Expression Of Cyclophilin Research Articles

Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D

Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here, we report that inhibition of complex I by rotenone is more effective at PTP inhibition than cyclosporin A in tissues that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sensitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone, the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration; and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably (i) genetic ablation of cyclophilin D or its displacement with cyclosporin A restored PTP inhibition by rotenone in tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et al. (2008) J. Biol. Chem. 283, 26307–26311]. These results indicate that inhibition of complex I by rotenone or metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism; and that cells can modulate their PTP response to complex I inhibition by modifying the expression of cyclophilin D, a finding that has major implications for pore modulation in vivo.

Leptin anti‐apoptotic mechanisms in LPS‐induced PC12 cells

Evidence indicates that leptin has neuroprotective properties as it protects neurons from a variety of neurotoxic agents. However, leptin mechanisms have not been well studied. This study evaluated the anti‐apoptotic effects of leptin in LPS‐induced PC12 cells. Cells were pretreated with varying doses of leptin for 2 hours before stimulation with LPS for 48 hours. The results showed that LPS decreased cell viability, while pretreatment with leptin increased it in a dose‐dependent manner, as measured with MTT assay. Furthermore, western blot analysis showed a dose‐dependent reduction in the expression of cyclophilin D (CypD), an important component in the apoptosis pathway. The anti‐apoptotic effect of leptin was confirmed by the decrease in the expression of pro‐apoptotic protein Bad, and the increase in the expression of anti‐apoptotic protein BcL2. The present study indicates that leptin or its analogs, through inhibition of apoptosis may be promising novel therapeutic agents for the treatment of neurodegenerative diseases such as AD.

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

175 EFFECT OF HEAT STRESS ON GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS (BOS TAURUS VS. BOS INDICUS)

Heat stress (HS) reduces the production of bovine embryos, especially taurine embryos, which are not adapted to heat. However, little is known about the competence of embryos produced under HS in breeds adapted or not adapted to heat. The aim of this study was to compare the gene expression of PLAC8, HSF1, COX2 and CDX2, related to competence and implantation, in bovine in vitro-produced embryos (Bos taurus vs Bos indicus), submitted or not submitted to HS. Oocytes from Nelore (zebu) and Jersey (taurine) cows were aspirated by ovum pickup, in vitro-matured in TCM-199 medium with bicarbonate containing 10% FCS, 2 μg mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 20 μg mL–1 of FSH and 10 IU mL–1 of LH for 22 h at 38.5°C in 5% CO2 in air. Matured oocytes were fertilized with semen from Nelore (n = 6) and Jersey (n = 6) bulls, respectively, at 38.5°C in 5% CO2 in air. The fertilization medium was TALP-IVF supplemented with 6 mg mL–1 of fatty acid-free BSA, 2 μL mL–1 of pyruvate, 75 μg mL–1 of gentamicin, 11 μg mL–1 of heparin and 44 μL mL–1 of penicillamine, hypotaurine and epinephrine. The day of fertilization was considered Day 0. Twelve hours post-insemination, presumptive zygotes were denuded and randomly divided into 2 groups, nonstressed or stressed and both were in vitro cultured at 38.5°C in 90% N2, 5% CO2 and 5% O2 in SOFaaci medium supplemented with 5% FCS, 5% BSA and 0,2% sodium pyruvate. In the stressed group, 96-h post-insemination embryos were subjected to HS of 41°C for 6 consecutive hours and then returned to 38.5°C. On Day 7, pools with 5 blastocysts [Nelore (n = 9); Nelore HS (n = 7); Jersey (n = 5); Jersey HS (n = 5)] were subjected to RNA extraction (RNeasy, Qiagen Inc., Valencia, CA, USA). The expression of target genes was analysed by real-time reverse transcription PCR with oligo-dT in reverse transcription and bovine specific-primers in PCR. The expression of cyclophilin A was used as an internal control. The mean mRNA levels of target genes among groups were compared by parametric ANOVA, followed by orthogonal contrast. Heat stress reduced (P < 0.05) mRNA expression of CDX2 and PLAC8 in both breeds; additionally, the expression of these genes was higher in the zebu breed when compared with the taurine breed. Messenger RNA expression of COX2 did not differ between groups, under HS or not, in both the Jersey and Nelore breeds. Moreover, HS reduced the mRNA expression of HSF1 (P < 0.05) in Nelore groups, but not in Jersey groups. The highest levels of PLAC8 and CDX2 in nonstressed Nelore embryos indicate better competence and a higher capacity of implantation of these embryos when compared with Jersey and HS embryos in both breeds. Moreover, low HSF1 levels in stressed Nelore embryos indicate the thermotolerance ability of this breed. In conclusion, the data indicate that HS alters the pattern of gene expression in Nelore and Jersey in vitro-produced bovine embryos. This research was supported by FAPESP.

