and enamel shavings. FAM20C localized intracellularly within ameloblasts and odontoblasts in a pattern consistent with Golgi localization. Western blots detected FAM20C in the EOE extracts but not in the enamel matrix. We conclude that FAM20C is not a constituent of the enamel extracellular matrix and functions intracellularly within ameloblasts. PMID:23703840

Wang, Shih-Kai; Samann, Andrew C.; Hu, Jan C-C.; Simmer, James P.

2013-01-01

232

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas  

PubMed Central

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in Cas substrate domain. These tyrosine phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas-Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-lengh Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects. PMID:20603213

Roselli, Severine; Wallez, Yann; Wang, Lei; Vervoort, Virginie; Pasquale, Elena B

2010-01-01

233

Hepatic sirtuin 1 is dispensable for fibrate-induced peroxisome proliferator-activated receptor-? function in vivo.  

PubMed

Peroxisome proliferator-activated receptor-? (PPAR?) mediates metabolic remodeling, resulting in enhanced mitochondrial and peroxisomal ?-oxidation of fatty acids. In addition to the physiological stimuli of fasting and high-fat diet, PPAR? is activated by the fibrate class of drugs for the treatment of dyslipidemia. Sirtuin 1 (SIRT1), an important regulator of energy homeostasis, was downregulated in fibrate-treated wild-type mice, suggesting PPAR? regulation of Sirt1 gene expression. The impact of SIRT1 loss on PPAR? functionality in vivo was assessed in hepatocyte-specific knockout mice that lack the deacetylase domain of SIRT1 (Sirt1(?Liv)). Knockout mice were treated with fibrates or fasted for 24 h to activate PPAR?. Basal expression of the PPAR? target genes Cyp4a10 and Cyp4a14 was reduced in Sirt1(?Liv) mice compared with wild-type mice. However, no difference was observed between wild-type and Sirt1(?Liv) mice in either fasting- or fibrate-mediated induction of PPAR? target genes. Similar to the initial results, there was no difference in fibrate-activated PPAR? gene induction. To assess the relationship between SIRT1 and PPAR? in a pathophysiological setting, Sirt1(?Liv) mice were maintained on a high-fat diet for 14 wk, followed by fibrate treatment. Sirt1(?Liv) mice exhibited increased body mass compared with control mice. In the context of a high-fat diet, Sirt1(?Liv) mice did not respond to the cholesterol-lowering effects of the fibrate treatment. However, there were no significant differences in PPAR? target gene expression. These results suggest that, in vivo, SIRT1 deacetylase activity does not significantly impact induced PPAR? activity. PMID:24496310

Bonzo, Jessica A; Brocker, Chad; Jiang, Changtao; Wang, Rui-Hong; Deng, Chu-Xia; Gonzalez, Frank J

2014-04-01

234

Creation of Nonischemic Functional Mitral Regurgitation by Annular Dilatation and Nonplanar Modification in a Chronic In Vivo Swine Model  

PubMed Central

Background Mechanisms and treatments of nonischemic functional mitral regurgitation (NIMR) are not fully established in part due to a lack of proper large animal models. We developed a novel technique of NIMR creation in a swine model by making multiple small incisions in the mitral annulus. Methods and Results Ex-vivo experiments using isolated swine hearts (n=10) showed a 15% increase in annular area (6.8 to 7.8cm2) after 16 incisions were made along the posterior mitral annulus of a pressurized left ventricle (LV). In an in vivo swine model (n=7, 46.4±2.2kg) NIMR was created by making 14-26 2mm incisions in the atrial aspect of the mitral annulus using a cardioport video-assisted imaging system in the beating heart. Animals were sacrificed at 4 weeks (n=4) and 6 weeks (n=3). Three-dimensional (3D) echocardiography was obtained before and immediately after NIMR creation, and at euthanasia; vena contracta area (VCA), mitral annular dimension, LV volume, and inter-papillary muscle distance were measured. The mitral annular incisions resulted in mild-moderate mitral regurgitation and an increased VCA. NIMR creation altered mitral valve (MV) geometry by decreasing mitral annular nonplanarity and increasing annular area, primarily in the anteroposterior dimension. NIMR creation did not significantly change LV volume or inter-papillary muscle distance. Longer follow-up period did not significantly affect these outcomes. Conclusions NIMR can successfully be created in a beating-heart swine model and results in dilatation and 3D changes in mitral annular geometry. This model can enhance the experimental validation of new valve repair devices and techniques. PMID:24030417

Yamauchi, Haruo; Feins, Eric N.; Vasilyev, Nikolay V.; Shimada, Shogo; Zurakowski, David; del Nido, Pedro J.

2013-01-01

235

Lack of in vitro effect of aglepristone on IFN-? and IL-4 production by resting and mitogen-activated T cells of luteal bitches  

PubMed Central

Background Aglepristone (RU534) is an antiprogestin used for pregnancy termination, parturition induction and conservative pyometra treatment in bitches. Its molecular structure is similar to mifepristone, an antiprogestin used in human medicine. Mifepristone has been shown to suppress proliferation and cytokine production by T cells, whereas the effect of aglepristone on T cell function remains elusive. The purpose of this project was to investigate the in vitro influence of RU534 on IFN-? and IL-4 synthesis by peripheral blood T cells isolated from healthy bitches (N = 16) in luteal phase. The peripheral blood mononuclear cells (PBMCs) were incubated with three different dosages of aglepristone, or dimethyl sulfoxide (DMSO), with or without mitogen. The production of cytokines by resting or mitogen-activated T cells was determined by intercellular staining and flow cytometry analysis or ELISA assay, respectively. Results Our results showed no statistically significant differences in the percentage of IFN-? and IL-4-synthesizing CD4+ or CD8+ resting T cells between untreated and aglepristone-treated cells at 24 and 48 hours post treatment. Moreover, mitogen-activated PBMCs treated with RU534 displayed similar concentration of IFN-? and IL-4 in culture supernatants to those observed in mitogen-activated DMSO-treated PBMCs. Presented results indicate that administration of aglepristone for 48 hours has no influence on IFN-? and IL-4 synthesis by resting and mitogen-activated T cells isolated from diestral bitches. Conclusions We conclude that antiprogestins may differentially affect T cell function depending on the animal species in which they are applied. PMID:24284004

2013-01-01

236

In Vivo Assessment of Endothelial Function in Human Lower Extremity Arteries  

PubMed Central

Objective Endothelial function has been measured in preclinical studies, in human brachial and coronary arteries, but not in lower extremity arteries affected by atherosclerosis. We describe a novel, first-in-man, evaluation of endothelial function of the superficial femoral arteries (SFA) in patients with peripheral arterial disease (PAD). Methods Patients with PAD (n=25) requiring lower extremity angiography were enrolled. Endothelial dependent relaxation (EDR) was measured using intravascular ultrasound and a Doppler Flow wire after the infusion of acetylcholine (Ach). IVUS derived virtual histology (IVUS-VH) of the same vessel was calculated. Endothelial independent relaxation (EIR) was measured with infusion of nitroglycerin (NTG, 200 µg). Levels of nitric oxide (NOx) and serum metabolites were determined by laboratory analysis. Results Patients (mean age 62, 48% male) had a history of hypertension (80%), coronary disease (36%), and diabetes (40%). The mean SFA diameter was 5.2 ± 1 mm (range 3.2–6.9 mm). Patients tolerated Ach infusion with no side effects or adverse events. EDR increased over baseline for all patients with Ach infusion 10?6-10?4. Diameter (0.5% at Ach 10?4) and area (1.8% at Ach 10?4) changes in the diseased SFA were modest and insignificant. But, average peak velocity of blood flow (APV) significantly increased 26, 46 and 63% with Ach infusion 10?6-10?4. Calculations of limb volumetric flow (Q, mL/s, 68%, Ach 10?4) were significantly increased after Ach infusion. Lower extremity NOx levels were slightly lower than systemic venous levels (P = .04). NTG infusion indicated normal smooth muscle responsiveness (3% diameter, 9% area, and 116% velocity change over baseline). IVUS-VH plaque stratification indicated predominantly fibrous morphology (46%; necrotic core, 29%; calcium, 18%). Atheroma burden was 14.9 ± 5.5 mm3/cm and did not correlate with endothelial responsiveness. Conclusions Endothelial function can be measured directly in human lower extremity arteries at the sites of vascular disease. Despite extensive atherosclerosis, endothelial function is still intact. These data support the application of regional endothelial-specific biological therapies in patients with PAD. PMID:23830159

Kashyap, Vikram S.; Lakin, Ryan O.; Feiten, Lindsay E.; Bishop, Paul; Sarac, Timur P.

2013-01-01

237

Delta-catenin is required for the maintenance of neural structure and function in mature cortex in vivo  

PubMed Central

Delta (™)-catenin is a brain specific member of the adherens junction complex that localizes to the post-synaptic and dendritic compartments. This protein is likely critical for normal cognitive function; its hemizygous loss is linked to the severe mental retardation syndrome, Cri-du-Chat, and it directly interacts with Presenilin-1 (PS1), the protein most frequently mutated in familial Alzheimer's disease. Mice lacking normal ™-catenin display severe impairments in learning and memory tasks and synaptic plasticity. Here we examine dendritic structure and cortical function in vivo in mice lacking ™-catenin. We find that in cerebral cortex of 5-week-old mice dendritic complexity, spine density, and cortical responsiveness are similar between mutant and littermate controls; thereafter, mutant mice experience progressive dendritic retraction, a reduction in spine density and stability, and concomitant reductions in cortical responsiveness. Our results indicate that ™-catenin regulates the maintenance of dendrites and dendritic spines in mature cortex but does not appear to be necessary for the initial establishment of these structures during development. PMID:19914181

Matter, Cheryl; Pribadi, Mochtar; Liu, Xin; Trachtenberg, Joshua T.

2009-01-01

238

Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo  

PubMed Central

Stem cell factor (SCF), also known as mast cell growth factor, kit ligand, and steel factor, is the ligand for the tyrosine kinase receptor (SCFR) that is encoded by the c-kit proto-oncogene. We analyzed the effects of recombinant human SCF (r-hSCF, 5-50 micrograms/kg/day, injected subcutaneously) on mast cells and melanocytes in a phase I study of 10 patients with advanced breast carcinoma. A wheal and flare reaction developed at each r-hSCF injection site; by electron microscopy, most dermal mast cells at these sites exhibited extensive, anaphylactic-type degranulation. A 14-d course of r-hSCF significantly increased dermal mast cell density at sites distant to those injected with the cytokine and also increased both urinary levels of the major histamine metabolite, methyl- histamine, and serum levels of mast cell alpha-tryptase. Five subjects developed areas of persistent hyperpigmentation at r-hSCF injection sites; by light microscopy, these sites exhibited markedly increased epidermal melanization and increased numbers of melanocytes. The demonstration that r-hSCF can promote both the hyperplasia and the functional activation of human mast cells and melanocytes in vivo has implications for our understanding of the role of endogenous SCF in health and disease. These findings also indicate that the interaction between SCF and its receptor represents a potential therapeutic target for regulating the numbers and functional activity of both mast cells and cutaneous melanocytes. PMID:8676090

1996-01-01

239

A USPL functional system with articulated mirror arm for in-vivo applications in dentistry  

NASA Astrophysics Data System (ADS)

Ultra-short pulsed laser (USPL) systems for dental application have overcome many of their initial disadvantages. However, a problem that has not yet been addressed and solved is the beam delivery into the oral cavity. The functional system that is introduced in this study includes an articulated mirror arm, a scanning system as well as a handpiece, allowing for freehand preparations with ultra-short laser pulses. As laser source an Nd:YVO4 laser is employed, emitting pulses with a duration of tp < 10 ps at a repetition rate of up to 500 kHz. The centre wavelength is at 1064 nm and the average output power can be tuned up to 9 W. The delivery system consists of an articulated mirror arm, to which a scanning system and a custom made handpiece are connected, including a 75 mm focussing lens. The whole functional system is compact in size and moveable. General characteristics like optical losses and ablation rate are determined and compared to results employing a fixed setup on an optical table. Furthermore classical treatment procedures like cavity preparation are being demonstrated on mammoth ivory. This study indicates that freehand preparation employing an USPL system is possible but challenging, and accompanied by a variety of side-effects. The ablation rate with fixed handpiece is about 10 mm3/min. Factors like defocussing and blinding affect treatment efficiency. Laser sources with higher average output powers might be needed in order to reach sufficient preparation speeds.

Schelle, Florian; Meister, Jörg; Dehn, Claudia; Oehme, Bernd; Bourauel, Christoph; Frentzen, Mathias

240

Phenotyping mouse pulmonary function in vivo with the lung diffusing capacity.  

PubMed

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models. PMID:25590416

Limjunyawong, Nathachit; Fallica, Jonathan; Ramakrishnan, Amritha; Datta, Kausik; Gabrielson, Matthew; Horton, Maureen; Mitzner, Wayne

2015-01-01

241

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial Antigen Presenting Cells (aAPC)  

PubMed Central

Summary Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial Antigen Presenting Cells (aAPC) to reliably expand antigen specific CTL. However for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and in treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth. PMID:18563409

Durai, Malarvizhi; Krueger, Christine; Ye, Zhaohui; Cheng, Linzhao; Mackensen, Andreas; Oelke, Mathias; Schneck, Jonathan P

2009-01-01

242

EFFECT OF OIL COMBUSTION PARTICLE BIOAVAILABLE CONSTITUENTS ON EX VIVO VASCULAR FUNCTION OF AORTAS RECOVERED FROM NORMAL AND TYPE 2 DIABETIC RATS  

EPA Science Inventory

Effect of Oil Combustion Particle Bioavailable Constituents on Ex Vivo Vascular Function of Aortae Recovered from Healthy and Early Type 2 Diabetic Rats KL Dreher1, SE Kelly2, SD Proctor2, and JC Russell2. 1National Health and Environmental Effects Laboratory, US EPA, RTP, NC;...

243

A model genetic system for testing the in vivo function of peptide toxins.  

PubMed

We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a. PMID:17141372

Tedford, Hugo W; Maggio, Francesco; Reenan, Robert A; King, Glenn

2007-01-01

244

In Vivo Approaches to Dissecting the Function of RNA Helicases in Eukaryotic Ribosome Assembly  

PubMed Central

In eukaryotes, ribosome biogenesis involves the nucleolar transcription and processing of pre-ribosomal RNA molecules (pre-rRNA) in a complex pathway requiring the participation of myriad protein and ribonucleoprotein factors. Through efforts aimed at categorizing and characterizing these factors, at least 20 RNA helicases have been shown to interact with or participate in the activities of the major ribosome biogenesis complexes. Unfortunately, little is known about the enzymatic properties of most of these helicases, and less is known about their roles in ribosome biogenesis and pre-rRNA maturation. This chapter presents approaches for characterizing RNA helicases involved in ribosome biogenesis. Included are methods for depletion of specific protein targets, with standard protocols for assaying the typical ribosome biogenesis defects that may result. Procedures and rationales for mutagenic studies of target proteins are discussed, as well as several approaches for identifying protein–protein interactions in order to determine functional context and potential cofactors of RNA helicases. PMID:22713326

Rawling, David C.; Baserga, Susan J.

2013-01-01

245

Development of optical neuroimaging to detect drug-induced brain functional changes in vivo  

NASA Astrophysics Data System (ADS)

Deficits in prefrontal function play a crucial role in compulsive cocaine use, which is a hallmark of addiction. Dysfunction of the prefrontal cortex might result from effects of cocaine on neurons as well as from disruption of cerebral blood vessels. However, the mechanisms underlying cocaine's neurotoxic effects are not fully understood, partially due to technical limitations of current imaging techniques (e.g., PET, fMRI) to differentiate vascular from neuronal effects at sufficiently high temporal and spatial resolutions. We have recently developed a multimodal imaging platform which can simultaneously characterize the changes in cerebrovascular hemodynamics, hemoglobin oxygenation and intracellular calcium fluorescence for monitoring the effects of cocaine on the brain. Such a multimodality imaging technique (OFI) provides several uniquely important merits, including: 1) a large field-of-view, 2) high spatiotemporal resolutions, 3) quantitative 3D imaging of the cerebral blood flow (CBF) networks, 4) label-free imaging of hemodynamic changes, 5) separation of vascular compartments (e.g., arterial and venous vessels) and monitoring of cortical brain metabolic changes, 6) discrimination of cellular (neuronal) from vascular responses. These imaging features have been further advanced in combination with microprobes to form micro-OFI that allows quantification of drug effects on subcortical brain. In addition, our ultrahigh-resolution ODT (?ODT) enables 3D microangiography and quantitative imaging of capillary CBF networks. These optical strategies have been used to investigate the effects of cocaine on brain physiology to facilitate the studies of brain functional changes induced by addictive substance to provide new insights into neurobiological effects of the drug on the brain.

Du, Congwu; Pan, Yingtian

2014-03-01

246

Assessing human 5-HT function in vivo with pharmacoMRI.  

