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The present study investigated the segmental discrepancy of the rat distal colonic anion transport induced by luminal forskolin and the possible underlying mechanisms using short-circuit current recording technique and comparative quantity real-time PCR analysis. Forskolin-induced I(SC) in the segment next to lymph node (DC(1)) and the segment 4 cm away from lymph node (DC(4)) were 4.09+/-0.66 muA/cm(2) and 18.84+/-3.18 muA/cm(2) (n=13), respectively, which were blocked by luminal Cl(-) channel blocker, glybenclamide (1 mM) (n=5, p<0.01), as well as removal of extracellular Cl(-) and HCO(3)(-) in both DC(1) and DC(4) (n=5, p<0.001). Furthermore luminal pretreatment with K(+) blockers, TEA (5 mM) and glybenclamide (100 muM) didn't affect forskolin and bumetanide-enhanced I(SC). Reducing serosal Cl(-) concentration increased forskolin-induced I(SC) by 90% in DC(1) but decreased forskolin-induced I(SC) in DC(4) by 50%. Furthermore, pretreatment with serosal bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter, enhanced forskolin-induced I(SC) by 87% in DC(1), from 4.09+/-0.66 muA/cm(2) to 7.65+/-0.53 muA/cm(2) (n=6, p<0.01), but inhibited forskolin-induced I(SC) by 50% in DC(4), from 29.19+/-4.51 muA/cm(2) to 15.06+/-4.10 muA/cm(2) (n=6, p<0.05). Pretreatment with luminal amiloride (10 muM), an inhibitor of ENaC, and serosal 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (200 muM), an inhibitor of NBC, significantly inhibited the forskolin-induced I(SC) in DC(1) (n=6, p<0.05), but not in DC(4). Real-time PCR analysis did not show the significant differences between the two segments in the expression amounts of CFTR and NKCC mRNAs, however the expression of NBC mRNA in DC(4) was significantly higher than that in DC(1). In conclusion, the rat distal colon manifests a segmental discrepancy in anion transport stimulated by luminal forskolin. HCO(3)(-) might be predominantly involved in the forskolin-induced anion secretion in DC(1), but Cl(-) might be the main component of the anion secretion in DC(4).

Objective: To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury., Methods: Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method., Results: There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group., Conclusion: Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.

Objective: To observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury., Methods: Male SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting., Results: nNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells., Conclusion: Ligustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.

Inactivation of the von Hippel-Lindau tumour suppressor in renal cell carcinoma (RCC) leads to failure of proteolytic regulation of the alpha subunits of hypoxia-inducible factor (HIF), constitutive upregulation of the HIF complex, and overexpression of HIF target genes. However, recent studies have indicated that in this setting, upregulation of the closely related HIF-alpha isoforms, HIF-1alpha and HIF-2alpha, have contrasting effects on tumour growth, and activate distinct sets of target genes. To pursue these findings, we sought to elucidate the mechanisms underlying target gene selectivity for HIF-1alpha and HIF-2alpha. Using chromatin immunoprecipitation to probe binding to hypoxia response elements in vivo, and expression of chimaeric molecules bearing reciprocal domain exchanges between HIF-1alpha and HIF-2alpha molecules, we show that selective activation of HIF-alpha target gene expression is not dependent on selective DNA-binding at the target locus, but depends on non-equivalent C-terminal portions of these molecules. Our data indicate that post-DNA binding mechanisms that are dissimilar for HIF-1alpha and HIF-2alpha determine target gene selectivity in RCC cells.