Differential Expression of Cyclophilin A and EMMPRIN in Developing Molars of Rats

A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.

The higher susceptibility of congenital analbuminemic rats to Ca2+-induced mitochondrial permeability transition is associated with the increased expression of cyclophilin D and nitrosothiol depletion

Congenital analbuminemia is a rare autosomal recessive disorder characterized by a trace level of albumin in blood plasma and mild clinical symptoms. Analbuminemic patients generally present associated abnormalities, among which dyslipidemia is a hallmark. In this study, we show that mitochondria isolated from different tissues (liver, heart and brain) from 3-month-old analbuminemic rats (NAR) present a higher susceptibility to Ca2+-induced mitochondrial permeability transition (MPT), as assessed by either Ca2+-induced mitochondrial swelling, dissipation of membrane potential or mitochondrial Ca2+ release. The Ca2+ retention capacity of the liver mitochondria isolated from 3-month-old NAR was about 50% that of the control. Interestingly, the assessment of this variable in 21-day-old NAR indicated that the mitochondrial Ca2+ retention capacity was preserved at this age, as compared to age-matched controls, which indicates that a reduced capacity for mitochondrial Ca2+ retention is not a constitutive feature. The search for putative mediators of MPT sensitization in NAR revealed a 20% decrease in mitochondrial nitrosothiol content and a 30% increase in cyclophilin D expression. However, the evaluation of other variables related to mitochondrial redox status showed similar results between the controls and NAR, i.e., namely the contents of reduced mitochondrial membrane protein thiol groups and total glutathione, H2O2 release rate, and NAD(P)H reduced state. We conclude that the higher expression of cyclophilin D, a major component of the MPT pore, and decreased nitrosothiol content in NAR mitochondria may underlie MPT sensitization in these animals.

P43 Cellular protein cyclophilin A is involved in hepatitis B virus replication and its inhibition with DEB025 (alisporivir) or NIM811 demonstrates antiviral activity in vitro

IntroductionCyclophilins are ubiquitously expressed intracellular proteins that have enzymatic activity—peptidyl-prolyl cis-trans isomerase, and are involved in regulation of mitochondrial/cytosolic transport in the cells. DEB025 (Alisporivir) and NIM811 are non-immunosuppressive cyclophilin...

Molecular cloning, expression, and characterization of cyclophilin A from Clonorchis sinensis

This study described the recognization, cloning, and recombinant expression of cyclophilin A-like gene from Clonorchis sinensis adult complementary DNA library (CsCyPA) and its expression and secretion in adult. Western blotting demonstrated the recombinant CsCyPA could be recognized by sera of clonorchiasis patients and a sole protein of the same size in the excretory-secretory antigens of in vitro cultured adult could be recognized by antiserum raised against the recombinant CsCyPA. Immunohistochemistry demonstrated that the CsCyPA was secreted in scattered vesicles from subtegumental parenchyma cells to the surface of tegument and mainly released from the tegument. ELISA showed the serum levels of IgG against CsCyPA in clonorchiasis patients negatively correlated with worm loads. This study suggested that C. sinensis adult in biliary ducts could release CsCyPA without signal peptide through nonclassical secretory pathway into the liver and might play a role in inflammation and biliary epithelium proliferation and adenomatoid hyperplasia.

Expression of cyclophilin A and CD147 during skin aging.

To investigate the role of cyclophilin A (CypA) and CD147 in the process of skin aging. Twenty cases of tissue samples from junior group(<15 years old), middle age group(30-40 years old)or old age group (>65 years old) were collected from photophobic and exposal parts of skin, respectively. Immunohistochemistry (IHC) and in situ hybridization (ISH) were carried out to semi-quantitatively detect the expression level of CyPA and CD147. IHC demonstrated that both CyPA and CD147 were expressed in both photophobic and exposal parts of normal human skin in all 3 groups. The expression levels of both CyPA and CD147 were increased with increase in age. There were significant differences in both CyPA and CD147 expression among 3 groups (P<0.05). CyPA and CD147 were also positively correlated in all 3 groups. Similar results were achieved by ISH. The interaction between CD147 and CyPA might play an important role in the process of skin aging.