PubMed

A number of novel ways of using magnetic resonance imaging (MRI) to visualise the action of drugs on animal and human brain (pharmacoMRI or phMRI) are becoming established tools in translational psychopharmacology. Using drugs with known pharmacology it is possible to investigate how neurotransmitter systems are involved in neural systems engaged by other processes, such as cognitive challenge (modulation phMRI) or to examine the acute effects of the drug itself in the brain (challenge phMRI). In this article we discuss the principles behind phMRI and review studies investigating the effect of serotonin (5-HT) manipulations. 5-HT modulation phMRI studies show the involvement of 5-HT in a broad range of neural processes ranging from motor function through 'cold' cognition, such as memory and response inhibition, to emotional processing. We highlight findings in brain areas that show some consistency or complementarity across studies, such as the ventrolateral orbitofrontal cortex where modulation by 5-HT is task-specific, and the amygdala in emotional processing where 5-HT is predominantly inhibitory. 5-HT challenge phMRI is promising but as yet few studies have been carried out. New ways of analysing phMRI data include connectivity analysis which holds the promise of going beyond identifying isolated areas of activation/modulation to understanding functional circuits and their neurochemistry. 5-HT phMRI now needs to be taken into patient populations and methods of investigating treatment effects need to be developed. If this is successful then phMRI will provide a genuinely exciting opportunity for the rapid development of better treatments for psychiatric conditions. PMID:18621068

Anderson, I M; McKie, S; Elliott, R; Williams, S R; Deakin, J F W

2008-11-01

247

Brain basis of early parent–infant interactions: psychology, physiology, and in vivo functional neuroimaging studies  

PubMed Central

Parenting behavior critically shapes human infants’ current and future behavior. The parent–infant relationship provides infants with their first social experiences, forming templates of what they can expect from others and how to best meet others’ expectations. In this review, we focus on the neurobiology of parenting behavior, including our own functional magnetic resonance imaging (fMRI) brain imaging experiments of parents. We begin with a discussion of background, perspectives and caveats for considering the neurobiology of parent–infant relationships. Then, we discuss aspects of the psychology of parenting that are significantly motivating some of the more basic neuroscience research. Following that, we discuss some of the neurohormones that are important for the regulation of social bonding, and the dysregulation of parenting with cocaine abuse. Then, we review the brain circuitry underlying parenting, proceeding from relevant rodent and nonhuman primate research to human work. Finally, we focus on a study-by-study review of functional neuroimaging studies in humans. Taken together, this research suggests that networks of highly conserved hypothalamic–midbrain–limbic–paralimbic–cortical circuits act in concert to support aspects of parent response to infants, including the emotion, attention, motivation, empathy, decision-making and other thinking that are required to navigate the complexities of parenting. Specifically, infant stimuli activate basal forebrain regions, which regulate brain circuits that handle specific nurturing and caregiving responses and activate the brain’s more general circuitry for handling emotions, motivation, attention, and empathy – all of which are crucial for effective parenting. We argue that an integrated understanding of the brain basis of parenting has profound implications for mental health. PMID:17355399

Swain, James E.; Lorberbaum, Jeffrey P.; Kose, Samet; Strathearn, Lane

2015-01-01

248

Accelerated endometrial maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of gonadotropin-releasing hormone agonists versus antagonists  

Microsoft Academic Search

ObjectiveTo evaluate the endometrium obtained during the luteal phase of controlled ovarian hyperstimulation (COH) cycles utilizing gonadotropin-releasing hormone (GnRH) antagonists, and to compare these findings with those obtained in cycles utilizing a GnRH agonist and with artificial cycles among recipients.

Peyman Saadat; Robert Boostanfar; Cristin C Slater; David E Tourgeman; Frank Z Stanczyk; Richard J Paulson

2004-01-01

249

Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.  

PubMed

Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary PMID:23971190

Ziolkowska, A; Mlynarczuk, J; Kotwica, J

2013-01-01

250

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows.  

PubMed

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day-10 (D-10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N = 42) or not (small follicle-small CL group; SF-SCL; N = 41) on D-10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 ± 0.33 mm vs. 10.76 ± 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 ± 0.28 pg/mL vs. 1.27 ± 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 ± 0.25 ng/mL vs. 2.62 ± 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes. PMID:24507960

Mesquita, F S; Pugliesi, G; Scolari, S C; França, M R; Ramos, R S; Oliveira, M; Papa, P C; Bressan, F F; Meirelles, F V; Silva, L A; Nogueira, G P; Membrive, C M B; Binelli, M

2014-04-01

251

Structural basis for the MukB-topoisomerase IV interaction and its functional implications in vivo  

PubMed Central

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB–topo IV complex to 2.3?Å resolution. The structure shows that the so-called ‘hinge' region of MukB forms a heterotetrameric assembly with a C-terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement. PMID:24097060

Vos, Seychelle M; Stewart, Nichole K; Oakley, Martha G; Berger, James M

2013-01-01

252

Dynamic noninvasive monitoring of renal function in vivo by fluorescence lifetime imaging  

NASA Astrophysics Data System (ADS)

Kidneys normally filter the blood of excess salts and metabolic products, such as urea, while retaining plasma proteins. In diseases such as multiple myeloma and diabetes mellitus, the renal function is compromised and protein escapes into the urine. In this study, we present the use of fluorescence lifetime imaging (FLI) to image excess serum protein in urine (proteinuria). The near-infrared fluorescent dye LS-288 has distinct lifetimes when bound to protein versus free in solution, providing contrast between the protein-rich viscera and the mostly protein-free bladder. FLI with LS-288 in mice revealed that fluorescence lifetime (FLT) differences in the bladder relative to surrounding tissues was due to the fractional contributions of the bound and unbound dye molecules. The FLT of LS-288 decreased in the case of proteinuria while fluorescence intensity was unchanged. The results show that FLI can be useful for the dynamic imaging of protein-losing nephropathy due to diabetes mellitus and other renal diseases and suggest the potential use of the FLI to distinguish tumors from fluid-filled cysts in the body.

Goiffon, Reece J.; Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Achilefu, Samuel

2009-03-01

253

Formulation of Functionalized PLGA-PEG Nanoparticles for In Vivo Targeted Drug Delivery  

PubMed Central

Nanoparticle (NP) size has been shown to significantly effect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly (d,l-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs, including: 1) polymer concentration, 2) drug loading, 3) water miscibility of solvent, and 4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient = 0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the Prostate Specific Membrane Antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of PCa. The surface functionalization of NPs with the A10 PSMA aptamer significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA aptamer (a 3.77-fold increase at 24 hrs; NP-Apt 0.83% ± 0.21% vs. NP 0.22% ± 0.07% of injected dose per gram of tissue; mean ± s.d., n = 4, p = 0.002). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies. PMID:17055572

Cheng, Jianjun; Teply, Benjamin A.; Sherifi, Ines; Sung, Josephine; Luther, Gaurav; Gu, Frank X.; Levy-Nissenbaum, Etgar; Radovic-Moreno, Aleksandar F.; Langer, Robert; Farokhzad, Omid C.

2009-01-01

254

Interaction between Saccharomyces cerevisiae glutaredoxin 5 and SPT10 and their in vivo functions.  

PubMed

Glutaredoxin 5 (Grx5) is a monothiol member of the Grx family that comprises two dithiol and three monothiol members. Using a yeast two-hybrid system, we isolated a Grx5-binding protein, SPT10, which has been previously suggested to act as a global transcriptional regulator of specific histone genes. We find that among the five members of the Grx family and two members of the thioredoxin (Trx) family (Trx1 and Trx2), Grx5 alone interacts with SPT10 via an intermolecular disulfide linkage between Cys60 of Grx5 and Cys385 of SPT10. To evaluate the physiological function of the Grx5/SPT10 interaction, we investigated the phenotypes of three null mutant strains (Grx5?, SPT10?, and Grx5?SPT10?). Taken together, the results show that all of these phenotypes are probably a consequence of the disruption of the interaction between Grx5 and SPT10. From this study, we suggest an interaction between Grx5 and SPT10 via intermolecular disulfide linkage and propose a model for a role of Grx5 in the regulation of protein expression under the control of SPT10. PMID:22326886

Oh, Young-Mee; Hong, Seung-Keun; Yeon, Jeong-Tae; Cha, Mee-Kyung; Kim, Il-Han

2012-05-01

255

Husbandry Factors and the Resumption of Luteal Activity in Open and Zero-Grazed Dairy Cows in Urban and Peri-Urban Kampala, Uganda  

PubMed Central

Contents The study investigated the influence of selected husbandry factors on interval to resumption of post-partum cyclicity among dairy cows in urban and peri-urban Kampala. A prospective study of 85 day post-partum period of 59 dairy cows in open (n = 38) and zero grazing (n = 21) systems was conducted on 24 farms. Cows of parity 1–6 were recruited starting 15–30 days post-partum. Progesterone (P4) content in milk taken at 10–12 day intervals was analysed using ELISA. The cow P4 profiles were classified into ‘normal’ (< 56 days), ‘delayed’ (> 56 days), ‘ceased’ or ‘prolonged’ (if started < 56 days but with abnormal P4 displays) resumption of luteal activity and tested for association with husbandry and cow factors. Of the 59 cows, luteal activity in 81.4% resumed normally and in 18.6%, delayed. Only 23.7% maintained regular luteal activity, while the others had ceased (10.2%), prolonged (37.3%) or unclear luteal activity (20.3%). There were no differences between open and zero-grazed cows. Milk production was higher (p < 0.05) in zero than open grazing, in urban than peri-urban and in cows fed on brew waste (p < 0.001) compared with mill products and banana peels. Results suggest that luteal activity resumes normally in a majority of cows, although only a minority experienced continued normal cyclicity once ovulation had occurred, in the two farming systems irrespective of feed supplements or water, and that supplementing with brew waste is beneficial for milk production. PMID:24930481

Kanyima, BM; Båge, R; Owiny, DO; Ntallaris, T; Lindahl, J; Magnusson, U; Nassuna-Musoke, MG

2014-01-01

256

Bisphenol A Exposure during Adulthood Causes Augmentation of Follicular Atresia and Luteal Regression by Decreasing 17?-Estradiol Synthesis via Downregulation of Aromatase in Rat Ovary  

PubMed Central

Background: Bisphenol A (BPA) has been detected in human body fluids, such as serum and ovarian follicular fluids. Several reports indicated that BPA exposure is associated with the occurrence of several female reproductive diseases resulting from the disruption of steroid hormone biosynthesis in the adult ovary. Objective: We hypothesized that long-term exposure to low concentrations of BPA disrupts 17?-estradiol (E2) production in granulosa cells via an alteration of steroidogenic proteins in ovarian cells. Methods: Adult female rats received BPA for 90 days by daily gavage at doses of 0, 0.001, or 0.1 mg/kg body weight. We determined serum levels of E2, testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). We also analyzed the expressions of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3?-hydroxysteroid dehydrogenase isomerase (3?-HSD), and aromatase cytochrome P450 (P450arom) in the ovary. Results: Exposure to BPA significantly decreased E2 serum concentration, which was accompanied by augmented follicular atresia and luteal regression via increase of caspase-3–associated apoptosis in ovarian cells. After BPA exposure, P450arom and StAR protein levels were significantly decreased in granulosa cells and theca-interstitial (T-I) cells, respectively. However, P450scc and 3?-HSD protein levels remained unchanged. The increase in LH levels appeared to be associated with the decreased synthesis of T in T-I cells after BPA exposure via homeostatic positive feedback regulation. Conclusions: BPA exposure during adulthood can disturb the maintenance of normal ovarian functions by reducing E2. The steroidogenic proteins StAR and P450arom appear to be targeted by BPA. PMID:23512349

Lee, Seung Gee; Chung, Jin-Yong; Park, Ji-Eun; Oh, Seunghoon; Yoon, Yong-Dal; Yoo, Ki Soo; Yoo, Young Hyun

2013-01-01

257

ANTIOXIDANT FUNCTIONS FOR THE HEMOGLOBIN ?93 CYSTEINE RESIDUE IN ERYTHROCYTES AND IN THE VASCULAR COMPARTMENT IN VIVO  

PubMed Central

The ?93 Cysteine (?93Cys) residue of hemoglobin is conserved in vertebrates but its function in the red blood cell (RBC) remains unclear. Since this residue is present at concentrations more than two orders of magnitude higher than enzymatic components of the RBC antioxidant network, a role in the scavenging of reactive species was hypothesized. Initial studies utilizing mice that express human hemoglobin with either Cys (B93C) or Ala (B93A) at the ?93 positions, demonstrated that loss of the ?93Cys did not affect activities nor expression of established components of the RBC antioxidant network (catalase, superoxide dismutase, peroxiredoxin-2, glutathione peroxidase, GSH:GSSG ratios). Interestingly, exogenous addition to RBC of reactive species that are involved in vascular inflammation demonstrated a role for the ?93Cys in hydrogen peroxide and chloramine consumption. To simulate oxidative stress and inflammation in vivo, mice were challenged with LPS. Notably, LPS induced a greater degree of hypotension and lung injury in B93A versus B93C mice, which was associated with greater formation of RBC reactive species and accumulation of DMPO-reactive epitopes in the lung. These data suggest that the ?93Cys is an important effector within the RBC antioxidant network contributing to the modulation of tissue injury during vascular inflammation. PMID:23159546

Vitturi, Dario A.; Sun, Chiao-Wang; Harper, Victoria M; Thrash-Williams, Bessy; Cantu-Medellin, Nadiezhda; Chacko, Balu K.; Peng, Ning; Dai, Yanying; Michael Wyss, J.; Townes, Tim; Patel, Rakesh P.

2013-01-01

258

Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions  

PubMed Central

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3? end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo. PMID:19221196

Björk, Petra; Jin, ShaoBo; Zhao, Jian; Singh, Om Prakash; Persson, Jan-Olov; Hellman, Ulf

2009-01-01

259

Nature, source and function of pigments in tardigrades: in vivo raman imaging of carotenoids in Echiniscus blumi.  

PubMed

Tardigrades are microscopic aquatic animals with remarkable abilities to withstand harsh physical conditions such as dehydration or exposure to harmful highly energetic radiation. The mechanisms responsible for such robustness are presently little known, but protection against oxidative stresses is thought to play a role. Despite the fact that many tardigrade species are variously pigmented, scarce information is available about this characteristic. By applying Raman micro-spectroscopy on living specimens, pigments in the tardigrade Echiniscus blumi are identified as carotenoids, and their distribution within the animal body is visualized. The dietary origin of these pigments is demonstrated, as well as their presence in the eggs and in eye-spots of these animals, together with their absence in the outer layer of the animal (i.e., cuticle and epidermis). Using in-vivo semi-quantitative Raman micro-spectroscopy, a decrease in carotenoid content is detected after inducing oxidative stress, demonstrating that this approach can be used for studying the role of carotenoids in oxidative stress-related processes in tardigrades. This approach could be thus used in further investigations to test several hypotheses concerning the function of these carotenoids in tardigrades as photo-protective pigments against ionizing radiations or as antioxidants defending these organisms against the oxidative stress occurring during desiccation processes. PMID:23185564

Bonifacio, Alois; Guidetti, Roberto; Altiero, Tiziana; Sergo, Valter; Rebecchi, Lorena

2012-01-01

260

Loss of Tsc1 in vivo impairs hippocampal mGluR-LTD and increases excitatory synaptic function  

PubMed Central

The autism-spectrum disorder Tuberous Sclerosis Complex (TSC) is caused by mutations in the Tsc1 or Tsc2 genes whose protein products form a heterodimeric complex that negatively regulates mTOR-dependent protein translation. Although several forms of synaptic plasticity, including metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD), depend on protein translation at the time of induction, it is unknown if these forms of plasticity require signaling through the Tsc1/2 complex. To examine this possibility, we postnatally deleted Tsc1 in vivo in a subset of hippocampal CA1 neurons using viral delivery of Cre recombinase in mice. We found that hippocampal mGluR-LTD was abolished by loss of Tsc1, whereas a protein synthesis-independent form of NMDA receptor-dependent LTD was preserved. Additionally, AMPA and NMDA receptor mediated excitatory postsynaptic currents (EPSCs) and miniature spontaneous EPSC frequency were enhanced in Tsc1 KO neurons. These changes in synaptic function occurred in the absence of alterations in spine density, morphology, or pre-synaptic release probability. Our findings indicate that signaling through Tsc1/2 is required for the expression of specific forms of hippocampal synaptic plasticity as well as the maintenance of normal excitatory synaptic strength. Furthermore, these data suggest that perturbations of synaptic signaling may contribute to the pathogenesis of TSC. PMID:21677170

Bateup, Helen S.; Takasaki, Kevin T.; Saulnier, Jessica L.; Denefrio, Cassandra L.; Sabatini, Bernardo L.

2011-01-01

261

Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).  

PubMed

The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp. PMID:24120486

Olsen, Christel M; Pemula, Anand Kumar; Braaen, Stine; Sankaran, Krishnan; Rimstad, Espen

2013-11-19

262

Gold nanoparticle-DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins.  

PubMed

Although the delivery of biologically functional protein(s) into mammalian cells could be of tremendous value to biomedical research, the development of such technology has been hindered by the lack of a safe and effective delivery method. Here, we present a simple, efficient, and versatile gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system, with nanoblock-like properties, that allows any recombinant protein to be loaded without additional modifications and delivered into mammalian living systems. AuNP-Apt-based protein delivery system was able to deliver various proteins into variety of cell types in vitro without showing cytotoxicity. This AuNP-Apt system was also effective for the local and systemic targeted delivery of proteins in vivo. A local injection of the AuNP-Apt loaded with the apoptosis-inducing BIM protein efficiently inhibited the growth of xenograft tumors in mice. Furthermore, an intravenous injection of AuNP-Apt loaded with both epidermal growth factor (EGF) and BIM resulted in the targeted delivery of BIM into a xenograft tumor derived from EGF receptor-overexpressing cancer cells with no detectable systemic toxicity. Our findings show that this system can serve as an innovative platform for the development of protein-based biomedical applications. PMID:25450403

Ryou, Sang-Mi; Yeom, Ji-Hyun; Kang, Hyo Jung; Won, Miae; Kim, Jin-Sik; Lee, Boeun; Seong, Maeng-Je; Ha, Nam-Chul; Bae, Jeehyeon; Lee, Kangseok

2014-12-28

263

Endocrine control of ovarian function in dogs and other carnivores  

Microsoft Academic Search

Ovarian function in dogs is minimally but successfully evolved and adapted for fertility, and represents a basic model for examining the more complex evolution of ovarian activity in other carnivores and mammals in general. Canids are monoestrous, polytocous, spontaneous ovulators with a spontaneous luteal function producing progesterone for the duration of a normal 2-month pregnancy and unaffected by hysterectomy. They

P. W. Concannon; V. D. Castracane; M. Temple; A. Montanez

264

Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells  

PubMed Central

Background Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion. PMID:24168020

2013-01-01

265

In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression  

SciTech Connect

Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 {mu}M, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [{sup 3}H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [{sup 3}H]-digoxin polarized transport attained at 50 {mu}M of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 {mu}M) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [{sup 3}H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 {mu}g/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.