Objective: To analyze the clinical manifestations of affected individuals in a family of congenital fibrosis of the extraocular muscles (CFEOM) with juvenile canities., Methods: All affected and unaffected individuals were retrospectively analyzed in this study. The clinical features include genetic aspects, sex, age, ptosis, restriction of eye movement, aberrant innervation and surgical procedures, were evaluated., Results: This pedigree was inherited as autosomal dominant. There were 14 cases suffering from congenital fibrosis of extraocular muscles in four generations. They had congenital blepharoptosis, head-tilt, chin lift and primary gaze fixed in a hypotrophic position. But vertical and horizontal positions of the eye and restriction of eye movement were different among affected individuals. Some of them also had pupillary abnormally, aberrant innervation and juvenile canities. Inferior rectus recession improved hypotropia in patients with infraducted eyes and chin elevation. Horizontal muscle recession corrected horizontal strabismus satisfactorily in most cases. Ptosis was repaired by frontalis sling or levator resection., Conclusions: This is the first report of CFEOM associated with juvenile canities. There was phenotypic heterogeneity in this CFEOM pedigree. So the phenotype alone is not sufficient to distinguish among the 3 genotypically distinct CFEOM syndromes. The combination of clinical characteristics and genetic analysis are the basis for the establishment of diagnosis.

Congenital cranial dysinnervation disorders (CCDDs) are congenital non-progressive, sporadic or familial abnormalities of cranial musculature that result from developmental abnormalities of, or the complete absence of, one or more cranial nerves with primary or secondary muscle dysinnervation. These disorders include vertical disorders of ocular motility, horizontal disorders of ocular motility and disorders with abnormalities of facial motility that were previously referred to as "congenital fibrosis syndromes". The advances in clinical and molecular genetics of congenital cranial dysinnervation disorders are reviewed.

The heterodimeric transcription factor HIF (hypoxia-inducible factor) is central to the regulation of gene expression by oxygen. Three oxygen-dependent prolyl hydroxylase enzymes [PHD1 (prolyl hydroxylase domain 1), PHD2 and PHD3] control the abundance of HIF. In the presence of oxygen, they hydroxylate specific proline residues in HIF-alpha, allowing recognition by pVHL (von Hippel-Lindau protein) and subsequent ubiquitylation and proteasomal destruction. The precise roles and regulation of these enzymes are therefore of particular importance in understanding the physiological and pathological responses to hypoxia. In the present study, we define the existence of two species of PHD1 and provide evidence that they are generated by alternative translational initiation. We demonstrate that these alternative forms are both biologically active with similar HIF prolyl hydroxylase activity but that they differ in their responses to oestrogen, cell confluence and proteasomal inhibition. We show that the two PHD1 species are subject to proteolytic regulation but differ markedly in their protein stability. Though each isoform has the potential to interact with members of the Siah (seven in absentia homologue) ubiquitin ligase family, genetic studies indicated that other proteolytic mechanisms are responsible for control of stability under the conditions examined. The data define the existence of a further level of control in the pathway that regulates cellular responses to hypoxia.

Hypoxia-inducible factor (HIF) regulates expression of genes involved in adaptation to hypoxia and ischemia. Three prolyl hydroxylases (PHD1-3) underlie oxygen-regulated destruction of HIFalpha chains. We have investigated the organ distribution of the PHDs in the rat, their regulation by hypoxia and changes in local expression after experimental myocardial infarction using RNase protection assays, in situ hybridization and immunohistochemistry. mRNAs of all isoforms were detectable in heart, liver, kidney, brain, testis and lung. In normal animals, highest levels for PHD2 mRNA and PHD3 mRNA were found in myocardium, whereas PHD1 mRNA was detected predominantly in the testis. PHD1 mRNA was constitutively expressed. PHD2 mRNA was induced by hypoxia in the liver and PHD3 mRNA in liver, testis and heart. Overall our results show that PHD2 mRNA is ubiquitously expressed in normal animals, in keeping with a general role in oxygen sensing. PHD1 and 3 mRNA distributions suggest particular roles in testis and heart, respectively. In a model of myocardial infarction, in situ hybridization showed periischemic enhancement for PHD2 mRNA and PHD3 mRNA, but not PHD1 mRNA. Immunostaining of PHD2 and 3 in infarcted hearts showed enhanced protein expression, maximal 7 days after infarction. Levels were strongest in regions neighboring areas of HIF staining but also partially overlapped with these zones. Inducibility of PHD2 and 3 by hypoxia and ischemia in vivo has important implications both for the pathophysiology of conditions where oxygen supply is deranged and for attempts to manipulate the HIF system therapeutically.