N‐methyl‐4‐isoleucine cyclosporine attenuates CCl4‐induced liver fibrosis in rats by interacting with cyclophilin B and D

N-methyl-4-isoleucine cyclosporine (NIM811), a new analogue of cyclosporine A, can inhibit collagen deposition in vitro and reduce liver necrosis in a bile-duct-ligation animal model. However, whether NIM811 effects on CCl(4) -induced rat liver fibrosis, and the related mechanism has not been determined. A liver fibrosis model was induced in Wistar rats using CCl(4) for 6 weeks. Meanwhile, two different doses of NIM811 (low-dose 10 mg/kg and high-dose 20 mg/kg) were given to the CCl(4) -treated rats. Liver fibrosis was then evaluated according to histopathological scoring and liver hydroxyproline content. Serum alanine aminotransferase, aspartate aminotransferase and albumin levels, expression of matrix metalloproteinase-13, tissue inhibitor of metalloproteinase-1, α-smooth muscle actin and cyclophilin B and D in liver tissue were determined. Cyclophilin B and D were also studied in an hepatic stellate cell line. Hydroxyproline content was decreased in both NIM811 groups compared with the model (P < 0.05). Liver necrosis and fibrosis were also attenuated in the NIM811 groups. NIM811 suppressed the expression of tissue inhibitor of metalloproteinase-1, transforming growth factor beta mRNA and α-smooth muscle actin protein in liver tissue. Expression of cyclophilin B in the fibrosis model was increased compared with the normal group (P < 0.05), and was decreased significantly in the low-dose NIM811 treatment group (P < 0.05), which indicated that cyclophilin B might have a profibrotic effect. In vitro studies revealed that cyclophilin B and/or D knockout were associated with collagen inhibition. NIM811 attenuates liver fibrosis in a CCl(4)-induced rat liver fibrosis model, which may be related to binding with cyclophilin B and D.

Cyclophilin D controls mitochondrial pore–dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice

Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif(-/-) mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif(-/-) mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif(-/-) mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif(-/-) mice was associated with an alteration in MPTP-mediated Ca(2+) efflux resulting in elevated levels of mitochondrial matrix Ca(2+) and enhanced activation of Ca(2+)-dependent dehydrogenases. Elevated matrix Ca(2+) led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca(2+) levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.

Molecular basis for age-related changes in ileum: Involvement of Bax/caspase-dependent mitochondrial apoptotic signaling

Previous studies indicate that in the elderly, a morphological change in the small intestine is accompanied by apoptosis. However, currently little information is available on the molecular events leading up to the apoptotic process in aged ileum. Our current study assessed mitochondrial apoptotic signaling along with key factors known to be involved in mitochondrial permeabilization in rat ileum. Experimentations were carried out utilizing Sprague–Dawley rats at 6 and 24 months of age. The histological analysis showed a significant loss in thickness of the intestinal mucosa during aging, which was accompanied by higher reactive species. Molecular analysis revealed the mitochondrial translocation of Bax showed a significant increase with aging. However, the expression of cyclophilin D, adenine nucleotide translocator, and the voltage-dependent anion channel that regulates the mitochondria permeability transition pore decreased or remained unchanged. Furthermore, the expression of caspase 3 was enhanced in aged ileum with increased DNA fragmentation, while nuclear translocation of apoptosis-inducing factor and endonuclease G were decreased with aging. In conclusion, our findings indicate that the mitochondrial translocation of Bax by increased oxidative stress may result in cell death through caspase-dependent apoptosis in aged ileum, thereby leading to a decrease in intestinal mucosal thickness during aging.

Influence of High Temperature during Grain Filling on the Accumulation of Storage Proteins and Grain Quality in Rice (Oryza sativa L.)