Han Yi [Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 (Singapore); Chin Tan, Theresa May [Department of Biochemistry, National University of Singapore, 18 Science Drive 4, 117543 (Singapore); Lim, Lee-Yong [Pharmacy, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009 (Australia)], E-mail: limly@cyllene.uwa.edu.au

2008-08-01

266

The gold nanoparticle size and exposure duration effect on the liver and kidney function of rats: In vivo  

PubMed Central

Nanoparticles (NPs) offer a great possibility for biomedical application, not only to deliver pharmaceutics, but also to be used as novel diagnostic and therapeutic approaches. Currently, there are no data available regarding to what extent the degree of the toxicity and the accumulation of gold nanoparticles (GNPs) are present in in vivo administration. This study aimed to address the GNP size and exposure duration effect on the liver and kidney function of rats: in vivo. Methods A total of 30 healthy male Wistar-Kyoto rats of the same age (12 weeks old) and weighing 220–240 g of King Saud University colony were used. Animals were randomly divided into groups, two GNP-treated rat groups and one control group (CG). The 50 ?l of 10 and 50 nm GNPs was intraperitoneally administered in rats for exposure duration of 3 days. Then, several biochemical parameters such as aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alanine transaminase (ALT), alkaline phosphatase (ALP), urea (UREA) and creatinine (CREA) were evaluated. Results In this study, the AST values increased with the administration of 10 and 50 nm GNPs compared with the control. The AST values significantly increased with 10 nm GNPs compared with 50 nm GNPs and control. The GGT and ALT values decreased with the administration of 10 and 50 nm GNPs compared with the control. The GGT and ALT values significantly decreased with 50 nm GNPs compared with 10 nm GNPs and control. The ALP values significantly decreased with the administration of 10 and 50 nm GNPs compared with the control. The decrease in ALP values with 10 nm GNPs was higher than those compared with 50 nm GNPs. In this study, the levels of UREA and CREA values increased in a non significant manner after the administration of 10 and 50 nm GNPs compared with the control. Conclusions This study demonstrates that the increase in the enzymes AST and the decrease in ALP are smaller GNPs (10 nm) size-dependent for exposure duration of 3 days; while the decrease in the enzymes GGT and ALT are bigger GNPs (50 nm) size-dependent. The levels of UREA and CREA values indicated no significant changes with the administration of 10 and 50 nm GNPs for exposure duration of 3 days compared with the control. The administration of 10 and 50 nm GNPs for short exposure duration of 3 days induced only significant variations with some liver enzymes while kidney showed no significant variations. This study suggests that synthesis and metabolism of GNPs as well as the protection of the liver will be more important issues for medical applications of gold-based nanomaterials in future. PMID:23961234

Abdelhalim, Mohamed Anwar K.; Abdelmottaleb Moussa, Sherif A.

2013-01-01

267

Non-Coding RNAs Regulating Morphine Function: With Emphasis on the In vivo and In vitro Functions of miR-190  

PubMed Central

Non-coding RNAs (ncRNAs), especially microRNAs, are reported to be involved in a variety of biological processes, including several processes related to drug addiction. It has been suggested that the biological functions of opioids, one typical type of addictive drugs, are regulated by ncRNAs. In the current review, we examine a variety of mechanisms through which ncRNAs could regulate ?-opioid receptor (OPRM1) activities and thereby contribute to the development of opioid addiction. Using miR-23b as an example, we present the possible ways in which ncRNA-mediated regulation of OPRM1 expression could impact opioid addiction. Using miR-190 as an example, we demonstrate the critical roles played by ncRNAs in the signal cascade from receptor to systemic responses, including the possible modulation of adult neurogenesis and in vivo contextual memory. After discussing the possible targets of ncRNAs involved in the development of opioid addiction, we summarize the mechanisms underlying the interaction between ncRNAs and opioid addiction and present suggestions for further study. PMID:22715342

Zheng, Hui; Law, Ping-Yee; Loh, Horace H.

2012-01-01

268

Affinity for, and localization of, PEG-functionalized silica nanoparticles to sites of damage in an ex vivo spinal cord injury model  

PubMed Central

Background Traumatic spinal cord injury (SCI) leads to serious neurological and functional deficits through a chain of pathophysiological events. At the molecular level, progressive damage is initially revealed by collapse of plasma membrane organization and integrity produced by breaches. Consequently, the loss of its role as a semi-permeable barrier that generally mediates the regulation and transport of ions and molecules eventually results in cell death. In previous studies, we have demonstrated the functional recovery of compromised plasma membranes can be induced by the application of the hydrophilic polymer polyethylene glycol (PEG) after both spinal and brain trauma in adult rats and guinea pigs. Additionally, efforts have been directed towards a nanoparticle-based PEG application. The in vivo and ex vivo applications of PEG-decorated silica nanoparticles following CNS injury were able to effectively and efficiently enhance resealing of damaged cell membranes. Results The possibility for selectivity of tetramethyl rhodamine-dextran (TMR) dye-doped, PEG-functionalized silica nanoparticles (TMR-PSiNPs) to damaged spinal cord was evaluated using an ex vivo model of guinea pig SCI. Crushed and nearby undamaged spinal cord tissues exhibited an obvious difference in both the imbibement and accumulation of the TMR-PSiNPs, revealing selective labeling of compression-injured tissues. Conclusions These data show that appropriately functionalized nanoparticles can be an efficient means to both 1.) carry drugs, and 2.) apply membrane repair agents where they are needed in focally damaged nervous tissue. PMID:22979980

2012-01-01

269

Application of an amine functionalized biopolymer in the colonic delivery of glycyrrhizin: a design and in vivo efficacy study.  

PubMed

In our current study, a newer amine functionalized guar gum derivative was studied for its efficacy in colonic drug delivery. Glycyrrhizic acid mono-ammonium salt was used as the model drug. Drug-loaded microparticles were formulated by ionic crosslinking using sodium tripolyphosphate. The Scanning Electron Microscopic study revealed spherical particles of sizes from 4.9 ± 3.8 ?m to 6.9 ± 3.9 ?m. The FT-IR studies presented a possible interaction between the drug and the polymer. The drug was encapsulated in amorphous form as observed from the powder X-Ray Diffraction studies. A cumulative drug release study was carried out in simulated gastric, intestinal, and colonic fluids. The cumulative drug release studies presented a burst release followed by a sustained release of the drug in simulated colonic fluid containing rat cecal contents. The drug-polymer ratio was optimised using a 3(2) factorial design by taking the amounts of glycyrrhizic acid (X1) and guar gum alkyl amine (X2) as the independant variables. The percent cumulative drug release at 240 mins (Q240), 720 mins (Q720), and at 1,440 mins (Q1440) were considered as the dependant variables. The efficacy of the optimized formulation was studied in a 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis model. The tissue's nitric oxide, malondialdehyde, and myeloperoxidase activities were found to be much lower in the microparticle-treated group compared to free drug-treated group. The histology of the colonic tissue from the treated group of animals revealed almost no infiltration of inflammatory cells in the tissue for the microparticle-treated group of animals. The synthesized amine derivative of guar gum was found to be better in vitro with a better in vivo efficacy in the colonic delivery of glycyrrhizic acid monoammonium salt and can be considered as a newer modified biopolymer for colonic drug delivery. PMID:24482776

Kumar De, Amit; Datta, Sriparna; Mukherjee, Arup

2013-12-01

270

In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients  

PubMed Central

Context: Lamin A/C (LMNA) gene variations have been reported in more than one third of genotyped families with dilated cardiomyopathy (DCM). However, the relationship between LMNA mutation and the development of DCM is poorly understood. Methods and results: We found that end stage DCM patients carrying LMNA mutations displayed either dramatic ultrastructural changes of the cardiomyocyte nucleus (D192G) or nonspecific changes (R541S). Overexpression of the D192G lamin C dramatically increased the size of intranuclear speckles and reduced their number. This phenotype was only partially reversed by coexpression of the D192G and wild type lamin C. Moreover, the D192G mutation precludes insertion of lamin C into the nuclear envelope when co-transfected with the D192G lamin A. By contrast, the R541S phenotype was entirely reversed by coexpression of the R541S and wild type lamin C. As lamin speckle size is known to be correlated with regulation of transcription, we assessed the SUMO1 distribution pattern in the presence of mutated lamin C and showed that D192G lamin C expression totally disrupts the SUMO1 pattern. Conclusion: Our in vivo and in vitro results question the relationship of causality between LMNA mutations and the development of heart failure in some DCM patients and therefore, the reliability of genetic counselling. However, LMNA mutations producing speckles result not only in nuclear envelope structural damage, but may also lead to the dysregulation of cellular functions controlled by sumoylation, such as transcription, chromosome organisation, and nuclear trafficking. PMID:16061563

Sylvius, N; Bilinska, Z; Veinot, J; Fidzianska, A; Bolongo, P; Poon, S; McKeown, P; Davies, R; Chan, K; Tang, A; Dyack, S; Grzybowski, J; Ruzyllo, W; McBride, H; Tesson, F

2005-01-01

271

Application of an Amine Functionalized Biopolymer in the Colonic Delivery of Glycyrrhizin: A Design and In Vivo Efficacy Study  

PubMed Central

In our current study, a newer amine functionalized guar gum derivative was studied for its efficacy in colonic drug delivery. Glycyrrhizic acid mono-ammonium salt was used as the model drug. Drug-loaded microparticles were formulated by ionic crosslinking using sodium tripolyphosphate. The Scanning Electron Microscopic study revealed spherical particles of sizes from 4.9 ± 3.8 ?m to 6.9 ± 3.9 ?m. The FT-IR studies presented a possible interaction between the drug and the polymer. The drug was encapsulated in amorphous form as observed from the powder X-Ray Diffraction studies. A cumulative drug release study was carried out in simulated gastric, intestinal, and colonic fluids. The cumulative drug release studies presented a burst release followed by a sustained release of the drug in simulated colonic fluid containing rat cecal contents. The drug-polymer ratio was optimised using a 32 factorial design by taking the amounts of glycyrrhizic acid (X1) and guar gum alkyl amine (X2) as the independant variables. The percent cumulative drug release at 240 mins (Q240), 720 mins (Q720), and at 1,440 mins (Q1440) were considered as the dependant variables. The efficacy of the optimized formulation was studied in a 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis model. The tissue’s nitric oxide, malondialdehyde, and myeloperoxidase activities were found to be much lower in the microparticle-treated group compared to free drug-treated group. The histology of the colonic tissue from the treated group of animals revealed almost no infiltration of inflammatory cells in the tissue for the microparticle-treated group of animals. The synthesized amine derivative of guar gum was found to be better in vitro with a better in vivo efficacy in the colonic delivery of glycyrrhizic acid monoammonium salt and can be considered as a newer modified biopolymer for colonic drug delivery. PMID:24482776

Kumar De, Amit; Datta, Sriparna; Mukherjee, Arup

2013-01-01

272

Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects  

PubMed Central

Background Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. Methodology/Principal Findings The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. Conclusions/Significance Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue. PMID:22384088

Christensen, Britt; Lundby, Carsten; Jessen, Niels; Nielsen, Thomas S.; Vestergaard, Poul F.; Møller, Niels; Pilegaard, Henriette; Pedersen, Steen B.; Kopchick, John J.; Jørgensen, Jens Otto L.

2012-01-01

273

166 in vivo model to examine the long-lasting effects of acute di-(2-ethylhexyl) phthalate (dehp) exposure on ovarian function in bovine.  

PubMed

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer. Its metabolites have been shown to have adverse effects on reproduction and development in laboratory animals. However, the mechanisms by which they induce infertility remain elusive. We established an experimental model in which lactating Holstein cows were synchronized (GnRH-PG-GnRH) and tube-fed with DEHP (100mgkg(-1) per day; n=4) or water (n=5), for 3 days. Urine and plasma samples were collected before (Day 0), during (Days 2 and 4), and after (Days 11, 19, and 24) treatment initiation. For each group, and on each day, samples were pooled and analysed to determine DEHP metabolite concentrations (MEHP, 5OH-MEHP, 5oxo-MEHP, 2cx-MMHP, 5cx-MEPP) by liquid chromatography-mass spectrometry/mass spectrometry. Incorporation of DEHP resulted in relatively high metabolite concentrations on the exposure days (acute phase; Days 1-4), which decreased dramatically through the subsequent period (chronic phase; Days >5). For example, the average plasma MEHP concentration in the control group was 0.012±0.0017µM throughout the experimental period. In the treated group, it increased to 47.7±8.9µM in the acute phase, then decreased to 0.045±0.02µM. To examine the effects on ovarian function, cows were resynchronized during the chronic phase, and monitored by ultrasonography scanner (SSD-900, Aloka, Tokyo, Japan; 7.5MHz) to classify ovarian follicular dynamics. Follicular fluids of the dominant follicles were aspirated with an ultrasonic scanner connected to a vaginal sector transducer (Pie Medical Imaging BV, Maastricht, the Netherlands; 7.5MHz). Data were analysed using JMP-7 software (SAS Institute Inc., Cary, NC, USA, 2004). Differences among treatments were analysed by one-way ANOVA followed by Student's t-test. Findings revealed the pronounced effect of DEHP on the dominant follicles. The diameter (15.7±1.8 v. 10.4±1.8mm) and growth rate (0.88±0.15 v. 0.43±0.17mmday(-1)) of the first-wave dominant follicle were higher in the control cows (P<0.05). The developmental pattern of the second-wave dominant follicle differed between groups, with a higher growth rate during the follicular phase (1.99±0.19 v. 0.46±0.29mmday(-1); P<0.02) and a larger diameter of the preovulatory follicle (14.48±0.35 v. 9.67±1.85mm; P<0.01) in the control group. The pattern of corpus luteum growth and regression also differed between groups, expressed by higher volume (4.37×10(3)±0.27 v. 2.91×10(3)±0.31mm(3); P<0.05) in the control group. The proportion of dominant follicles that developed to follicular cysts (> 25mm) tended to be higher (75 v. 20%; P<0.09) in the treated group. The average oestradiol concentration in the follicular fluid of the preovulatory follicle was lower in the DEHP-treated group (361.6±130.5 v. 832.6±109.7ngmL(-1); P<0.05). However, the average progesterone concentration in the plasma during the luteal phase were normal (4.5-5.0ngmL(-1)) and did not differ between groups. The findings reveal the potential risk of DEHP exposure and its long-lasting effects on bovine ovarian function. These impairments are suggested to adversely affect fertility. PMID:25472215

Roth, Z; Hadas, R; Maor, Y; Kalo, D

2014-12-01

274

Longitudinal, 3D In Vivo Imaging of Sebaceous Glands by Coherent Anti-Stokes Raman Scattering Microscopy: Normal Function and Response to Cryotherapy.  

PubMed

Sebaceous glands perform complex functions, and they are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland's main function-excretion of sebum via the holocrine mechanism-can only be evaluated over time. We present a longitudinal, real-time alternative-the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes, were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three dimensions in vivo. PMID:25026458

Jung, Yookyung; Tam, Joshua; Jalian, H Ray; Anderson, R Rox; Evans, Conor L

2015-01-01

275

Integrating EMR-Linked and In Vivo Functional Genetic Data to Identify New Genotype-Phenotype Associations  

PubMed Central

The coupling of electronic medical records (EMR) with genetic data has created the potential for implementing reverse genetic approaches in humans, whereby the function of a gene is inferred from the shared pattern of morbidity among homozygotes of a genetic variant. We explored the feasibility of this approach to identify phenotypes associated with low frequency variants using Vanderbilt's EMR-based BioVU resource. We analyzed 1,658 low frequency non-synonymous SNPs (nsSNPs) with a minor allele frequency (MAF)<10% collected on 8,546 subjects. For each nsSNP, we identified diagnoses shared by at least 2 minor allele homozygotes and with an association p<0.05. The diagnoses were reviewed by a clinician to ascertain whether they may share a common mechanistic basis. While a number of biologically compelling clinical patterns of association were observed, the frequency of these associations was identical to that observed using genotype-permuted data sets, indicating that the associations were likely due to chance. To refine our analysis associations, we then restricted the analysis to 711 nsSNPs in genes with phenotypes in the On-line Mendelian Inheritance in Man (OMIM) or knock-out mouse phenotype databases. An initial comparison of the EMR diagnoses to the known in vivo functions of the gene identified 25 candidate nsSNPs, 19 of which had significant genotype-phenotype associations when tested using matched controls. Twleve of the 19 nsSNPs associations were confirmed by a detailed record review. Four of 12 nsSNP-phenotype associations were successfully replicated in an independent data set: thrombosis (F5,rs6031), seizures/convulsions (GPR98,rs13157270), macular degeneration (CNGB3,rs3735972), and GI bleeding (HGFAC,rs16844401). These analyses demonstrate the feasibility and challenges of using reverse genetics approaches to identify novel gene-phenotype associations in human subjects using low frequency variants. As increasing amounts of rare variant data are generated from modern genotyping and sequence platforms, model organism data may be an important tool to enable discovery. PMID:24949630

Mosley, Jonathan D.; Van Driest, Sara L.; Weeke, Peter E.; Delaney, Jessica T.; Wells, Quinn S.; Bastarache, Lisa; Roden, Dan M.; Denny, Josh C.

2014-01-01

276

Effect of Perinatal secondhand tobacco smoke exposure on in vivo and intrinsic airway structure/function in non-human primates  

SciTech Connect

Infants exposed to second hand smoke (SHS) experience more problems with wheezing. This study was designed to determine if perinatal SHS exposure increases intrinsic and/or in vivo airway responsiveness to methacholine and whether potential structural/cellular alterations in the airway might explain the change in responsiveness. Pregnant rhesus monkeys were exposed to filtered air (FA) or SHS (1 mg/m{sup 3} total suspended particulates) for 6 h/day, 5 days/week starting at 50 days gestational age. The mother/infant pairs continued the SHS exposures postnatally. At 3 months of age each infant: 1) had in vivo lung function measurements in response to inhaled methacholine, or 2) the right accessory lobe filled with agarose, precision-cut to 600 {mu}m slices, and bathed in increasing concentrations of methacholine. The lumenal area of the central airway was determined using videomicrometry followed by fixation and histology with morphometry. In vivo tests showed that perinatal SHS increases baseline respiratory rate and decreases responsiveness to methacholine. Perinatal SHS did not alter intrinsic airway responsiveness in the bronchi. However in respiratory bronchioles, SHS exposure increased airway responsiveness at lower methacholine concentrations but decreased it at higher concentrations. Perinatal SHS did not change eosinophil profiles, epithelial volume, smooth muscle volume, or mucin volume. However it did increase the number of alveolar attachments in bronchi and respiratory bronchioles. In general, as mucin increased, airway responsiveness decreased. We conclude that perinatal SHS exposure alters in vivo and intrinsic airway responsiveness, and alveolar attachments.