Aims: The cellular response to hypoxia includes the hypoxia inducible factor (HIF)-induced transcription of genes involved in diverse processes such as glycolysis, angiogenesis and the growth of experimental tumours. Regulation of the level of hypoxia inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) is a primary determinant of HIF activity. Recent biochemical and candidate gene approach studies have led to the discovery of three HIF-regulatory prolyl hydroxylases, PHD-1, -2 and -3 and an asparaginyl hydroxylase, also known as FIH (factor inhibiting HIF). In this study, we raised and characterized monoclonal antibodies against PHD-1, PHD-2, PHD-3 and FIH., Methods and Results: Immunohistochemistry of normal tissues with these monoclonal antibodies demonstrated a wide distribution in epithelial cells, stromal cells and leucocytes, with cytoplasmic staining predominating over nuclear staining. A preliminary study of tumours showed variable staining in tumour, stromal and inflammatory cells. While all tumour types showed some positive staining with each antibody, the overall pattern suggested a slight decrease in the amount of staining seen with PHD-1, -2 and -3 and an increase in FIH staining in neoplasia compared with corresponding normal tissues., Conclusions: These monoclonal antibodies will allow further larger scale studies to determine the significance of PHD and FIH expression in neoplasia.

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Hypoxia-inducible factor (HIF) is a transcriptional complex that is regulated by oxygen sensitive hydroxylation of its alpha subunits by the prolyl hydroxylases PHD1, 2 and 3. To better understand the role of these enzymes in directing cellular responses to hypoxia, we derived an assay to determine their specific activity in both native cell extracts and recombinant sources of enzyme. We show that all three are capable of high rates of catalysis, in the order PHD2=PHD3>PHD1, using substrate peptides derived from the C-terminal degradation domain of HIF-alpha subunits, and that each demonstrates similar and remarkable sensitivity to oxygen, commensurate with a common role in signaling hypoxia., (Copyright 2004 Federation of European Biochemical Societies)

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that directs a broad range of cellular responses to hypoxia. Recent studies have defined a set of 2-oxoglutarate and Fe(II)-dependent dioxygenases that modify HIF-alpha subunits by prolyl and asparaginyl hydroxylation. These processes potentially provide a dual system of control, down-regulating both HIF-alpha stability and transcriptional activity. Although genetic analyses in both primitive organisms and mammalian cells have demonstrated a critical role for the prolyl hydroxylase pathway in the regulation of HIF, analogous studies have not been performed on the HIF asparaginyl hydroxylase pathway, and its role in directing the expression of endogenous HIF transcriptional targets has not yet been clearly defined. Here we demonstrate, using small interfering RNA-mediated FIH suppression and controlled overexpression by a doxycycline-inducible system, that alterations in FIH expression in both directions have reciprocal effects on the expression of a range of HIF target genes. These effects were observed in normoxic and severely hypoxic cells but not anoxic cells. Evidence for FIH activity in severely hypoxic cells contrasted with results for the prolyl hydroxylase PHD2, suggesting that these enzymes display different oxygen dependence in vivo, with PHD2 requiring higher levels of oxygen for biological activity. Our results demonstrate an important physiological role for FIH in regulating HIF-dependent target genes over a wide range of oxygen tensions and indicate that inhibition of FIH has the potential to augment HIF target gene expression even in severe hypoxia.

Hypoxia-inducible factor (HIF) is a transcriptional regulator that plays a key role in many aspects of oxygen homeostasis. The heterodimeric HIF complex is regulated by proteolysis of its alpha-subunits, following oxygen-dependent hydroxylation of specific prolyl residues. Although three HIF prolyl hydroxylases, PHD1, PHD2, and PHD3, have been identified that have the potential to catalyze this reaction, the contribution of each isoform to the physiological regulation of HIF remains uncertain. Here we show using suppression by small interference RNA that each of the three PHD isoforms contributes in a non-redundant manner to the regulation of both HIF-1alpha and HIF-2alpha subunits and that the contribution of each PHD under particular culture conditions is strongly dependent on the abundance of the enzyme. Thus in different cell types, isoform-specific patterns of PHD induction by hypoxia and estrogen alter both the relative abundance of the PHDs and their relative contribution to the regulation of HIF. In addition, the PHDs manifest specificity for different prolyl hydroxylation sites within each HIF-alpha subunit, and a degree of selectively between HIF-1alpha and HIF-2alpha isoforms, indicating that differential PHD inhibition has the potential to selectively alter the characteristics of HIF activation.