The present study was performed to understand the effects of high temperature (HT) during filling on the expression of storage proteins and the quality of rice grains. HT (35/30 °C day/night) reduced the weight, amylose content, and flour gel consistency of grains. It increased the accumulation of all classes of storage proteins at early filling stage but decreased the accumulation of prolamins at maturation. For albumins, the expressions of cyclophilin 2, peroxiredoxin, and HSP16.9 were differentially enhanced by HT. For globulins, HT decreased the accumulation of globulin but increased that of glyoxalase I and peroxiredoxin. HT enhanced the transcription of genes for glutelins, prolamins, globulins, and protein disulfide isomerase at early filling stage but decreased the expression of these genes at a later stage. Low amounts of prolamins and globulins, as well as low pH value, were found in sound, immature, and dead kernels grown under HT. The relationships among HT, storage proteins, and grain quality are discussed.

A cyclophilin A CPR1 overexpression enhances stress acquisition in Saccharomyces cerevisiae.

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidant enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.

230 EXPRESSION OF mRNA ENCODING FGF10 AND COGNATE RECEPTORS (FGFR1B AND FGFR2B) AROUND FOLLICLE DEVIATION IN NELORE HEIFERS

A member of the FGF7 subfamily, FGF10 acts via FGFR2B and FGFR1B. In bovine antral follicles, FGF-10 was detected in oocytes and theca cells (TC). Levels of mRNA were negatively correlated with intrafollicular concentrations of estradiol, and FGF10 inhibited estradiol production from granulosa cells (GC). In Nellore (Bos indicus), morphological divergence occurs on average 2.5 days after ovulation, when dominant follicle diameter is around 6.0 mm. To gain insight into the involvement of the FGF10 system in the control of follicle selection, we assessed mRNA expression of FGF10 in TC and of FGFR1B and FGFR2B in GC from dominant and subordinate follicles around deviation in Nellore heifers. Thirteen Nellore heifers were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (n =4), 2.5 (n = 5), and 3 (n = 4) days after ovulation. Granulosa cells and TC were separated from the 2 largest follicles and submitted to total RNA extraction. mRNA abundance of CYP19 (aromatase), FGF10, FGFR1B, and FGFR2B was measured by real-time RT-PCR and normalized by the expression of cyclophilin A (CYCA) and GAPDH, for TC and GC, respectively. Dominant and subordinate follicles were considered those expressing the greatest and second-greatest abundance of CYP19 mRNA in GC within each heifer. Effects of follicle status and day on CYP19, FGF10, FGFR2B, and FGFR1B mRNA abundance were tested by ANOVA. On Day 2, FGFR2B mRNA abundance was greater in GC of subordinate follicles compared with dominant follicles (P = 0.006), and that of FGF10 in TC tended to exhibit the same pattern (P = 0.06). Follicle diameter was not different between dominant and subordinate follicles on Day 2 (5.5 ± 0 v. 5.12 ± 0.3 cm). On Day 2.5, FGF10 expression was greater in TC from subordinate follicles (P = 0.01), and FGFR2B expression in GC was no longer different between dominant and subordinate follicles. Follicle diameter was greater in dominant follicles on Day 2.5 (6.7 ± 0.2 v. 5.8 ± 0.3 cm; P = 0.04). On Day 3, no differences were observed between dominant and subordinate follicles for any of the genes assessed. mRNA expression of FGFR1B in GC did not change with follicle status or day. In conclusion, expression of FGF10 and FGFR2B was decreased in dominant follicles around morphological divergence, suggesting their involvement in the mechanisms controlling dominant follicle selection. As FGF10 inhibits estradiol production of GC, we propose that FGF10 and FGFR2B are suppressed in the dominant follicle to allow acquisition of full steroidogenic capacity. This research was supported by FAPESP.

212 EXPRESSION OF FIBROBLAST GROWTH FACTOR 18 (FGF18) AND COGNATE RECEPTORS (FGFR3C AND FGFR4) DURING LUTEAL DEVELOPMENT AND INDUCED LUTEOLYSIS IN CATTLE