Joad, Jesse P. [Department of Pediatrics, School of Medicine, University of California at Davis, Davis, California, 95616 (United States)], E-mail: jesse.joad@ucdmc.ucdavis.edu; Kott, Kayleen S.; Bric, John M.; Peake, Janice L.; Pinkerton, Kent E. [Department of Pediatrics, School of Medicine, University of California at Davis, Davis, California, 95616 (United States)

2009-02-01

277

Effects of "own" versus "alien" suckling on incidence of ovarian luteal activity, estrus and secretion of luteinizing hormone, oxytocin and prolactin in early postpartum beef cows  

E-print Network

) Newell H. McArthur (Member) Theodore H. Friend (Member) H. Russell Cross (Head of Department) May 1992 ABSTRACT Effects of "Own" versus "Alien" Suckling on Incidence of Ovarian Luteal Activity, Estrus and Secretion of Luteinizing Hormone... following suckling bouts on Experimental Days 2 and 4. Mean concentrations of serum prolactin in fall-calving primiparous heifers on Experimental Days 2 and 4 combined. Incidence of estrus and sexual activity in Alien, Own and Weaned cows. . 37 40 41...

Silveira, Patrice Auvern

1992-01-01

278

Luteal activity of Abadeh ecotype does in summer and winter and the effect of kisspeptin-10 on luteinizing hormone secretion in the anestrus does  

PubMed Central

The aims of the present study were to evaluate luteal activity in Abadeh ecotype goat during summer and winter and also the effect of a single dose kisspeptin-10 injection on the secretion of luteinizing hormone (LH) in female anestrous goats. In the first study, progesterone (P4) concentration in 10 goats in summer (n = 6) and winter (n = 4) were measured every other day. Moreover, in summer group, a male teaser goat was left in the herd on days of sampling for one hour. Goats with P4 concentration ?1 ng mL-1, at least two consecutive measurements, were considered with luteal activity. In the second study, the anestrous phase was confirmed by P4 measurement 20 and 10 days before the kisspeptin injection in five female Abadeh ecotype goats (4 to 5 years old). The goats were given a single IV injection of saline (2 mL) as control group and the same goats (1 hr after the last blood sampling) were given kisspeptin (1 ?g kg-1) as treatment group. The blood samples were collected at –60, –40, –20 and 0 min (before injection), and 10, 20, 30, 40, 50, 60, 80, 100, 120 and 140 min after the injection and LH concentration were measured. A single IV injection of 1 µg kg-1 of kisspeptin-10 did not stimulate the release of LH in female anestrous goats. In summer, in the presence of teaser goat, luteal activity was seen in all goats. In the absence of male goat in winter, some goats showed luteal activity and others showed anestrus. PMID:25610575

Arjmand, Mohammad; Mirzaei, Abdolah; Jafarzadeh Shirazi, Mohammad Reza; Tamadon, Amin; Salehi, Mohammad Saied; Saeb, Mehdi; Namavar, Mohammad Reza; Zandi, Mohammad Reza; Shahheidari, Hojatollah; Moradi, Sara

2014-01-01

279

Relationships between thyroid hormones and serum energy metabolites with different patterns of postpartum luteal activity in high-producing dairy cows.  

PubMed

This study investigated the relationships of thyroid hormones, serum energy metabolites, reproductive parameters, milk yield and body condition score with the different patterns of postpartum luteal activity in the postpartum period. A total of 75 multiparous healthy (free of detectable reproductive disorders) Holstein dairy cows (mean peak milk yield = 56.5 ± 7.0 kg/day) were used in this study. Transrectal ultrasound scanning and blood sample collection were performed twice weekly. Serum concentrations of progesterone (P4) were measured twice weekly and beta-hydroxybutyrate (BHBA), non-esterified fatty acids, thyroxine (T4), 3,30,5-tri-iodothyronine (T3), free thyroxine (fT4) and free 3,30,5-tri-iodothyronine (fT3) were measured every 2 weeks from the 1st to the 8th week postpartum. On the basis of the serum P4 profile of the cows, 25 (33.4%) had normal luteal activity (NLA), whereas 30 (40%), 10 (13.3%), 6 (8%) and 4 (5.3%) had prolonged luteal phase (PLP), delayed first ovulation (DOV), anovulation (AOV) and short luteal phase, respectively. Serum T4 concentrations in PLP cows were higher than that in NLA cows at the 3rd week postpartum and did not change during the period of study, whereas in the NLA cows the concentrations increased (P < 0.05). Further, the least square (LS) mean of serum fT4 concentrations in the DOV and AOV cows were significantly lower than in the NLA cows during the study period (P < 0.05). In addition, the AOV cows had higher LS mean serum BHBA and T4 concentrations than the NLA cows in early weeks postpartum (P < 0.05). In conclusion, the serum thyroid hormones' profile differs in high-producing dairy cows showing PLP, AOV and DOV in comparison with the postpartum NLA cows. PMID:23217229

Kafi, M; Tamadon, A; Saeb, M; Mirzaei, A; Ansari-Lari, M

2012-08-01

280

Greater overall olfactory performance, explicit wanting for high fat foods and lipid intake during the mid-luteal phase of the menstrual cycle.  

PubMed

Increases in energy, lipid and carbohydrate intakes during the luteal phase have been previously observed. However, it is not known whether this is due to phase-dependent variations in the reward value of certain foods. Moreover, increases in olfactory sensitivity have been proposed and may be involved in these changes in food reward. Therefore, we examined olfactory performance and the reward value of foods varying in fat content and taste. Seventeen women (Body mass index: 22.3±1.6 kg/m(2); Body fat-DXA: 28.5±6.8%) were recruited to participate in 3 identical sessions, performed during distinct phases of the menstrual cycle - early follicular/menstruation, late follicular/ovulation and mid-luteal - verified by plasma sex-steroid hormones and oral temperature. Food preference, implicit wanting, and explicit wanting and liking for visual food cues, varying in fat content and taste, were measured with a validated experimental platform involving a forced choice computer task. Odour threshold, odour discrimination, odour identification and total odour scores were measured using odourized pens. Ad libitum energy and macronutrient intake was measured with a validated food menu. Results showed greater total odour scores (p<0.05), explicit wanting for high fat foods (p<0.05) and lipid intake (p<0.05) during the mid-luteal phase. Inter-correlations between these variables were non-significant. These findings support previous observations of increased lipid intake during the luteal phase and provide evidence for phase-dependent variation in overall olfactory performance and explicit wanting for high fat foods. PMID:23458628

McNeil, Jessica; Cameron, Jameason D; Finlayson, Graham; Blundell, John E; Doucet, Éric

2013-03-15

281

Naturally occurring mastitis effects on timing of ovulation, steroid and gonadotrophic hormone concentrations, and follicular and luteal growth in cows.  

PubMed

The effects of naturally occurring subclinical chronic or clinical short-term mastitis on time of ovulation, plasma steroid and gonadotropin concentrations, and follicular and luteal dynamics were examined in 73 lactating Holstein cows. Cows were sorted by milk somatic cell count and bacteriological examination into an uninfected group (n=22), a clinical mastitis group (n=9; events occurring 20+/-7 d before the study), and a subclinical chronic mastitis group (n=42). In addition, uninfected and mastitic cows were further sorted by their estrus to ovulation (E-O) interval. About 30% of mastitic cows (mainly subclinical) manifested an extended E-O interval of 56+/-9.2h compared with 28+/-0.8h in uninfected cows and 29+/-0.5h in the other 70% of mastitic cows. In mastitic cows with extended E-O interval, the concentration of plasma estradiol at onset of estrus was lower than that of uninfected cows or mastitic cows that exhibited normal E-O intervals (3.1+/-0.4, 5.8+/-0.5, and 5.5+/-0.5 pg/mL, respectively). The disruptive effect of mastitis on follicular estradiol probably does not involve alterations in gonadotropin secretion because any depressive effects of mastitis on pulsatile LH concentrations were not detected. Cortisol concentrations did not differ among groups. The preovulatory LH surge in mastitic cows with delayed ovulation varied among individuals, being lower, delayed, or with no surge noted compared with the normal LH surge exhibited by uninfected cows or mastitic cows with normal E-O interval (6.8+/-0.7 ng/mL). The diameter of the second-wave dominant follicle was larger and the number of medium follicles was smaller in uninfected and subclinical cows with normal intervals compared with subclinical cows with extended intervals (13.4+/-0.5 vs. 10.9+/-0.9mm, and 3.8+/-0.2 vs. 6.7+/-0.14 follicles, respectively). Mid-luteal progesterone concentrations were similar in uninfected and mastitic cows. These results indicate for the first time that around 30% of cows with subclinical chronic mastitis exhibit delayed ovulation that is associated with low plasma concentrations of estradiol and a low or delayed preovulatory LH surge. PMID:20172211

Lavon, Y; Leitner, G; Voet, H; Wolfenson, D

2010-03-01

282

P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.  

PubMed

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position. PMID:21295466

Isaacs, Richard C A; Newton, Christina L; Cutrona, Kellie J; Mercer, Swati P; Dorsey, Bruce D; McDonough, Colleen M; Cook, Jacquelynn J; Krueger, Julie A; Lewis, S Dale; Lucas, Bobby J; Lyle, Elizabeth A; Lynch, Joseph J; Miller-Stein, Cynthia; Michener, Maria T; Wallace, Audrey A; White, Rebecca B; Wong, Bradley K

2011-03-01

283

Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization.  

PubMed

Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages. Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching. A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart. The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses. Overall, 7 of 33 patients had normal T cell function. Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers. HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL. Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment. PMID:10915073

Fogelman, I; Davey, V; Ochs, H D; Elashoff, M; Feinberg, M B; Mican, J; Siegel, J P; Sneller, M; Lane, H C

2000-08-01

284

Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo.  

PubMed

Graphene oxide (GO) has attracted intensive interest in the biomedical field in recent years. We investigate whether the use of functional graphene oxide as an efficient delivery system for delivering specific molecular antitumor therapeutics in vivo could achieve a more excellent antitumor effect. Constitutive activation of signal transducer and activator of transcription 3 (Stat3) promotes survival in a wide spectrum of human cancers. In this paper, we study the in vivo behavior of graphene oxide chemically functionalized with polyethylenimine and polyethylene glycol (GO-PEI-PEG) as a plasmid-based Stat3-specific small interfering RNA (siRNA) carrier in mouse malignant melanoma. The in vivo results indicate significant regression in tumor growth and tumor weight after plasmid-based Stat3 siRNA delivered by GO-PEI-PEG treatment. Moreover, there was no significant side effect from GO-PEI-PEG treatment according to histological examination and blood chemistry analysis in mice. Thus, our work is the first success of using GO-PEI-PEG as a promising carrier for plasmid Stat3 siRNA delivery and down-regulation of Stat3 by a polymer-mediated vehicle and suggests the great promise of graphene in biomedical applications such as cancer treatment. PMID:23425941

Yin, Di; Li, Yang; Lin, Hang; Guo, Baofeng; Du, Yanwei; Li, Xin; Jia, Huijie; Zhao, Xuejian; Tang, Jun; Zhang, Ling

2013-03-15

285

A New Functional Suppressor tRNA/ Aminoacyl-tRNA Synthetase Pair for the in Vivo  

E-print Network

analyzed biochemical data available for tRNATyr/TyrRS pairs from a variety of organisms. This analysis, together with in vivo complemen- tation assays, has afforded a new orthogonal tRNACUA Tyr /TyrRS pair cannot be amino- acylated by bacterial synthetases, nor do their TyrRS aminoacylate bacterial t

Magliery, Thomas J.

286

In Vivo Evidence of Increased nNOS Activity in Acute MPTP Neurotoxicity: A Functional Pharmacological MRI Study  

PubMed Central

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin commonly used to produce an animal model of Parkinson's disease. Previous studies have suggested a critical role for neuronal nitric oxide (NO) synthase- (nNOS-) derived NO in the pathogenesis of MPTP. However, NO activity is difficult to assess in vivo due to its extremely short biological half-life, and so in vivo evidence of NO involvement in MPTP neurotoxicity remains scarce. In the present study, we utilized flow-sensitive alternating inversion recovery sequences, in vivo localized proton magnetic resonance spectroscopy, and diffusion-weighted imaging to, respectively, assess the hemodynamics, metabolism, and cytotoxicity induced by MPTP. The role of NO in MPTP toxicity was clarified further by administering a selective nNOS inhibitor, 7-nitroindazole (7-NI), intraperitoneally to some of the experimental animals prior to MPTP challenge. The transient increase in cerebral blood flow (CBF) in the cortex and striatum induced by systemic injection of MPTP was completely prevented by pretreatment with 7-NI. We provide the first in vivo evidence of increased nNOS activity in acute MPTP-induced neurotoxicity. Although the observed CBF change may be independent of the toxicogenesis of MPTP, this transient hyperperfusion state may serve as an early indicator of neuroinflammation. PMID:24069609

Siow, Tiing Yee; Chen, Chiao-Chi V.; Wan, Nina; Chow, Kai-Ping N.

2013-01-01

287

Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy  

PubMed Central

Bone marrow mononuclear cells (BMC) from patients with ischemic cardiomyopathy (ICMP) show a reduced neovascularization capacity in vivo. NO plays an important role in neovascularization, and NO bioavailability is typically reduced in patients with ICMP. We investigated whether the impaired neovascularization capacity of ICMP patient-derived progenitor cells can be restored by pretreatment with the novel endothelial NO synthase (eNOS) transcription enhancer AVE9488 (AVE). Ex vivo pretreatment of BMC from patients with ICMP with AVE significantly increased eNOS mRNA expression by 2.1-fold (P < 0.05) and eNOS activity as assessed by ESR by >3-fold (P < 0.05). The increased eNOS expression was associated with an enhanced migratory capacity in vitro (P < 0.01) and improved neovascularization capacity of the infused BMC in an ischemic hind limb model in vivo (P < 0.001). The improvement in ischemic limb perfusion after infusion of AVE-pretreated BMC resulted in an increase in swimming time (P < 0.05). The enhancement of limb perfusion by AVE-treated BMC was abrogated by ex vivo pretreatment with the eNOS inhibitor NG-nitro-l-arginine methyl ester. Consistently, AVE showed no effect on the impaired migratory capacity of BMC derived from eNOS-deficient mice, documenting the specific involvement of NO. The reduced neovascularization capacity of BMC from patients with ICMP may limit their therapeutic potential in cell therapy studies. Here, we show that pharmacological enhancement of eNOS expression with AVE at least partially reverses the impaired functional activity of BMC from ICMP patients, highlighting the critical role of NO for progenitor cell function. PMID:16983080

Sasaki, Ken-ichiro; Heeschen, Christopher; Aicher, Alexandra; Ziebart, Thomas; Honold, Joerg; Urbich, Carmen; Rossig, Lothar; Koehl, Ulrike; Koyanagi, Masamichi; Mohamed, Annisuddin; Brandes, Ralf P.; Martin, Hans; Zeiher, Andreas M.; Dimmeler, Stefanie

2006-01-01

288

Luteal cell steroidogenesis in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.  

PubMed

The primary aim of this study was to determine the possible cause of slow or delayed embryonic development in Cynopterus sphinx by investigating morphological and steroidogenic changes in the corpus luteum (CL) and circulating hormone concentrations during two pregnancies of a year. This species showed delayed post-implantational embryonic development during gastrulation of the first pregnancy. Morphological features of the CL showed normal luteinization during both pregnancies. The CL did not change significantly in luteal cell size during the delay period of the first pregnancy as compared with the second pregnancy. The circulating progesterone and 17beta-estradiol concentrations were significantly lower during the period of delayed embryonic development as compared with the same stage of embryonic development during the second pregnancy. We also showed a marked decline in the activity of 3beta-hydroxysteroid dehydrogenase, P450 side chain cleavage enzyme, and steroidogenic acute regulatory peptide in the CL during the delay period. This may cause low circulating progesterone and estradiol synthesis and consequently delay embryonic development. What causes the decrease in steroidogenic factors in the CL during the period of delayed development in C. sphinx is under investigation. PMID:19186043

Meenakumari, Karukayil J; Banerjee, Arnab; Krishna, Amitabh

2009-01-01

289

Preserved endothelium-dependent dilatation of the coronary microvasculature at the early phase of diabetes mellitus despite the increased oxidative stress and depressed cardiac mechanical function ex vivo  

PubMed Central

Background There has been accumulating evidence associating diabetes mellitus and cardiovascular dysfunctions. However, most of the studies are focused on the late stages of diabetes and on the function of large arteries. This study aimed at characterizing the effects of the early phase of diabetes mellitus on the cardiac and vascular function with focus on the intact coronary microvasculature and the oxidative stress involved. Materials and methods Zucker diabetic fatty rats and their lean littermates fed with standard diet A04 (Safe) were studied at the 11th week of age. Biochemical parameters such as glucose, insulin and triglycerides levels as well as their oxidative stress status were measured. Their hearts were perfused ex vivo according to Langendorff and their cardiac activity and coronary microvascular reactivity were evaluated. Results Zucker fatty rats already exhibited a diabetic state at this age as demonstrated by the elevated levels of plasma glucose, insulin, glycated hemoglobin and triglycerides. The ex vivo perfusion of their hearts revealed a decreased cardiac mechanical function and coronary flow. This was accompanied by an increase in the overall oxidative stress of the organs. However, estimation of the active form of endothelial nitric oxide synthase and coronary reactivity indicated a preserved function of the coronary microvessels at this phase of the disease. Diabetes affected also the cardiac membrane phospholipid fatty acid composition by increasing the arachidonic acid and n-3 polyunsaturated fatty acids levels. Conclusions The presence of diabetes, even at its beginning, significantly increased the overall oxidative stress of the organs resulting to decreased cardiac mechanical activity ex vivo. However, adaptations were adopted at this early phase of the disease regarding the preserved coronary microvascular reactivity and the associated cardiac phospholipid composition in order to provide a certain protection to the heart. PMID:23530768

2013-01-01

290

Tissue Engineering Special Feature: A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo  

NASA Astrophysics Data System (ADS)

A microvascular network is critical for the survival and function of most tissues. We have investigated the potential of neural progenitor cells to augment the formation and stabilization of microvascular networks in a previously uncharacterized three-dimensional macroporous hydrogel and the ability of this engineered system to develop a functional microcirculation in vivo. The hydrogel is synthesized by cross-linking polyethylene glycol with polylysine around a salt-leached polylactic-co-glycolic acid scaffold that is degraded in a sodium hydroxide solution. An open macroporous network is formed that supports the efficient formation of tubular structures by brain endothelial cells. After subcutaneous implantation of hydrogel cocultures in mice, blood flow in new microvessels was apparent at 2 weeks with perfused networks established on the surface of implants at 6 weeks. Compared to endothelial cells cultured alone, cocultures of endothelial cells and neural progenitor cells had a significantly greater density of tubular structures positive for platelet endothelial cell adhesion molecule-1 at the 6-week time point. In implant cross sections, the presence of red blood cells in vessel lumens confirmed a functional microcirculation. These findings indicate that neural progenitor cells promote the formation of endothelial cell tubes in coculture and the development of a functional microcirculation in vivo. We demonstrate a previously undescribed strategy for creating stable microvascular networks to support engineered tissues of desired parenchymal cell origin. microvasculature | neural stem cells | polymer | scaffold

Ford, Millicent C.; Bertram, James P.; Royce Hynes, Sara; Michaud, Michael; Li, Qi; Young, Michael; Segal, Steven S.; Madri, Joseph A.; Lavik, Erin B.