The vasodilator hydralazine, used clinically in cardiovascular therapy, relaxes arterial smooth muscle by inhibiting accumulation of intracellular free Ca2+ via an unidentified primary target. Collagen prolyl hydroxylase is a known target of hydralazine. We therefore investigated whether inhibition of other members of this enzyme family, namely the hypoxia-inducible factor (HIF)-regulating O2-dependent prolyl hydroxylase domain (PHD) enzymes, could represent a novel mechanism of action. Hydralazine induced rapid and transient expression of HIF-1alpha and downstream targets of HIF (endothelin-1, adrenomedullin, haem oxygenase 1, and vascular endothelial growth factor [VEGF]) in endothelial and smooth muscle cells and induced endothelial cell-specific proliferation. Hydralazine dose-dependently inhibited PHD activity and induced nonhydroxylated HIF-1alpha, evidence for HIF stabilization specifically by inhibition of PHD enzyme activity. In vivo, hydralazine induced HIF-1alpha and VEGF protein in tissue extracts and elevated plasma VEGF levels. In sponge angiogenesis assays, hydralazine increased stromal cell infiltration and blood vessel density versus control animals. Thus, hydralazine activates the HIF pathway through inhibition of PHD activity and initiates a pro-angiogenic phenotype. This represents a novel mechanism of action for hydralazine and presents HIF as a potential target for treatment of ischemic disease.

Hypoxia-inducible factor-1 (HIF) is regulated by oxygen-dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways. We have used co-purification and mass spectrometry to identify proteins that interact with PHD3. The cytosolic chaperonin TRiC was found to copurify with PHD3 in extracts from several cell types. Our results indicate that PHD3 is a TRiC substrate, providing another step at which PHD3 activity may be regulated.

Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha--10 residues downstream of Pro-564--is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.

The effect of melatonin on age-related thymic involution and peripheral immune dysfunctions was investigated. Exogenous melatonin was administered through the drinking water (15 microg/ml) of 22-month-old female C57BL mice for 60 consecutive days. Our results show that melatonin distinctly reversed the age-related thymic involution as revealed by the notable increase of thymus weight, total number of thymocytes and percentage of thymocytes at G2+S phases. More strikingly, spleen weight, total number of splenocytes and some peripheral immune capacity such as mitogen responsiveness and NK cell activity were also significantly recovered by 60 days of melatonin application in aged mice. Our findings demonstrate that even when the melatonin supplementation begins late in life, the age-related thymic involution and peripheral immune dysfunctions can be restored at least partially in old mice.

In this study, the effect of EGb 761, a standard extract of Ginkgo biloba leaf, on thymocyte apoptosis and age-related thymic atrophy and on peripheral immune dysfunctions was investigated in mice. When primary culture of thymocytes was preincubated with 100 microg/ml EGb 761 before their exposure to hydroxyl radicals (*OH) generated by Fe(2+)-mediated Fenton reaction, apoptotic cell death induced by *OH was distinctly prevented as determined by DNA laddering, the TUNEL assay and flow cytometric analysis. Furthermore, oral EGb 761 administration (about 1.5 mg/day/mouse) for 60 consecutive days led to a significant thymic regrowth in 22-month-old mice as revealed by the increment of thymus weight and total numbers of thymocytes. Partial recovery of peripheral immune capacities such as mitogen responsiveness and NK cell activity were also found in the old mice after 60 days of EGb 761 supplementation. Taken together, our study indicates that in addition to its protective and rescuing abilities on neurodegenerative disorders and cardiovascular diseases, EGb 761 was also found active in the rejuvenation of degenerated thymus and accordingly the strengthening of the immune system. These beneficial effects of EGb 761 on immune system are based on its antioxidant properties as well as the cell proliferation-stimulating effect.