Fibroblast growth factor receptor 2 (FGFR2) has been shown to induce luteinization in granulosa cells, luteal angiogenesis, and luteal growth. Alternative splicing of 4 genes give rise to 7 subtypes of fibroblast growth factor receptors (FGFR) with varying affinity for different fibroblast growth factors (FGF). Fibroblast growth factor receptor 2 and FGF18 efficiently activate FGFR3C and FGFR4 and may act in cooperation in tissues expressing these receptors. We aimed to determine mRNA expression patterns for FGF18, FGFR3C, and FGFR4 during bovine luteal development and following induced luteolysis. In addition, we assessed FGF18 localization in the bovine CL. Bovine CL were obtained from abattoir ovaries and classed into 4 stages of development: stage 1 =corpus hemorragicum; stage 2 = developing CL; stage 3 = mature or early functional luteolysis CL; and stage 4 = structural luteolysis. To assess FGF18 and FGFR mRNA expression during induced luteolysis, adult cows (Bos taurus Holstein-Friesians) were injected with the PGF2 analogue cloprostenol (500 mg i.m. Intervet, Unterschleissheim, Germany) during the mid-luteal phase of the cycle (Days 8-12). Corpus luteum were collected by transvaginal ovariectomy at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PGF2 injection. Tissue samples were submitted to total RNA extraction. Expression of FGF18, FGFR3C, and 4 mRNA during the bovine CL lifespan and induced luteolysis were measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophilin was used as internal control. The effect of developmental stage and time post-PGF2 on gene expression was tested by ANOVA, followed by Tukey- Kramer HSD test. Immunohistochemical analysis was performed with a commercial human antibody (anti-FGF18; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Fibroblast growth factor 18, FGFR3C, and FGFR4 mRNA was detected in all 4 developmental stages; FGF18 mRNA abundance was higher in stage 3 (2.89 ± 0.05; mean ± SEM) compared with stages 1 (0.3 ± 0.27), 2 (0.56 ± 1.27), and 4 (0.99 ± 0.32). Fibroblast growth factor 18 and FGFR4 mRNA expression did not significantly change during induced luteolysis. Fibroblast growth factor receptor 3C mRNA abundance peaked 4 h after PGF2 injection and significantly decreased at 24 h post-treatment in comparison with peak levels. Immunohistochemical analysis revealed the presence of FGF18 in small and large luteal cells and in blood vessels. In conclusion, the mRNA expression patterns of FGF18 and its receptors suggest their participation in the control of luteal differentiation, particularly during functional luteolysis. The localization of FGF18 protein to blood vessels suggests it may play a role in the control of angiogenesis in the bovine CL. Supported by CAPES/FAPESP.

Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type

BackgroundHIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA) protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA) and TRIM5α can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A – CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5α by the target cells.ResultsViral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5α expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity.ConclusionMultiple interactions between host-cell factors and Gag can strongly affect HIV-1 infectivity, and these vary according to target cell type and the origin of the Gag sequence. Two of the cellular activities involved appear to be modulated in opposite directions by CypA-CA interactions, and Gag sequences determine the intrinsic sensitivity of a given virus to each of these cellular activities.