2006-02-01

291

The level of DLDB/CHIP controls the activity of the LIM homeodomain protein apterous: evidence for a functional tetramer complex in vivo.  

PubMed

The LIM homeodomain (LIM-HD) protein Apterous (Ap) and its cofactor DLDB/CHIP control dorso- ventral (D/V) patterning and growth of Drosophila wing. To investigate the molecular mechanisms of Ap/CHIP function we altered their relative levels of expression and generated mutants in the LIM1, LIM2 and HD domains of Ap, as well as in the LIM-interacting and self-association domains of CHIP. Using in vitro and in vivo assays we found that: (i) the levels of CHIP relative to Ap control D/V patterning; (ii) the LIM1 and LIM2 domains differ in their contributions to Ap function; (iii) Ap HD mutations cause weak dominant negative effects; (iv) overexpression of ChipDeltaSAD mutants mimics Ap lack-of-function, and this dominant negative phenotype is caused by titration of Ap because it can be rescued by adding extra Ap; and (v) overexpression of ChipDeltaLID mutants also causes an Ap lack-of-function phenotype, but it cannot be rescued by extra Ap. These results support the model that the Ap-CHIP active complex in vivo is a tetramer. PMID:10835358

Rincón-Limas, D E; Lu, C H; Canal, I; Botas, J

2000-06-01

292

Luteal-phase support in assisted reproduction treatment: real-life practices reported worldwide by an updated website-based survey.  

PubMed

An updated worldwide web-based survey assessed the real-life clinical practices regarding luteal-phase supplementation (LPS) in assisted reproduction. This survey looked for changes since a former survey conducted nearly 3years earlier. The survey questions were: If you support the luteal phase, when do you start the regimen you are using?; Which agent/route is your treatment of choice to support the luteal phase?; If you use vaginal progesterone, which formulation do you use?; and How long you continue progesterone supplementation if the patient conceived? Data were obtained from 408 centres (82 countries) representing 284,600 IVF cycles/year. The findings were: (i) most practitioners (80% of cycles) start LPS on the day of egg collection; (ii) in >90%, a vaginal progesterone product is used (77% as a single agent and 17% in combination with i.m. progesterone), while human chorionic gonadotrophin as a single agent for LPS is not being used at all; and (iii) in 72% of cycles, LPS is administered until 8-10weeks' gestation or beyond. When compared with the initial survey, the results of this survey are encouraging as there is a clear shift towards a more unified and evidence-based approach to LPS in IVF cycles. This updated worldwide web-based survey assessed the actual real-life clinical practices regarding luteal-phase supplementation (LPS) in assisted reproduction. Specifically, this survey looked for changes since an initial survey conducted nearly 3years earlier. The survey included the following questions: If you support the luteal phase, when do you start the regimen you are using?; Which agent/route is your treatment of choice to support the luteal phase?; If you use vaginal progesterone, which formulation do you use?; and How long you continue progesterone supplementation if the patient conceived? Data from 408 centres in 82 countries representing a total of 284,600 IVF cycles/year were included. Most practitioners (80% of cycles) start LPS on the day of egg collection and in more than 90% a vaginal progesterone product is used for LPS (in 77% as a single agent and in 17% in combination with i.m. progesterone). As a single agent for LPS, human chorionic gonadotrophin is not being used at all. Regarding the duration of supplementation, in 72% of cycles, LPS is administered until 8-10weeks' gestation or beyond. When compared with the initial survey from 2009, the results of this updated survey are encouraging as there is a clear shift towards a more unified and evidence-based approach to luteal-phase support in IVF cycles. Nevertheless, although there is no firm evidence supporting the continuation of LPS after the demonstration of fetal heart beat on ultrasound, this remains the common practice of the majority of assisted reproduction centres worldwide. PMID:24447959

Vaisbuch, Edi; de Ziegler, Dominique; Leong, Milton; Weissman, Ariel; Shoham, Zeev

2014-03-01

293

Effect of human chorionic gonadotropin on luteal function and reproductive performance of high-producing lactating Holstein dairy cows.  

PubMed

The objectives were to evaluate effects of human chorionic gonadotropin (hCG) (3,300 IU i.m.) administered on d 5 after AI on CL number, plasma progesterone concentration, conception rate, and pregnancy loss in high-producing dairy cows. Following the synchronization of estrus and AI, 406 cows were injected with either hCG or saline on d 5 after AI in a randomized complete block design. Blood sampling and ovarian ultrasonography were conducted once between d 11 and 16 after AI. Pregnancy diagnoses were performed on d 28 by ultrasonography and on d 45 and 90 after AI by rectal palpation. Treatment with hCG on d 5 resulted in 86.2% of the cows with more than one CL compared with 23.2% in controls. Plasma progesterone concentrations were increased by 5.0 ng/mL in hCG-treated cows. The presence of more than one CL increased progesterone concentration in hCG-treated cows but not in controls. Conception rates were higher for hCG-treated cows on d 28 (45.8 > 38.7%), 45 (40.4 > 36.3%), and 90 (38.4 > 31.9%) after AI. Treatment with hCG improved conception rate in cows losing body condition between AI and d 28 after Al. Pregnancy losses were similar between treatment groups. Treatment with hCG on d 5 after AI induces accessory CL, enhances plasma progesterone concentration, and improves conception rate of high-producing dairy cows. PMID:11768118

Santos, J E; Thatcher, W W; Pool, L; Overton, M W

2001-11-01

294

In vivo measurement of the shape of the tissue-refractive-index correlation function and its applicationto detection of colorectal field carcinogenesis  

NASA Astrophysics Data System (ADS)

Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development.

Gomes, Andrew J.; Ruderman, Sarah; DelaCruz, Mart; Wali, Ramesh K.; Roy, Hemant K.; Backman, Vadim

2012-04-01

295

In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer’s disease model  

PubMed Central

Summary Loss of neurons after brain injury and in neurodegenerative disease is often accompanied by reactive gliosis and scarring, which are difficult to reverse with existing treatment approaches. Here, we show that reactive glial cells in the cortex of stab-injured or Alzheimer’s disease (AD) model mice can be directly reprogrammed into functional neurons in vivo using retroviral expression of a single neural transcription factor, NeuroD1. Following expression of NeuroD1, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and GABAergic neurons. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting that they integrated into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. Our studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide an alternative approach for repair of injured or diseased brain. PMID:24360883

Guo, Ziyuan; Zhang, Lei; Wu, Zheng; Chen, Yuchen; Wang, Fan; Chen, Gong

2014-01-01

296

Targeted Resequencing and Systematic In Vivo Functional Testing Identifies Rare Variants in MEIS1 as Significant Contributors to Restless Legs Syndrome  

PubMed Central

Restless legs syndrome (RLS) is a common neurologic condition characterized by nocturnal dysesthesias and an urge to move, affecting the legs. RLS is a complex trait, for which genome-wide association studies (GWASs) have identified common susceptibility alleles of modest (OR 1.2–1.7) risk at six genomic loci. Among these, variants in MEIS1 have emerged as the largest risk factors for RLS, suggesting that perturbations in this transcription factor might be causally related to RLS susceptibility. To establish this causality, direction of effect, and total genetic burden of MEIS1, we interrogated 188 case subjects and 182 control subjects for rare alleles not captured by previous GWASs, followed by genotyping of ?3,000 case subjects and 3,000 control subjects, and concluded with systematic functionalization of all discovered variants using a previously established in vivo model of neurogenesis. We observed a significant excess of rare MEIS1 variants in individuals with RLS. Subsequent assessment of all nonsynonymous variants by in vivo complementation revealed an excess of loss-of-function alleles in individuals with RLS. Strikingly, these alleles compromised the function of the canonical MEIS1 splice isoform but were irrelevant to an isoform known to utilize an alternative 3? sequence. Our data link MEIS1 loss of function to the etiopathology of RLS, highlight how combined sequencing and systematic functional annotation of rare variation at GWAS loci can detect risk burden, and offer a plausible explanation for the specificity of phenotypic expressivity of loss-of-function alleles at a locus broadly necessary for neurogenesis and neurodevelopment. PMID:24995868

Schulte, Eva C.; Kousi, Maria; Tan, Perciliz L.; Tilch, Erik; Knauf, Franziska; Lichtner, Peter; Trenkwalder, Claudia; Högl, Birgit; Frauscher, Birgit; Berger, Klaus; Fietze, Ingo; Hornyak, Magdolna; Oertel, Wolfgang H.; Bachmann, Cornelius G.; Zimprich, Alexander; Peters, Annette; Gieger, Christian; Meitinger, Thomas; Müller-Myhsok, Bertram; Katsanis, Nicholas; Winkelmann, Juliane

2014-01-01

297

Ovulation, In Vivo Emotion Regulation Problems, and Sexual Risk Recognition Deficits  

PubMed Central

Objective To examine associations between menstrual cycle phase, negative mood, sexual risk recognition deficits (assessed via an analogue risk vignette), and in vivo emotion dysregulation. Participants Participants were 714 college women recruited between February 2007 and December 2009. Methods Participants were randomly assigned to a negative or neutral mood induction and instructed to identify sexual risk during an audiotaped sexual coercion vignette. Participants reported menstrual cycle information, in vivo emotional nonacceptance, and attention during the vignette. Results In the negative mood condition, ovulation was associated with longer risk recognition latencies relative to the luteal and follicular phases of the menstrual cycle. Increased in vivo emotional nonacceptance and decreased attention to the vignette mediated associations between ovulation and risk recognition deficits in the negative mood condition. Conclusions Sexual assault risk reduction programs could provide psychoeducation regarding negative mood during ovulation and emphasize emotional acceptance and attention to external stimuli when distressed. PMID:25158013

Walsh, Kate; DiLillo, David

2013-01-01

298

Cathelicidin Host Defence Peptide Augments Clearance of Pulmonary Pseudomonas aeruginosa Infection by Its Influence on Neutrophil Function In Vivo  

PubMed Central

Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens. PMID:24887410

Beaumont, Paula E.; McHugh, Brian; Gwyer Findlay, Emily; Mackellar, Annie; Mackenzie, Karen J.; Gallo, Richard L.; Govan, John R. W.; Simpson, A. John; Davidson, Donald J.

2014-01-01

299

Impact of female cigarette smoking on circulating B cells in vivo: the suppressed ICOSLG , TCF3 , and VCAM1 gene functional network may inhibit normal cell function  

Microsoft Academic Search

As pivotal immune guardians, B cells were found to be directly associated with the onset and development of many smoking-induced\\u000a diseases. However, the in vivo molecular response of B cells underlying the female cigarette smoking remains unknown. Using\\u000a the genome-wide Affymetrix HG-133A GeneChip® microarray, we firstly compared the gene expression profiles of peripheral circulating\\u000a B cells between 39 smoking and

Feng Pan; Tie-Lin Yang; Xiang-Ding Chen; Yuan Chen; Ge Gao; Yao-Zhong Liu; Yu-Fang Pei; Bao-Yong Sha; Yan Jiang; Chao Xu; Robert R. Recker; Hong-Wen Deng

2010-01-01

300

Expression of HOXA11 in the mid-luteal endometrium from women with endometriosis-associated infertility  

PubMed Central

Background A decrease in HOXA11 expression in eutopic mid-secretory endometrium has been found in women with endometriosis-associated infertility. Methods Using Real-time quantitative PCR (RQ-PCR) and western blotting analysis we studied the HOXA11 transcript and protein levels in mid-luteal eutopic endometrium from eighteen infertile women with minimal endometriosis, sixteen healthy fertile women and sixteen infertile women with fallopian tubal occlusion from the Polish population. We also evaluated transcript levels of DNA methyltransferases DNMT1, DNMT3A and DNMT3B in these groups of women. Results There were significantly lower levels of HOXA11 transcripts (p = 0.003, p = 0.041) and protein (p = 0.004, p = 0.001) in women with endometriosis as compared to fertile women and infertile women with tubal occlusion. Moreover, we found significantly higher methylation levels of the CpG region in the first exon of HOXA11 in infertile women with endometriosis compared with fertile women (p < 0.001) and infertile women with tubal occlusion (p < 0.001). We also observed significantly increased levels of DNMT3A transcript in women with endometriosis than fertile women (p = 0.044) and infertile women with tubal occlusion (p = 0.047). However, we did not observe significant differences in DNMT1 and DNMT3B transcript levels between these investigated groups of women. Conclusions We confirmed that reduced HOXA11 expression may contribute to endometriosis-associated infertility. Moreover, we found that DNA hypermethylation can be one of the possible molecular mechanisms causing a decrease in HOXA11 expression in the eutopic mid-secretory endometrium in infertile women with endometriosis. PMID:22233680

2012-01-01

301

Effects of treatment with oral isosorbide dinitrate on platelet function in vivo; a double-blind placebo-controlled study in patients with stable angina pectoris.  

PubMed Central

1. A randomised double-blind placebo-controlled cross-over study was performed to investigate the effects of oral isosorbide dinitrate (ISDN; 20 mg twice daily for 2 weeks) on various aspects of platelet function in vivo in 20 patients with stable angina pectoris. Measurements were performed at rest and after platelet activation by physical exercise (bicycle ergometry). 2. Compared with placebo, treatment with ISDN significantly decreased systolic blood pressure at rest by 7 (-14 to -1) mm Hg (mean and 95% CI) and tended to increase exercise capacity by 7 (-1 to 14) W and attenuate perceived chest pain during maximal work. The dosage was high, as judged by side-effects reported (mainly headache). Compliance was good, as assessed by electronic counter equipped tablet bottles (Medication Event Monitoring System); only one patient had a compliance rate below 60%. 3. Exercise significantly increased platelet aggregability as measured by filtragometry ex vivo; the time taken for platelet aggregates in whole blood drawn directly from an antecubital vein to occlude a microfilter was significantly decreased from 155 to 95 s (antilog of mean log values). Platelet secretion in vivo also increased, as indicated by significant elevations of beta-thromboglobulin in plasma; from 22 to 35 ng ml-1 (P = 0.006). 4. ISDN treatment did not inhibit platelet function. Relative to placebo, filtragometry readings (ISDN/placebo ratios; mean and 95% CI) were not altered either at rest (1.05 (0.83 to 1.32)) or immediately after exercise (0.98 (0.80 to 1.20)). Similarly, beta TG in plasma was unaltered by ISDN treatment; 1.09 (0.98 to 1.21) at rest, and 1.04 (0.82 to 1.30) immediately after exercise.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7946939

Wallén, N H; Andersson, A; Hjemdahl, P

1994-01-01

302

A randomized, controlled trial comparing the efficacy and safety of aqueous subcutaneous progesterone with vaginal progesterone for luteal phase support of in vitro fertilization  