Analogues of the naturally occurring cyclic hydroxamate dealanylalahopcin, which is an inhibitor of procollagen prolyl-4-hydroxylase, were synthesised and shown to be inhibitors of the human hypoxia-inducible factor prolyl hydroxylases.

Hypoxia-inducible factor-1 (HIF) is a transcription factor central to oxygen homeostasis. It is regulated via its alpha isoforms. In normoxia they are ubiquitinated by the von Hippel-Lindau E3 ligase complex and destroyed by the proteasome, thereby preventing the formation of an active transcriptional complex. Oxygen-dependent enzymatic hydroxylation of either of two critical prolyl residues in each HIFalpha chain has recently been identified as the modification necessary for targeting by the von Hippel-Lindau E3 ligase complex. Here we demonstrate that polypeptides bearing either of these prolyl residues interfere with the degradative pathway, resulting in stabilization of endogenous HIFalpha chains and consequent up-regulation of HIF target genes. Similar peptides in which the prolyl residues are mutated are inactive. Induction of peptide expression in cell cultures affects physiologically important functions such as glucose transport and leads cocultured endothelial cells to form tubules. Coupling of these HIFalpha sequences to the HIV tat translocation domain allows delivery of recombinant peptide to cells with resultant induction of HIF-dependent genes. Injection of tat-HIF polypeptides in a murine sponge angiogenesis assay causes a markedly accelerated local angiogenic response and induction of glucose transporter-1 gene expression. These results demonstrate the feasibility of using these polypeptides to enhance HIF activity, opening additional therapeutic avenues for ischemic diseases.

Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-alpha. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-alpha abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-alpha. Since FIH contains certain motifs present in iron- and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-alpha fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

The effect of melatonin on age-related thymic involution and apoptosis induced by hydroxyl radicals (*OH) in mouse thymocyte cultures was investigated. Exogenous melatonin was administered in the drinking water (15 microg/mL) of 7-month-old male Balb/c mice for 40 consecutive days. Our results show that melatonin distinctly reversed the age-related thymic involution as revealed by the notable increase of cellular density, particularly the number of thymocytes, percentage of thymocytes at G2+S phases and the younger morphological appearance as a whole when compared with control animals. More strikingly, the recovery of these morphometric parameters were maintained for 30 days after the termination of melatonin administration suggesting that the re-established homeostasis by melatonin may last for a longer time. At the same time, when primary culture of thymocytes was preincubated with 200 microM melatonin before their exposure to hydroxyl radicals (*OH) generated by Fe(2+)-mediated Fenton reaction, apoptotic cell death induced by *OH was almost completely prevented as determined by both flow cytometric analysis and the TUNEL assay. DNA laddering assay also documented the inhibition of thymocyte apoptosis by melatonin. Furthermore, we found that the *OH-induced increment of caspase-3 activity in thymocytes was completely abolished by melatonin preincubation. Taken together, our study indicates that in addition to other mechanisms, melatonin may also directly act as an antioxidant via attenuating apoptotic thymocyte death caused by free radicals and stimulates thymocyte proliferation in thymus and thus to rejuvenate the degenerative organ.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade.

Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1alpha (HIF-1alpha). To analyze EPAS1 domains responsible for transactivation and oxygen-regulated function, we constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain, plus or minus the VP16 activation domain. Two transactivation domains were defined in EPAS1; a C-terminal domain (amino acids 828-870), and a larger internal domain (amino acids 517-682). These activation domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity. Two types of activity were defined. Sequences lying N-terminal to and overlapping the internal transactivation domain conferred regulated repression on the VP16 transactivator. Sequences lying C-terminal to this internal domain conveyed repression and oxygen-regulated activity on the native EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion. Fusions containing internal but not C-terminal regulatory domains manifested regulation of fusion protein level. Comparison of EPAS1 with HIF-1alpha demonstrated a similar organization for both proteins, and for the C terminus defined a conserved RLL motif critical for inducibility. Overall, EPAS1 sequences were less inducible than those of HIF-1alpha, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1alpha, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains.

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.