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

180 EXPRESSION OF CANDIDATE GENES FOR OOCYTE COMPETENCE IN BOVINE CUMULUS CELLS

Cumulus cells (CC) are closed connected to the oocytes through a gap junction network during follicular development and ovulation. This reciprocal functional interconnection is essential for oocyte growth, acquitision of competence, and complete maturation. Therefore, CC are a promising source of markers for predicting oocyte developmental potential without damaging the oocyte. There is a clear relationship between follicular size and oocyte competence, being those obtained from large follicles more developmentally competent in vitro. The aim of this study was to identify in CC genes candidates to be involved in the acquisition of competence, using the follicles size model to recovered cumulus–ooctye complexes (COCs) with various levels of competence. Follicles from slaughterhouse ovaries were dissected, selected, and distributed according to their diameter into 4 groups: (1) 1.0 to 3.0 mm, (2) 3.1 to 6.0 mm, (3) 6.1 to 8.0 mm, (4) ≥8.1 mm. The COCs were released by follicle rupture, morphologically classified, matured, fertilized, and cultured in vitro or denuded for the recovery of CC. Then, CC were frozen until gene expression analysis. The developmental potential of oocytes was evaluated by cleavage and blastocyst rates at 48 and 168 h post-insemination, respectively. Transcripts for FSH receptor, GH receptor, epidermal growth factor (EGF) receptor, pentraxin 3, and insulin-like growth factor II (IGF-II) were quantified by real-time RT-PCR and normalized by cyclophilin expression. Data from cleavage and blastocyst rates were analyzed by chi-square test. The relative abundance of mRNA for the 5 genes in CC were evaluated by ANOVA and Tukey’s test. Non-parametric test (Wilcoxon) was used when data were not normally distributed. The results are presented as mean ± SEM, and P &lt; 0.05 was considered statistically different. Cleavage and blastocyst rates were higher in oocytes originated from groups 3 (86 and 62%) and 4 (87 and 60%) than those obtained from groups 1 (20%) and 2 (34%). The lowest level of expression for FSH receptor was observed in CC from 1.0–3.0 mm follicles (1.1 ± 0.07) and the greatest in CC from ≥8.1-mm follicles (2.1 ± 0.09), whereas the expression in the intermediary groups 2 (1.6 ± 0.1) and 3 (1.6 ± 0.2) did not show any difference from the other groups. Similar pattern of expression was observed for EFG receptor gene, except that CC from group 3 (1.7 ± 0.2) already presented a significant increase in mRNA level compared to group 1 (0.9 ± 0.1). Relative abundance of transcript for GH receptor rose gradually in CC according to follicular size, being the higher expression detected in the largest follicles and the lower in the smallest follicles, with means of 0.8 ± 0.1, 2.3 ± 0.6, 2.7 ± 0.6, and 3.9 ± 0.1 for groups 1, 2, 3, and 4, respectively. The other 2 candidates (pentraxin 3 and IGF-II) did not show any significant differences related to follicle size. The results indicate that increases in expression of receptor for FSH, EGF, and GH was coincident with the increase in follicle size and in oocyte developmental potential; therefore, they can be used as markers for bovine oocyte competence. However, additional studies are needed to confirm the markers identified. Financial support from EMBRAPA/CAPES.

Mitochondrial Defects and Dysfunction in Calcium Regulation in Glaucomatous Trabecular Meshwork Cells

Disruption in intracellular calcium ion (Ca(2+)) homeostasis has major effects on health. Persistent Ca(2+) overload induces mitochondrial permeability transition pore (MPTP) opening, which prompts mitochondrial release of calcium (mCICR) and reactive oxygen species (ROS) into the cytosol which, in turn, compromises mitochondrial function. This study was conducted to examine intracellular Ca(2+) levels and mitochondrial vulnerability to Ca(2+) stress in trabecular meshwork (TM) of individuals with primary open-angle glaucoma (POAG). Primary cultures of TM cells from POAG (GTM) and age-matched, nondiseased (NTM) eyes, obtained from postmortem donors eyes by standard surgical trabeculectomy, were treated with the following calcium regulators: the mitochondrial respiratory chain I inhibitor rotenone (ROT); the mitochondrial permeability transition pore (MPTP) inhibitors cyclosporine (Cys) and aristolochic acid (ArA); the Ca(2+) chelators BAPTA/AM or EDTA; the mitochondrial Ca(2+) uniporter inhibitor ruthenium red (RR); the Ca(2+)/Na(+) exchanger inhibitor trifluoperazine; and the inositol 1,4,5-triphosphate receptor type 3 (IP3R) inhibitors 2-aminoethoxydiphenyl borane (2-APB) and xestospongin C (Xe-C). Ca(2+) concentrations in the cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)](m)) were determined by confocal microscopy and flow cytometry with the fluorescent Ca(2+) indicators fluo-3/AM and rhod-2/AM, respectively. Mitochondrial membrane potential (DeltaPsim) was examined with the fluorescent probe tetramethylrhodamine ethyl ester (TMRE). The expression of cyclophilin D, a protein that induces MPTP opening was also measured. There was increased [Ca(2+)](c), [Ca(2+)](m), mCICR, MPTP opening, and expression of cyclophilin D and decreased DeltaPsim in POAG TM cells compared with control cells. ROT artificially exacerbated these conditions in GTM cells. Chelation of [Ca(2+)](c) and inhibition of IP3R and MPTP opening suppressed mitochondrial dysfunction and reduced the additional effects of ROT in GTM cells. POAG TM cells have defective mitochondrial function, which causes them to be abnormally vulnerable to Ca(2+) stress. The dysfunction in calcium regulation by these cells may contribute to the failure of this tissue to control IOP. Pharmacologic inhibitors of IP3R, MPTP opening, and cyclophilin D could have clinical implications for primary open-angle glaucoma.