PubMed Central

STUDY QUESTION Is the ongoing pregnancy rate with a new aqueous formulation of subcutaneous progesterone (Prolutex®) non-inferior to vaginal progesterone (Endometrin®) when used for luteal phase support of in vitro fertilization? SUMMARY ANSWER In the per-protocol (PP) population, the ongoing pregnancy rates per oocyte retrieval at 12 weeks of gestation were comparable between Prolutex and Endometrin (41.6 versus 44.4%), with a difference between groups of ?2.8% (95% confidence interval (CI) ?9.7, 4.2), consistent with the non-inferiority of subcutaneous progesterone for luteal phase support. WHAT IS KNOWN ALREADY Luteal phase support has been clearly demonstrated to improve pregnancy rates in women undergoing in vitro fertilization (IVF). Because of the increased risk of ovarian hyperstimulation syndrome associated with the use of hCG, progesterone has become the treatment of choice for luteal phase support. STUDY DESIGN, SIZE, DURATION This prospective, open-label, randomized, controlled, parallel-group, multicentre, two-arm, non-inferiority study was performed at eight fertility clinics. A total of 800 women, aged 18–42 years, with a BMI of ?30 kg/m2, with <3 prior completed assisted reproductive technology (ART) cycles, exhibiting baseline (Days 2–3) FSH of ?15 IU/L and undergoing IVF at 8 centres (seven private, one academic) in the USA, were enrolled from January 2009 through June 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS In total, 800 women undergoing IVF were randomized after retrieval of at least three oocytes to an aqueous preparation of progesterone administered subcutaneously (25 mg daily) or vaginal progesterone (100 mg bid daily). Randomization was performed to enrol 100 patients at each site using a randomization list that was generated with Statistical Analysis Software (SAS®). If a viable pregnancy occurred, progesterone treatment was continued up to 12 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE Using a PP analysis, which included all patients who received an embryo transfer (Prolutex = 392; Endometrin = 390), the ongoing pregnancy rate per retrieval for subcutaneous versus vaginal progesterone was 41.6 versus 44.4%, with a difference between groups of ?2.8% (95% CI ?9.7, 4.2), consistent with the non-inferiority of subcutaneous progesterone for luteal phase support. In addition, rates of initial positive ?-hCG (56.4% subcutaneous versus 59.0% vaginal; 95% CI ?9.5, 4.3), clinical intrauterine pregnancy with fetal cardiac activity (42.6 versus 46.4%; 95% CI ?10.8, 3.2), implantation defined as number of gestational sacs divided by number of embryos transferred (33.2 versus 35.1%; 95% CI ?7.6, 4.0), live birth (41.1 versus 43.1%; 95% CI ?8.9, 4.9) and take-home baby (41.1 versus 42.6%; 95% CI ?8.4, 5.4) were comparable. Both formulations were well-tolerated, with no difference in serious adverse events. Analysis with the intention-to-treat population also demonstrated no difference for any outcomes between the treatment groups. LIMITATIONS, REASONS FOR CAUTION The conclusions are limited to the progesterone dosing regimen studied and duration of treatment for the patient population examined in this study. WIDER IMPLICATIONS OF THE FINDINGS Subcutaneous progesterone represents a novel option for luteal phase support in women undergoing IVF who for personal reasons prefer not to use a vaginal preparation or who wish to avoid the side effects of vaginal or i.m. routes of administration. STUDY FUNDING/COMPETING INTERESTS The study was funded by Institut Biochimique SA (IBSA). CAJ, BC, ST and CJ are employees of IBSA. FH currently consults for IBSA. TRIAL REGISTRATION NUMBER NCT00828191. PMID:25100106

Baker, Valerie L.; Jones, Christopher A.; Doody, Kevin; Foulk, Russell; Yee, Bill; Adamson, G. David; Cometti, Barbara; DeVane, Gary; Hubert, Gary; Trevisan, Silvia; Hoehler, Fred; Jones, Clarence; Soules, Michael

2014-01-01

303

The Oncogenic EWS-FLI1 Protein Binds In Vivo GGAA Microsatellite Sequences with Potential Transcriptional Activation Function  

PubMed Central

The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis. PMID:19305498

Boeva, Valentina; Zynovyev, Andrei; Barillot, Emmanuel; Delattre, Olivier

2009-01-01

304

A Salmonella Typhimurium-Typhi Genomic Chimera: A Model to Study Vi Polysaccharide Capsule Function In Vivo  

PubMed Central

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi+), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi+ colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi+ and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi+ resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi? infected animals. C5.507 Vi+ infection stimulated reduced numbers of TNF-?, MIP-2 and perforin producing cells compared to SGB1 Vi?. The modulating effect associated with Vi was not observed in MyD88?/? and was reduced in TLR4?/? mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro. PMID:21829346

Clare, Simon; Goulding, David; Holt, Kathryn E.; Grant, Andrew J.; Mastroeni, Piero; Dougan, Gordon; Kingsley, Robert A.

2011-01-01

305

In vivo performance of a novel fluorinated magnetic resonance imaging agent for functional analysis of bile acid transport.  

PubMed

A novel trifluorinated cholic acid derivative, CA-lys-TFA, was designed and synthesized for use as a tool to measure bile acid transport noninvasively using magnetic resonance imaging (MRI). In the present study, the in vivo performance of CA-lys-TFA for measuring bile acid transport by MRI was investigated in mice. Gallbladder CA-lys-TFA content was quantified using MRI and liquid chromatography/tandem mass spectrometry. Results in wild-type (WT) C57BL/6J mice were compared to those in mice lacking expression of Asbt, the ileal bile acid transporter. (19)F signals emanating from the gallbladders of WT mice 7 h after oral gavage with 150 mg/kg CA-lys-TFA were reproducibly detected by MRI. Asbt-deficient mice administered the same dose had undetectable (19)F signals by MRI, and gallbladder bile CA-lys-TFA levels were 30-fold lower compared to WT animals. To our knowledge, this represents the first report of in vivo imaging of an orally absorbed drug using (19)F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI. PMID:24708306

Vivian, Diana; Cheng, Kunrong; Khurana, Sandeep; Xu, Su; Kriel, Edwin H; Dawson, Paul A; Raufman, Jean-Pierre; Polli, James E

2014-05-01

306

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate  

PubMed Central

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1? Asn in the RSRR?N cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase. PMID:18378898

Essalmani, Rachid; Zaid, Ahmed; Marcinkiewicz, Jadwiga; Chamberland, Ann; Pasquato, Antonella; Seidah, Nabil G.; Prat, Annik

2008-01-01

307

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate.  

PubMed

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1' Asn in the RSRR downward arrowN cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase. PMID:18378898

Essalmani, Rachid; Zaid, Ahmed; Marcinkiewicz, Jadwiga; Chamberland, Ann; Pasquato, Antonella; Seidah, Nabil G; Prat, Annik

2008-04-15

308

Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata  

SciTech Connect

Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns encountered in using heterologous systems are totally excluded.

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India)] [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India); Sanglard, Dominique [Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne (Switzerland)] [Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne (Switzerland); Prasad, Rajendra, E-mail: rp47jnu@gmail.com [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India)] [Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067 (India)

2011-01-07

309

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.  

PubMed

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity. PMID:11056386

Taguchi, S; Kuwasako, K; Suenaga, A; Okada, M; Momose, H

2000-11-01

310

Duration of luteal support (DOLS) with progesterone pessaries to improve the success rates in assisted conception: study protocol for a randomized controlled trial  

PubMed Central

Background Luteal support with progesterone is necessary for successful implantation of the embryo following egg collection and embryo transfer in an in-vitro fertilization (IVF) cycle. Progesterone has been used for as little as 2 weeks and for as long as 12 weeks of gestation. The optimal length of treatment is unresolved at present and it remains unclear how long to treat women receiving luteal supplementation. Design The trial is a prospective, randomized, double-blind, placebo-controlled trial to investigate the effect of the duration of luteal support with progesterone in IVF cycles. Following 2 weeks standard treatment and a positive biochemical pregnancy test, this randomized control trial will allocate women to a supplementary 8 weeks treatment with vaginal progesterone or 8 weeks placebo. Further studies would be required to investigate whether additional supplementation with progesterone is beneficial in early pregnancy. Discussion Currently at the Hewitt Centre, approximately 32.5% of women have a positive biochemical pregnancy test 2 weeks after embryo transfer. It is this population that is eligible for trial entry and randomization. Once the patient has confirmed a positive urinary pregnancy test they will be invited to join the trial. Once the consent form has been completed by the patient a trial prescription sheet will be sent to pharmacy with a stated collection time. The patient can then be randomized and the drugs dispensed according to pharmacy protocol. A blood sample will then be drawn for measurement of baseline hormone levels (progesterone, estradiol, free beta-human chorionic gonadotrophin, pregnancy-associated plasma protein-A, Activin A, Inhibin A and Inhibin B). The primary outcome measure is the proportion of all randomized women that continue successfully to a viable pregnancy (at least one fetus with fetal heart rate >100 beats/minute) on transabdominal/transvaginal ultrasound at 10 weeks post embryo transfer/12 weeks gestation (that is at the end of 8 weeks supplementary trial treatment). Trial registration ISRCTN05696887 PMID:22834768

2012-01-01

311

An endoderm-specific transcriptional enhancer from the mouse Gata4 gene requires GATA and homeodomain protein binding sites for function in vivo  

PubMed Central

Several transcription factors function in the specification and differentiation of the endoderm, including the zinc finger transcription factor GATA4. Despite its essential r ole in endoderm development, the transcriptional control of the Gata4 gene in the developing endoderm and its derivatives remains incompletely understood. Here, we identify a distal enhancer from the Gata4 gene, which directs expression exclusively to the visceral and definitive endoderm of transgenic mouse embryos. The activity of this enhancer is initially broad within the definitive endoderm but later restricts to developing endoderm-derived tissues, including pancreas, glandular stomach, and duodenum. The activity of this enhancer in vivo is dependent on evolutionarily-conserved HOX and GATA binding sites, which are bound by PDX-1 and GATA4, respectively. These studies establish Gata4 as a direct transcriptional target of homeodomain and GATA transcription factors in the endoderm and support a model in which GATA4 functions in the transcriptional network for pancreas formation. PMID:19777593

Rojas, Anabel; Schachterle, William; Xu, Shan-Mei; Black, Brian L.

2009-01-01

312

Combined optical coherence tomography and electroretinography system for in vivo simultaneous morphological and functional imaging of the rodent retina  

NASA Astrophysics Data System (ADS)

A combined ultrahigh resolution optical coherence tomography (UHROCT) and a electroretinography (ERG) system is presented for simultaneous imaging of the retinal structure and physiological response to light stimulation in the rodent eye. The 1060-nm UHROCT system provides ~3×5 ?m (axial×lateral) resolution in the rat retina and time resolution of 22 ?s. A custom-designed light stimulator integrated into the UHROCT imaging probe provides light stimuli with user-selected color, duration, and intensity. The performance of the combined system is demonstrated in vivo in healthy rats, and in a rat model of drug-induced outer retinal degeneration. Experimental results show correlation between the observed structural and physiological changes in the healthy and degenerated retina.

Moayed, Alireza Akhlagh; Hariri, Sepideh; Hyun, Chulho; Doran, Bruce; Kraft, Timothy W.; Boyd, Shelley; Bizheva, Kostadinka

2010-07-01

313

Integrating structural and functional imaging for computer assisted detection of prostate cancer on multi-protocol in vivo 3 Tesla MRI  

NASA Astrophysics Data System (ADS)

Screening and detection of prostate cancer (CaP) currently lacks an image-based protocol which is reflected in the high false negative rates currently associated with blinded sextant biopsies. Multi-protocol magnetic resonance imaging (MRI) offers high resolution functional and structural data about internal body structures (such as the prostate). In this paper we present a novel comprehensive computer-aided scheme for CaP detection from high resolution in vivo multi-protocol MRI by integrating functional and structural information obtained via dynamic-contrast enhanced (DCE) and T2-weighted (T2-w) MRI, respectively. Our scheme is fully-automated and comprises (a) prostate segmentation, (b) multimodal image registration, and (c) data representation and multi-classifier modules for information fusion. Following prostate boundary segmentation via an improved active shape model, the DCE/T2-w protocols and the T2-w/ex vivo histological prostatectomy specimens are brought into alignment via a deformable, multi-attribute registration scheme. T2-w/histology alignment allows for the mapping of true CaP extent onto the in vivo MRI, which is used for training and evaluation of a multi-protocol MRI CaP classifier. The meta-classifier used is a random forest constructed by bagging multiple decision tree classifiers, each trained individually on T2-w structural, textural and DCE functional attributes. 3-fold classifier cross validation was performed using a set of 18 images derived from 6 patient datasets on a per-pixel basis. Our results show that the results of CaP detection obtained from integration of T2-w structural textural data and DCE functional data (area under the ROC curve of 0.815) significantly outperforms detection based on either of the individual modalities (0.704 (T2-w) and 0.682 (DCE)). It was also found that a meta-classifier trained directly on integrated T2-w and DCE data (data-level integration) significantly outperformed a decision-level meta-classifier, constructed by combining the classifier outputs from the individual T2-w and DCE channels.

Viswanath, Satish; Bloch, B. Nicolas; Rosen, Mark; Chappelow, Jonathan; Toth, Robert; Rofsky, Neil; Lenkinski, Robert; Genega, Elizabeth; Kalyanpur, Arjun; Madabhushi, Anant

2009-02-01

314

Function of the asparagine 74 residue of the inhibitory ?-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo  

PubMed Central

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6?, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6? has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6??. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6? control transgene in the photoreceptors of +/Pde6gtm1 mice. This transgenic line was crossed with Pde6gtm1/Pde6gtm1 mice to generate animals able to synthesize only the transgenic mutant PDE6?. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6? on PDE6?? may be more dynamic and context-dependent than was replicated in vitro. PMID:21616145

Tsang, Stephen H.; Woodruff, Michael L.; Hsu, Chun Wei; Naumann, Matthew C.; Cilluffo, Marianne; Tosi, Joaquin; Lin, Chyuan-Sheng

2011-01-01

315

IMMUNE “TOLERANCE PROFILES” IN DONOR BONE MARROW INFUSED KIDNEY TRANSPLANT PATIENTS USING MULTIPLE EX VIVO FUNCTIONAL ASSAYS  

PubMed Central

Ex-vivo identification of donor specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living-related-donor-kidney transplant patients upto 11 years post-operatively, i.e., 20 haploidenticals with donor bone marrow cell (DBMC) infusions, 8 non-infused haploidentical controls (haplo-controls) and 11 HLA-identical controls (HLA-Id), using multiple ex vivo immune assays. None developed donor specific antibodies. The majority showed donor specific CTL unresponsiveness from year one onwards. 13/20 DBMC recipients became specifically donor MLR non-reactive. Depletion of donor cells in DBMC recipients still MLR reactive increased donor specific reactivity by 75±36% (p=0.04). Adding them back in low concentration caused antigen specific inhibition. The frequencies of ELISPOT granzyme-B and interferon-? producing cells somewhat paralleled the CTL and MLR responses. In the transvivo-DTH, 14/19 DBMC recipients demonstrated donor specific unresponsiveness and 16/19 showed “linked suppression”, vs 0/8 and 1/8 haplo-controls and vs. 6/10 and 1/10 HLA-ids respectively. Most importantly, when all 6 assays were performed simultaneously, 10/18 DBMC, 5/10 HLA-id but no haplo-controls were specifically donor unresponsive long-term. We propose that a cluster-analysis combining these assays will reveal tolerant recipients in whom IS minimization may safely be tested. This appears to have occurred in many DBMC infused recipients. PMID:20153397

Mathew, James M.; Ciancio, Gaetano; Burke, George W.; Garcia-Morales, Rolando O.; Rosen, Anne; Wang, Edward; Gomez, Carmen I.; Blomberg, Bonnie B.; Fuller, Laphalle; Esquenazi, Violet; Ricordi, Camillo; Miller, Joshua

2010-01-01

316

A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo  

PubMed Central

GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, the question as to whether they exist as monomers, dimers, or higher order structures in the body is unanswered. The GPCR dimer field has been dominated by techniques involving recombinant cell lines expressing mutant receptors, often involving the solubilization of the receptors. These techniques cannot be applied in vivo or even to primary cell cultures. This review will focus on a novel approach to exploring the functional properties of homodimers. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist (“inactivator”) to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. The data obtained using the 5-HT7 and 5-HT2A receptors are strongly supportive of a GPCR homodimer structure, with little evidence of monomer involvement in the function of these receptors. PMID:22119169

Teitler, Milt; Klein, Michael T.

2011-01-01

317

A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signalling Pathways during Early Xenopus  

E-print Network

and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-b/Nodal signalling pathways. Our gain-of-function screen is based

Amaya, Enrique

318

Mitochondria-targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed by in vivo 31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy.  

PubMed

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms. PMID:23482635

Righi, Valeria; Constantinou, Caterina; Mintzopoulos, Dionyssios; Khan, Nadeem; Mupparaju, S P; Rahme, Laurence G; Swartz, Harold M; Szeto, Hazel H; Tompkins, Ronald G; Tzika, A Aria

2013-06-01

319

Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function.  

PubMed Central

The Tat-responsive region (TAR) sequence is present at the 5' end of human immunodeficiency virus 1 mRNAs and as a cytoplasmic form of 58-66 nucleotides. TAR RNA blocks the activation and autophosphorylation of the double-stranded RNA-activated protein kinase in vitro. We show here that TAR RNA also prevents the double-stranded RNA-mediated inhibition of translation in a cell-free system. Mutagenic and structural analyses of TAR RNA indicate that a stem of at least 14 base pairs is required for this activity, whereas the loop and bulge required for transactivation by Tat are dispensable. Truncation of the RNA to 68 nucleotides results in the loss of translational rescue ability, suggesting that the short cytoplasmic TAR RNA produced by viral transcription in vivo may not have the capability to suppress activation of the kinase. However, because longer TAR transcripts stimulate expression in a transient assay in vivo, the TAR structure at the 5' end of viral mRNAs could still exert this function in cis. Images PMID:1360669

Gunnery, S; Green, S R; Mathews, M B

1992-01-01

320

Investigation of Functional Activity of Cells in Granulomatous Inflammatory Lesions from Mice with Latent Tuberculous Infection in the New Ex Vivo Model  

PubMed Central

The new ex vivo model system measuring functional input of individual granuloma cells to formation of granulomatous inflammatory lesions in mice with latent tuberculous infection has been developed and described in the current study. Monolayer cultures of cells that migrated from individual granulomas were established in the proposed culture settings for mouse spleen and lung granulomas induced by in vivo exposure to BCG vaccine. The cellular composition of individual granulomas was analyzed. The expression of the leukocyte surface markers such as phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and the expression of the costimulatory molecules CD80, CD83, and CD86 were tested as well as the production of proinflammatory cytokines (IFN? and IL-1?) and growth factors (GM-CSF and FGFb) for cells of individual granulomas. The colocalization of the phagocytic receptors and costimulatory molecules in the surface microdomains of granuloma cells (with and without acid-fast BCG-mycobacteria) has also been detected. It was found that some part of cytokine macrophage producers have carried acid-fast mycobacteria. Detected modulation in dynamics of production of pro-inflammatory cytokines, growth factors, and leukocyte surface markers by granuloma cells has indicated continued processes of activation and deactivation of granuloma inflammation cells during the latent tuberculous infection progress in mice. PMID:24198843

2013-01-01

321

Folic acid-functionalized up-conversion nanoparticles: toxicity studies in vivo and in vitro and targeted imaging applications  

NASA Astrophysics Data System (ADS)

Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents.Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity. Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa cells and can therefore serve as potential fluorescent contrast agents. Electronic supplementary information (ESI) available: Up-conversion luminescence spectra of UCNC-Er and UCNC-Er-FA, UCNC-Tm and UCNC-Tm-FA. Confocal luminescence imaging data collected as a series along the Z optical axis. See DOI: 10.1039/c4nr02312a

Sun, Lining; Wei, Zuwu; Chen, Haige; Liu, Jinliang; Guo, Jianjian; Cao, Ming; Wen, Tieqiao; Shi, Liyi

2014-07-01

322

Intranasal Administration of Human MSC for Ischemic Brain Injury in the Mouse: In Vitro and In Vivo Neuroregenerative Functions  

PubMed Central

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×106 or 2×106 hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×106 hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system. PMID:25396420

Donega, Vanessa; Nijboer, Cora H.; Braccioli, Luca; Slaper-Cortenbach, Ineke; Kavelaars, Annemieke; van Bel, Frank; Heijnen, Cobi J.

2014-01-01

323

Impact of the functional status of saeRS on in vivo phenotypes of Staphylococcus aureus?sarA mutants.  

PubMed

We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS(C)) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants. PMID:24779437

Beenken, Karen E; Mrak, Lara N; Zielinska, Agnieszka K; Atwood, Danielle N; Loughran, Allister J; Griffin, Linda M; Matthews, K Alice; Anthony, Allison M; Spencer, Horace J; Skinner, Robert A; Post, Ginell R; Lee, Chia Y; Smeltzer, Mark S

2014-06-01

324

Anatomical and Functional Images of in vitro and in vivo Tissues by NIR Time-domain Diffuse Optical Tomography  

NASA Astrophysics Data System (ADS)

Near infra-red (NIR) diffuse optical tomography (DOT) has gained much attention and it will be clinically applied to imaging breast, neonatal head, and the hemodynamics of the brain because of its noninvasiveness and deep penetration in biological tissue. Prior to achieving the imaging of infant brain using DOT, the developed methodologies need to be experimentally justified by imaging some real organs with simpler structures. Here we report our results of an in vitro chicken leg and an in vivo exercising human forearm from the data measured by a multi-channel time-resolved NIR system. Tomographic images were reconstructed by a two-dimensional image reconstruction algorithm based on a modified generalized pulse spectrum technique for simultaneous reconstruction of the µa and µs´. The absolute µa- and µs´-images revealed the inner structures of the chicken leg and the forearm, where the bones were clearly distinguished from the muscle. The ?µa-images showed the blood volume changes during the forearm exercise, proving that the system and the image reconstruction algorithm could potentially be used for imaging not only the anatomic structure but also the hemodynamics in neonatal heads.

Zhao, Huijuan; Gao, Feng; Tanikawa, Yukari; Homma, Kazuhiro; Onodera, Yoichi; Yamada, Yukio

325

A 19-year-old man with leucocyte adhesion deficiency. In vitro and in vivo studies of leucocyte function  

PubMed Central

We describe a male patient with leucocyte adhesion molecule deficiency (LAD) of moderate phenotype. Although diagnosis was made only 2 years before his death, the patient survived until 19 years of age. This enabled us to perform a number of novel investigations, both in vivo and in vitro, relating to his leucocyte biology. Monocytes cultured in vitro matured into morphologically normal, phagocytically capable macrophages, which were able to recognize aged `apoptotic' neutrophils. By injection of radiolabelled autologous neutrophils we demonstrated a prolonged neutrophil half-life, but normal margination, de-margination on exercise, and splenic pooling. Neutrophile adherence in vitro to vascular endothelium was normal. Histological examination of the patient's lungs at postmortem showed intravascular aggregation of polymorphonuclear leucocytes but a paucity of cells in the interstitium and alveolar spaces. These findings indicate that the peripheral blood leucocytosis commonly observed in these patients may be due to prolonged intravascular neutrophil survival, and suggest that CD11/18 molecules have an important role in facilitating neutrophil emigration from blood vessels at sites of inflammation. ImagesFig. 1Fig. 5Fig. 7 PMID:1673876

Davies, K. A.; Toothill, V. J.; Savill, J.; Hotchin, N.; Peters, A. M.; Pearson, J. D.; Haslett, C.; Burke, M.; Law, S. K. A.; Mercer, N. F. G.; Walport, M. J.; Webster, A. D. B.

1991-01-01

326

Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model  

E-print Network

Short hairpin RNAs (shRNAs) capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor-suppressor-gene loss in mice. By selecting for shRNAs capable of accelerating lymphomagenesis in a ...

Bric, Anka

327

In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication  

Microsoft Academic Search

Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase -endonuclease that participates

Sudha Sharma; Joshua A. Sommers; Robert M. Brosh

2004-01-01

328

Functional neuroanatomy of the human premotor oculomotor brainstem nuclei: insights from postmortem and advanced in vivo imaging studies  

Microsoft Academic Search

Considerable progress has been made recently in the field of the functional neuroanatomy of the primate oculomotor system,\\u000a which has also improved our understanding of the structure, organization and function of the human oculomotor system. In the\\u000a present review we provide for the first time an overview of the neuroanatomical basis of eye movement control in humans as\\u000a revealed by

Udo Rüb; Joanna C. Jen; Heiko Braak; Thomas Deller

2008-01-01

329

Characterization of the alpha-gamma and alpha-beta complex: evidence for an in vivo functional role of alpha-crystallin as a molecular chaperone  

NASA Technical Reports Server (NTRS)

Previous studies have demonstrated that in vitro, alpha-crystallin can protect other lens proteins against extensive denaturation and aggregation. The mechanism of this protection involves preferential binding of the partially denatured protein to a central region of the native alpha-crystallin complex. To test whether a similar phenomenon might occur in vivo, a high molecular weight aggregate (HMWA) fraction was isolated from the aged bovine lens. Negative staining of this preparation revealed the presence of particles of 13-14 nm diameter, characteristic of alpha-crystallin. Immunolocalization of the same particles using antiserum specific for gamma- and beta-crystallins demonstrated preferential binding of these crystallins to the central region of the alpha-crystallin complex. Together, these results provide evidence that in the intact lens, the alpha-crystallins are functionally important molecular chaperones.

Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1994-01-01

330

In vivo cranial suture function and suture morphology in the extant fish Polypterus: implications for inferring skull function in living and fossil fish  

Microsoft Academic Search

This study describes the mechanical role that cranial sutures play in fish during feeding. The long-term goal of our work is to establish relationships between suture form and function, so that functional inferences can be made from suture morphology in fossil taxa. To this end, strain gauges were surgically implanted across selected sutures in the skull roof of four individuals

Molly J. Markey; Russell P. Main; Charles R. Marshall

2006-01-01

331

Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo  

NASA Astrophysics Data System (ADS)

A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery.A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr00294b

Lin, Daoshu; Cheng, Qiang; Jiang, Qian; Huang, Yuanyu; Yang, Zheng; Han, Shangcong; Zhao, Yuning; Guo, Shutao; Liang, Zicai; Dong, Anjie

2013-05-01

332

BRCA2 is ubiquitinated in vivo and interacts with USP11, a deubiquitinating enzyme that exhibits prosurvival function in the cellular response to DNA damage.  

PubMed

Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination. PMID:15314155

Schoenfeld, Alan R; Apgar, Sarah; Dolios, Georgia; Wang, Rong; Aaronson, Stuart A

2004-09-01

333

BRCA2 Is Ubiquitinated In Vivo and Interacts with USP11, a Deubiquitinating Enzyme That Exhibits Prosurvival Function in the Cellular Response to DNA Damage  

PubMed Central

Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination. PMID:15314155

Schoenfeld, Alan R.; Apgar, Sarah; Dolios, Georgia; Wang, Rong; Aaronson, Stuart A.

2004-01-01

334

A Conserved Salt Bridge in the G-Loop of Multiple Protein Kinases is Important for Catalysis and for in Vivo Lyn Function  

PubMed Central

The glycine-rich G-loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases from plants to humans. By limiting G-loop flexibility, it controls ATP binding, catalysis and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn-/- mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G-loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein. PMID:19150426

Barouch-Bentov, Rina; Che, Jianwei; Lee, Christian C.; Yang, Yating; Herman, Ann; Jia, Yong; Velentza, Anastasia; Watson, James; Sternberg, Luise; Kim, Sunjun; Ziaee, Niusha; Miller, Andrew; Jackson, Carie; Fujimoto, Manabu; Young, Mike; Batalov, Serge; Liu, Yi; Warmuth, Markus; Wiltshire, Tim; Cooke, Michael P.; Sauer, Karsten

2009-01-01

335

RNA Interference Mutant Induction In Vivo Demonstrates the Essential Nature of Trypanosome Flagellar Function during Mammalian Infection?  

PubMed Central

We demonstrate that trypanosomes compromised in flagellar function are rapidly cleared from infected mice. Analysis of the PFR2 bloodstream RNA interference mutant revealed that defective cell motility occurred prior to cytokinesis failure. This validation provides a paradigm for the flagellum as a target for future assays and interventions against this human pathogen. PMID:17513568

Griffiths, Samantha; Portman, Neil; Taylor, Philip R.; Gordon, Siamon; Ginger, Michael L.; Gull, Keith

2007-01-01

336

JUNONIA COENIA DENSOVIRUS DERIVED VECTORS OFFER NEW AVENUES FOR EXPLORATION AND EXAMINATION OF POLYDNAVIRUS (PDV) GENE FUNCTION IN VIVO  

Technology Transfer Automated Retrieval System (TEKTRAN)

Endoparasitic wasps often utilize symbiotic polydnaviruses (PDVs) as a means to overcome and regulate host development and immunity. Genetic manipulations and functional investigations of PDV immunosuppressive activities have been difficult due to the inability to replicate PDV DNA outside host wasp...

337

The course and the anatomo-functional relationships of the optic radiation: a combined study with 'post mortem' dissections and 'in vivo' direct electrical mapping.  

PubMed

Even if different dissection, tractographic and connectivity studies provided pure anatomical evidences about the optic radiations (ORs), descriptions of both the anatomical structure and the anatomo-functional relationships of the ORs with the adjacent bundles were not reported. We propose a detailed anatomical and functional study with 'post mortem' dissections and 'in vivo' direct electrical stimulation (DES) of the OR, demonstrating also the relationships with the adjacent eloquent bundles in a neurosurgical 'connectomic' perspective. Six human hemispheres (three left, three right) were dissected after a modified Klingler's preparation. The anatomy of the white matter was analysed according to systematic and topographical surgical perspectives. The anatomical results were correlated to the functional responses collected during three resections of tumours guided by cortico-subcortical DES during awake procedures. We identified two groups of fibres forming the OR. The superior component runs along the lateral wall of the occipital horn, the trigone and the supero-medial wall of the temporal horn. The inferior component covers inferiorly the occipital horn and the trigone, the lateral wall of the temporal horn and arches antero-medially to form the Meyer's Loop. The inferior fronto-occipital fascicle (IFOF) covers completely the superior OR along its entire course, as confirmed by the subcortical DES. The inferior longitudinal fascicle runs in a postero-anterior and inferior direction, covering the superior OR posteriorly and the inferior OR anteriorly. The IFOF identification allows the preservation of the superior OR in the anterior temporal resection, avoiding post-operative complete hemianopia. The identification of the superior OR during the posterior temporal, inferior parietal and occipital resections leads to the preservation of the IFOF and of the eloquent functions it subserves. The accurate knowledge of the OR course and the relationships with the adjacent bundles is crucial to optimize quality of resection and functional outcome. PMID:25402811

Sarubbo, Silvio; De Benedictis, Alessandro; Milani, Paola; Paradiso, Beatrice; Barbareschi, Mattia; Rozzanigo, Umbero; Colarusso, Enzo; Tugnoli, Valeria; Farneti, Marco; Granieri, Enrico; Duffau, Hugues; Chioffi, Franco

2015-01-01

338

Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo  

PubMed Central

Mutations in LRRK2 cause a dominantly inherited form of Parkinson’s disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling. PMID:25288684

Dodson, Mark W.; Leung, Lok K.; Lone, Mohiddin; Lizzio, Michael A.; Guo, Ming

2014-01-01

339

Angiogenesis and vascular function in the ovary.  

PubMed

Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular-luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte-endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility. PMID:19786399

Robinson, R S; Woad, K J; Hammond, A J; Laird, M; Hunter, M G; Mann, G E

2009-12-01

340

Comparing the in Vivo Function of ?-Carboxysomes and ?-Carboxysomes in Two Model Cyanobacteria1[W][OPEN  

PubMed Central

The carbon dioxide (CO2)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO2 concentrations at the site of fixation, providing an advantage in low-CO2 environments. Cyanobacteria with Form-IA Rubisco contain ?-carboxysomes, and cyanobacteria with Form-IB Rubisco contain ?-carboxysomes. The two carboxysome types have arisen through convergent evolution, and ?-cyanobacteria and ?-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from ?-cyanobacteria (Cyanobium spp. PCC7001) and ?-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3?) transporter systems. Despite evolutionary and structural differences between ?-carboxysomes and ?-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external Ci and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and Ci pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic Ci, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm Ci. This finding has bearing on the viability of transplanting functional carboxysomes into the C3 chloroplast. PMID:24642960

Whitehead, Lynne; Long, Benedict M.; Price, G. Dean; Badger, Murray R.

2014-01-01

341

Ectodomains of the LDL Receptor-Related Proteins LRP1b and LRP4 Have Anchorage Independent Functions In Vivo  

Microsoft Academic Search

BackgroundThe low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation

Martin F. Dietrich; Louise van der Weyden; Haydn M. Prosser; Allan Bradley; Joachim Herz; David J. Adams; Pieter H. Reitsma

2010-01-01

342

Molecular Advances in Reporter Genes: The Need to Witness the Function of Stem Cells in Failing Heart in Vivo  

Microsoft Academic Search

Stem cells possess the ability to terminally differentiate in cell phenotypes belonging to several different lineages. Over\\u000a the last decade, transplant of adult stem cells into the injuried myocardium has been widely studied as a revolutionary approach\\u000a to promote the non-pharmacological improvement or replacement of the lost function. In spite of the tantalizing perspectives\\u000a and controversial results, several questions about

Silvia Agostini; Fabio A. Recchia; Vincenzo Lionetti

343

Further Development of a Tissue Engineered Muscle Repair Construct In Vitro for Enhanced Functional Recovery Following Implantation In Vivo in a Murine Model of Volumetric Muscle Loss Injury  

PubMed Central

Volumetric muscle loss (VML) can result from trauma and surgery in civilian and military populations, resulting in irrecoverable functional and cosmetic deficits that cannot be effectively treated with current therapies. Previous work evaluated a bioreactor-based tissue engineering approach in which muscle derived cells (MDCs) were seeded onto bladder acellular matrices (BAM) and mechanically preconditioned. This first generation tissue engineered muscle repair (TEMR) construct exhibited a largely differentiated cellular morphology consisting primarily of myotubes, and moreover, significantly improved functional recovery within 2 months of implantation in a murine latissimus dorsi (LD) muscle with a surgically created VML injury. The present report extends these initial observations to further document the importance of the cellular phenotype and composition of the TEMR construct in vitro to the functional recovery observed following implantation in vivo. To this end, three distinct TEMR constructs were created by seeding MDCs onto BAM as follows: (1) a short-term cellular proliferation of MDCs to generate primarily myoblasts without bioreactor preconditioning (TEMR-1SP), (2) a prolonged cellular differentiation and maturation period that included bioreactor preconditioning (TEMR-1SPD; identical to the first generation TEMR construct), and (3) similar treatment as TEMR-1SPD but with a second application of MDCs during bioreactor preconditioning (TEMR-2SPD); simulating aspects of “exercise” in vitro. Assessment of maximal tetanic force generation on retrieved LD muscles in vitro revealed that TEMR-1SP and TEMR-1SPD constructs promoted either an accelerated (i.e., 1 month) or a prolonged (i.e., 2 month postinjury) functional recovery, respectively, of similar magnitude. Meanwhile, TEMR-2SPD constructs promoted both an accelerated and prolonged functional recovery, resulting in twice the magnitude of functional recovery of either TEMR-1SP or TEMR-1SPD constructs. Histological and molecular analyses indicated that TEMR constructs mediated functional recovery via regeneration of functional muscle fibers either at the interface of the construct and the native tissue or within the BAM scaffolding independent of the native tissue. Taken together these findings are encouraging for the further development and clinical application of TEMR constructs as a VML injury treatment. PMID:22439962

Corona, Benjamin T.; Machingal, Masood A.; Criswell, Tracy; Vadhavkar, Manasi; Dannahower, Ashley C.; Bergman, Christopher; Zhao, Weixin

2012-01-01

344

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire  

PubMed Central

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a ‘liquid biopsy’ with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immunocytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0–50) CTCs in breast cancer (n=12) and 16 (2–515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment. PMID:22825490

SAUCEDO-ZENI, NADIA; MEWES, STEFFI; NIESTROJ, ROBERT; GASIOROWSKI, LUKASZ; MURAWA, DAVID; NOWACZYK, PIOTR; TOMASI, TATIANA; WEBER, EKKEHARD; DWORACKI, GRZEGORZ; MORGENTHALER, NILS G.; JANSEN, HEIKE; PROPPING, CORINNA; STERZYNSKA, KAROLINA; DYSZKIEWICZ, WOJCIECH; ZABEL, MACIEJ; KIECHLE, MARION; REUNING, UTE; SCHMITT, MANFRED; LÜCKE, KLAUS

2012-01-01

345

Absolute abundance and function of intestinal drug transporters: a prerequisite for fully mechanistic in vitro-in vivo extrapolation of oral drug absorption.  

PubMed

The use of whole body physiological-based pharmacokinetic (PBPK) models linked with in vitro-in vivo extrapolation (IVIVE) of kinetic parameters from laboratory experiments, has become embedded within many of the pharmaceutical industry and is used even as part of regulatory submissions. These include the influence of transporter proteins on drug disposition, a subject for which we have witnessed an increasing awareness. A combination of the development of high-powered analytical techniques and antibody-based technology, together with a realization that an understanding of absolute transporter protein abundances together with activity can potentially enhance the modelling of transporter kinetics by PBPK-IVIVE link models. This review summarizes the mechanistic approaches to integrate suitable non-biased in vitro transporter kinetic data relevant to the intestine (i.e. 'intrinsic' K(i) , 'intrinsic' K(m) ), by in vitro system modelling for these kinetic inputs with the advantages of, and challenges for, generating these data for input into PBPK models. This step is considered as a prerequisite for mechanistic modelling of the oral absorption for drugs that are substrates for transporters. Various approaches are provided to integrate intestinal transporter expression into PBPK models with a perspective on the incorporation of the absolute abundance/activity of transporters to enhance the predictive power of the models. We define the key intestinal tissue and functional expression-based scaling factors required. The objective is to use these for facilitating the extrapolation from in vitro intestinal transporter assays to the in vivo system, using absolute quantification methodologies. The models could be used to elucidate the complex relationship and relative importance of metabolizing enzymes and transporters in drug disposition and toxicity. PMID:22927116

Harwood, M D; Neuhoff, S; Carlson, G L; Warhurst, G; Rostami-Hodjegan, A

2013-01-01

346

Ultrathin sP(EO-stat-PO) hydrogel coatings are biocompatible and preserve functionality of surface bound growth factors in vivo.  

PubMed

Hydrogel coatings prepared from reactive star shaped polyethylene oxide based prepolymers (NCO-sP(EO-stat-PO)) minimize unspecific protein adsorption in vitro, while proteins immobilized on NCO-sP(EO-stat-PO) coatings retain their structure and biological function. The aim of the present study was to assess biocompatibility and the effect on early osseointegrative properties of a NCO-sP(EO-stat-PO) coating with additional RGD-peptides and augmentation with bone morphogenetic protein-4 (BMP) used on a medical grade high-density polyethylene (HDPE) base under in vivo circumstances. For testing of biocompatibility dishes with large amounts of bulk NCO-sP(EO-stat-PO) were implanted subcutaneously into 14 Wistar rats. In a second set-up functionalization of implants with ultrathin surface layers by coating ammonia-plasma treated HDPE with NCO-sP(EO-stat-PO), functionalization with linear RGD-peptides, and augmentation with RGD and BMP-4 was analyzed. Therefore, implants were placed subcutaneously in the paravertebral tissue and transcortically in the distal femur of another 14 Wistar rats. Both tests revealed no signs of enhanced inflammation of the surrounding tissue analyzed by CD68, IL-1ß-/TNF-?-antibody staining, nor systemic toxic reactions according to histological analysis of various organs. The mean thickness of the fibrous tissue surrounding the femoral implants was highest in native HDPE-implants and tended to be lower in all NCO-sP(EO-stat-PO) modified implants. Micro-CT analysis revealed a significant increase of peri-implant bone volume in RGD/BMP-4 coated samples. These results demonstrate that even very low amounts of surface bound growth factors do have significant effects when immobilized in an environment that retains their biological function. Hence, NCO-sP(EO-stat-PO)-coatings could offer an attractive platform to improve integration of orthopedic implants. PMID:23801500

Neuerburg, Carl; Recknagel, Stefan; Fiedler, Jörg; Groll, Jürgen; Moeller, Martin; Bruellhoff, Kristina; Reichel, Heiko; Ignatius, Anita; Brenner, Rolf E

2013-10-01

347

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice  

PubMed Central

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied. The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle. PMID:23426237

Hakim, Chady H.; Wasala, Nalinda B.; Duan, Dongsheng

2013-01-01

348

Effect of Hyperkalemia and Hemolysis Caused by Hyperacute Rejection on Cardiac Function in Pig to Human Ex Vivo Xenogeneic Cardiac Perfusion Model  

PubMed Central

Background and Objectives Hyperacute rejection (HAR) is a major obstacle to successful xenotransplantation of vascularized organs. This study was conducted to observe the effect of hemolysis of perfused human whole blood on pig heart function, and determine the major risk factors for preservation of xenoperfused cardiac function using ex-vivo pig to human xenogeneic cardiac perfusion model. Materials and Methods Harvested pig hearts were perfused with normal human whole blood (group 1), two different types of pre-treated human whole blood (group 2: immunoglobulins were depleted by plasmapheresis, group 3: pre-treated with plasmapheresis, GAS914, cobra venom factor (CVF) and steroid), and normal porcine whole blood as control (group 4) for 3 hours. Results Duration of heart beat was significantly prolonged in group 2 and group 3. Histological examination showed widespread HAR features but was gradually delayed in groups 2 and 3 compared to group 1. The absolute levels of serum creatine kinase-MB and Troponin I increased gradually, and was lower in group 3. Serum hemoglobin levels were rapidly increased in groups 3 and 4, compared to group 1. Extracellular potassium level increased sharply from the beginning of blood perfusion in groups 1, 2 and 3, compared to group 4. Conclusion Pretreatment of human whole blood, including immunoglobulin depletion, CVF and steroid reduced and delayed the destruction of pig myocardium by HAR. However, the increased extracellular potassium levels in groups 1, 2 and 3 reflected that these treatments could not prohibit myocardial injury by HAR. PMID:21519511

Kim, Jun Seok; Lee, Hak-Mo; Oh, Byoung Chol; Lim, Hong-Gook

2011-01-01

349

Overexpression of Mitogen-activated Protein Kinase Kinase 6 in the Heart Improves Functional Recovery from Ischemia in Vitro and Protects against Myocardial Infarction in Vivo*  

PubMed Central

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, ?B-crystallin (?BC), in cultured cardiac myocytes, we examined ?BC levels in the mouse hearts. The level of ?BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in ?BC levels in the mitochondrial fractions of TG mouse hearts but no increase in ?BC levels in any of the other fractions analyzed. These alterations in ?BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts. PMID:15492008

Martindale, Joshua J.; Wall, Jason A.; Martinez-Longoria, Diana M.; Aryal, Prafulla; Rockman, Howard A.; Guo, Yiru; Bolli, Roberto; Glembotski, Christopher C.

2013-01-01

350

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.  

PubMed

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications. PMID:22817267

Paul, Arghya; Shao, Wei; Abbasi, Sana; Shum-Tim, Dominique; Prakash, Satya

2012-09-01

351

Engineered anti-CD70 antibody with multiple effector functions exhibits in vitro and in vivo antitumor activities.  

PubMed

Antigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is highly restricted but is aberrantly expressed in hematologic malignancies including non-Hodgkin lymphoma (NHL), Hodgkin disease, and multiple myeloma. In addition, solid tumors such as renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, meduloblastoma, and glioblastoma express high levels of this antigen. To functionally target CD70-expressing cancers, a murine anti-CD70 monoclonal antibody was engineered to contain human IgG1 constant domains. The engineered antibody retained the binding specificity of the murine parent monoclonal antibody and was shown to induce Fc-mediated effector functions including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis in vitro. Further, administration of this antibody significantly prolonged survival of severe combined immunodeficient (SCID) mice bearing CD70+ disseminated human NHL xenografts. Survival of these mice was dependent upon the activity of resident effector cells including neutrophils, macrophages, and natural killer (NK) cells. These data suggest that an anti-CD70 antibody, when engineered to contain human IgG1 constant domains, possesses effector cell-mediated antitumor activity and has potential utility for anticancer therapy. PMID:17038522

McEarchern, Julie A; Oflazoglu, Ezogelin; Francisco, Leigh; McDonagh, Charlotte F; Gordon, Kristine A; Stone, Ivan; Klussman, Kerry; Turcott, Eileen; van Rooijen, Nico; Carter, Paul; Grewal, Iqbal S; Wahl, Alan F; Law, Che-Leung

2007-02-01

352

p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo  

PubMed Central

Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-? and GPV. We recently demonstrated that tumor necrosis factor-? converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

2010-01-01

353

In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication.  

PubMed

Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase-endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN-FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing. PMID:15282207

Sharma, Sudha; Sommers, Joshua A; Brosh, Robert M

2004-10-01

354

Cell-specific in vivo functions of glycosphingolipids: lessons from genetic deletions of enzymes involved in glycosphingolipid synthesis.  

PubMed

Glycosphingolipids (GSLs) are believed to be involved in many cellular events including trafficking, signaling and cellular interactions. Over the past decade considerable progress was made elucidating the function of GSLs by generating and exploring animal models with GSL-deficiency. Initial studies focused on exploring the role of complex sialic acid containing GSLs (gangliosides) in neuronal tissue. Although complex gangliosides were absent, surprisingly, the phenotype observed was rather mild. In subsequent studies, several mouse models with combinations of gene-deletions encoding GSL-synthesizing enzymes were developed. The results indicated that reduction of GSL-complexity correlated with severity of phenotypes. However, in these mice, accumulation of precursor GSLs or neobiosynthesized GSL-series seemed to partly compensate the loss of GSLs. Thus, UDP-glucose:ceramide glucosyltransferase (Ugcg), catalyzing the basic step of the glucosylceramide-based GSL-biosynthesis, was genetically disrupted. A total systemic deletion of Ugcg caused early embryonic lethality. Therefore, Ugcg was eliminated in a cell-specific manner using the cre/loxP-system. New insights into the cellular function of GSLs were gained. It was demonstrated that neurons require GSLs for differentiation and maintenance. In keratinocytes, preservation of the skin barrier depends on GSL synthesis and in enterocytes of the small intestine GSLs are involved in endocytosis and vesicular transport. PMID:23473748

Jennemann, Richard; Gröne, Hermann-Josef

2013-04-01

355

Antibody limits in vivo murid herpesvirus-4 replication by IgG Fc receptor-dependent functions  

PubMed Central

Antibody is an important antiviral defence. However, it is considered to do little against human gamma-herpesviruses, which establish predominantly latent infections regulated by T cells. One limitation on analysing these infections has been that latency is already well-established at clinical presentation; early infection may still be accessible to antibody. Here, using murid herpesvirus-4 (MuHV-4), we tested the impact of adoptively transferred antibody on early gamma-herpesvirus infection. Immune sera and neutralizing and non-neutralizing monoclonal antibodies (mAbs) all reduced acute lytic MuHV-4 replication. The reductions, even by neutralizing mAbs, were largely or completely dependent on host IgG Fc receptors. Therefore, passive antibody can blunt acute gamma-herpesvirus lytic infection, and does this principally by IgG Fc-dependent functions rather than by neutralization. PMID:19625459

Wright, Debbie E.; Colaco, Susanna; Colaco, Camilo; Stevenson, Philip G.

2009-01-01

356

In Vivo Effect of Mutant ELOVL4 on the Expression and Function of Wild-Type ELOVL4  

PubMed Central

Purpose. Mutations in the elongation of very long chain fatty acids 4 (ELOVL4) gene cause human Stargardt's macular dystrophy 3 (STGD3), a juvenile onset dominant form of macular degeneration. To understand the role of the ELOVL4 protein in retinal function, several mouse models have been developed by using transgenic (TG), knock-in (Elovl4+/mut), and knockout (Elovl4+/?) approaches. Here we analyzed quantitatively the ELOVL4 protein and its enzymatic products (very long chain saturated fatty acid [VLC-FA] and VLC–polyunsaturated fatty acid [VLC-PUFA]) in the retinas of 8 to 10-week-old TG1+, TG2+, and Elovl4+/mut mice that harbor the mutant ELOVL4 and compared them to their wild-type littermates and Elovl4+/? that do not express the mutant protein. We also analyzed skin from these mice to gain insight into the pathogenesis resulting from the ELOVL4 mutation. Methods. ELOVL4 protein localization in the retina was determined by immunohistochemistry. Levels of wild-type ELOVL4 protein in skin and retinas were determined by Western blotting. Total lipids from skin and retinas were measured by gas chromatography–mass spectrometry (GC-MS). Retinal glycerophosphatidylcholines (PC) were analyzed by tandem mass spectrometry. Results. Immunohistochemical and Western analysis indicated that wild-type ELOVL4 protein was reduced in heterozygous Elovl4+/mut and Elovl4+/? retinas, but not in TG2+ retinas. We found that VLC-FA was reduced by 50% in the skin of Elovl4+/? and by 60% to 65% in Elovl4+/mut. We found VLC-PUFA levels at ?50% in both the retinas, and wild-type levels of VLC-PUFA in TG2+ retinas. Conclusions. We conclude that the presence of the mutant ELOVL4 does not affect the function of wild-type ELOVL4 in the fully developed 8- to 10-week-old retinas. PMID:24644051

Mandal, Nawajes A.; Tran, Julie-Thu A.; Zheng, Lixin; Wilkerson, Joseph L.; Brush, Richard S.; McRae, Joel; Agbaga, Martin-Paul; Zhang, Kang; Petrukhin, Konstantin; Ayyagari, Radha; Anderson, Robert E.

2014-01-01

357

Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy.  

PubMed

Reperfusion injury of the heart is a severe complication of angioplasty treatment of acute myocardial ischemia, for which no therapeutics are currently available. The present study aimed to identify whether and how a novel protein, netrin-1, induces cardioprotection in vivo during ischemia/reperfusion (I/R) injury. Wild type (WT) C57BL6/J mice were subjected to a 30min coronary occlusion followed by a 24h reperfusion with vehicle (normal saline), netrin-1, UO126 (MEK1/2 inhibitor), PTIO (nitric oxide/NO scavenger), netrin-1/UO126 or netrin-1/PTIO intraventricularly. Some were injected of netrin-1 via tail vein. Netrin-1 at 5?g/kg induced a substantial reduction in infarct size (19.7±5.0% from 41.3±1.8% in the controls), and markedly improved cardiac function as measured by ejection fraction and fractional shortening from echocardiography. Experiments with mice deficient in netrin-1 receptor DCC (deleted in colorectal cancer, DCC+/-), or reperfusion with netrin-1/UO126 or netrin-1/PTIO, attenuated the protective effects of netrin-1, implicating intermediate roles of DCC, ERK1/2 and NO. Netrin-1 induced phosphorylation of ERK1/2 and eNOS was abolished in DCC+/-mice. Electron spin resonance (ESR) determination of NO production from isolated left ventricles demonstrated that netrin-1 improves NO bioavailability, which was attenuated by UO126 or in DCC+/-mice, suggesting upstream roles of DCC and ERK1/2 in NO production. Netrin-1 further reduced mitochondrial swelling and mitochondrial superoxide production, which was absent when co-treated with PTIO or UO126, or in DCC+/-mice, indicating critical roles of DCC, ERK1/2 and NO in preserving mitochondrial integrity. In a permanent coronary ligation model of myocardial infarction (MI) to assess post-MI remodeling, netrin-1 abolished the marked increase in autophagy. In summary, our data demonstrate robust cardioprotective effect of netrin-1 in vivo, as shown by reduced infarct size and improved cardiac function. Mechanistically, this protection is mediated by netrin-1 receptor DCC, and NO dependent preservation of mitochondria. This work clearly establishes a therapeutic potential of netrin-1 for acute treatment of MI, perhaps also for chronic post-MI remodeling. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. PMID:24928309

Bouhidel, Jalaleddinne Omar; Wang, Ping; Siu, Kin Lung; Li, Hong; Youn, Ji Youn; Cai, Hua

2015-02-01

358

Effect of exogenous progesterone supplementation in the early luteal phase post-insemination on pregnancy per artificial insemination in Holstein-Friesian cows.  

PubMed

One of the main determining factors of pregnancy per artificial insemination (P/AI) is an optimum concentration of progesterone (P4) in the early luteal phase. This study examined the effects of P4 supplementation on P/AI in lactating Holstein-Friesian cows. A total of 453 cows in 8 spring-calving herds were used in the study. Following AI, cows were randomly assigned to 1 of 2 treatment groups: (1) no subsequent treatment (control; n=221); (2) insertion of a Controlled Internal Drug Release device (CIDR) from day 4 to day 9 post-estrus (supplemented; n=232). Pregnancy per AI was determined by transrectal ultrasonography at day 30 following AI. Insertion of a CIDR increased concentrations of milk P4 in supplemented cows by 4.78ng/mL between day 4 and 4.5 in comparison with a 0.55ng/mL increase in control cows. Progesterone supplementation from day 4 to 9 after AI decreased P/AI by 12 percentage points (56 vs 44%). There was a positive linear and quadratic relationship between P/AI and milk concentration of P4 on day 4 post-estrus in control cows. An optimum concentration of 2.5ng/mL on day 4 was calculated from the logistic regression curve to achieve a probability of P/AI of 65%. When both treatments groups were included in the analysis, there was no association between P/AI and concentrations of P4 on day 4. The results of the study indicate that supplementation with P4 initiated in the early luteal phase had a negative effect on P/AI in dairy cows. PMID:25205297

Parr, M H; Crowe, M A; Lonergan, P; Evans, A C O; Rizos, D; Diskin, M G

2014-11-10

359

A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae.  

PubMed

BackgroundThe second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence.ResultsComplementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization.ConclusionsThese results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae. PMID:25343965

McKee, Robert W; Kariisa, Ankunda; Mudrak, Benjamin; Whitaker, Courtney; Tamayo, Rita

2014-10-25

360

In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach.  

PubMed

Different optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP-aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca(2+)-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca(2+)-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca(2+)-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca(2+)-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium. PMID:23287020

Minocci, Daiana; Carbognin, Elena; Murmu, Meena Sriti; Martin, Jean-René

2013-07-01

361

Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo  

PubMed Central

Summary Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. We, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS–bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. We propose that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision. PMID:23264735

Efetova, Marina; Petereit, Linda; Rosiewicz, Kamil; Overend, Gayle; Haußig, Florian; Hovemann, Bernhard T.; Cabrero, Pablo; Dow, Julian A. T.; Schwärzel, Martin

2013-01-01

362

Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo.  

PubMed

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2(-/-)IL-2R?c(-/-) mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRP?; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRP?, we introduced functional CD47/SIRP? interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRP? interactions, human T cells accumulated as CD4(+) or CD8(+) single-positive, naive, resting T cells in the presence of functional CD47/SIRP? interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRP? is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo. PMID:21788504

Legrand, Nicolas; Huntington, Nicholas D; Nagasawa, Maho; Bakker, Arjen Q; Schotte, Remko; Strick-Marchand, Hélène; de Geus, Sandra J; Pouw, Stephan M; Böhne, Martino; Voordouw, Arie; Weijer, Kees; Di Santo, James P; Spits, Hergen

2011-08-01

363