Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

MARINE CRYPTOMONAD STARCH FROM AUTOLYSIS OF GLYCEROL-GROWN CHROOMONAS SALINA1

The extracellular starch released by autolysis of glycerol-grown Chroomonas salina (Wislouch) Butcher was purified and chemically characterized. It contained 99%D-glucose, formed a blue complex (λmax at 605 nm) with iodine, and showed an optical rotation value more positive than that of potato starch. Methylation analysis established the presence of 1,4- and 1,4,6-glucoside linkages in the proportion 15:1. These properties indicate the cryptomonad starch to be an iodophilic α-(1,4)-glucan, composed of ca. 30% amylose with amylopectin, and broadly resembling potato, Chilomonas and other unicellular algal starches.     

High-dose mode of death in Tribolium

This study reports the first demonstration within a single insect genus (Tribolium) of both the acute, or lethal-midlethal, dose-independent pattern of mortality, and the hyperacute, dose-dependent pattern, after appropriate doses of ionizing radiation. This demonstration provides resolution of apparently contradictory reports of insect responses in terms of doses required to cause lethality and those based on survival time as a function of dose. A dose-dependent mortality pattern was elicited in adult Tribolium receiving high doses, viz., 300 Gy or greater; its time-course was complete in 10 days, before the dose-independent mortality began. Visual observations of heavily-irradiated Tribolium suggested neural and/or neuromuscular damage, as had been previously proposed by others for lethally-irradiated wasps, flies, and mosquitoes. Results of experiments using fractionated high doses supported the suggestion that the hyperacute or high-dose mode of death is the result of damage to nonproliferative tissues. Relative resistance of a strain to the hyperacute or high-dose mode of death is not necessarily correlated with resistance to the midlethal mode, which is believed to be the result of damage to the proliferative cells of the midgut.

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Résumé Les résultats de nombreuses études des réactions des insectes adultes de différents groupes à l'irradiation s'opposent quant à l'importance de la dose provoquant la létalité et quant aux modalités de la mort. Les diptères et les guèpes impliquent des doses très élevées,-des centaines de Gy-, ne présentent aucune période caractérisant la mort par irradiation, et décèdent de plus en plus tôt avec l'augmentation des doses. Beaucoup d'autres insectes succombent à des doses (milétalelétale) beaucoup plus faibles,-de quelques Gy à des dizaines-, et quelle que soit la dose meurent au bout d'un temps voisin.Au cours de cette étude, nous avons pu observer que ces deux types de mortalité peuvent être provoqués chez le même genre d'insecte (Tribolium), avec des doses convenables d'irradiation . Un syndrôme caractéristique a été provoqué avec des doses très élevées, de 300 Gy ou plus,-à ces doses la mort est obtenue en 10 jours après l'irradiation. L'absence de syndrôme caractéristique se produit avec des doses inférieures ou égales à 80 Gy; la mort a lieu alors entre 10 et 16 jours en fonction de la dose.Les différences entre les deux types de décès indiquent deux processus de mort par irradiation. La manifestation d'une désorientation et d'une perte de coordination motrice chez les Tribolium fortement irradiés suggère des altérations neurales et/ou neuromusculaires comme cause/s de ce type de mort provoquée par des doses élevées. L'implication de tissus sans prolifération a été confortée par les résultats d'expériences utilisant de hautes doses fractionnées. Le type de mort milétal est considéré après de nombreuses observations indépendantes, comme le résultat d'atteintes à la prolifération des cellules de l'intestin moyen.Les données contradictoires sur les réactions des insectes aux irradiations proviennent d'abord de l'absence de connaissances sur le lieu des dégâts. Les diptères sont connus maintenant, après différentes études, comme perdant complètement l'aptitude au renouvellement cellulaire, et présentent ainsi un type de mort avec dose élevée. Beaucoup d'autres insectes adultes ont un renouvellement cellulaire de l'intestin moyen limité, et ainsi présentent le type de mort milétal. Le type de mort, dit haute dose, peut être induit dans cette dernière catégorie d'insectes par une irradiation suffisamment forte, et, dans le cas du Tribolium le déroulement de la mort se produit alors de deux façons bien distinctes.

     

Differential expression of AP-2gamma and AP-2alpha during human trophoblast differentiation.

Previous studies from our laboratory demonstrated that AP-2alpha induces the expression of the hPL and hCG genes in cultured trophoblast cells. In the current study, we have shown by transient transfection studies that AP-2gamma, which is the product of a separate gene from AP-2alpha, also stimulates hPL and hCGbeta promoter activities. However, AP-2gamma mRNA levels during in vitro differentiation of human cytotrophoblast cells were strikingly different than those of AP-2alpha mRNA levels, with AP-2alpha increasing and AP-2gamma markedly decreasing during the differentiation process. The amount of AP-2gamma protein binding to AP-2 elements on the hPL promoter, as determined by supershift assays, also markedly decreased during the differentiation process. These findings strongly suggest that AP-2gamma action in cytotrophoblast cells is repressed by a co-factor(s) that inhibits AP-2gamma action or is prevented by the absence of a co-factor(s) that is essential for AP-2gamma action.     

Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line.

The ratio between apoptotic promoters and repressors in the Bcl-2 family determines the chemosensitivity of cells to apoptotic stimuli. This study examines the chemoresistance of a transfected human hepatoblastoma HepG2 cell-line during Taxol and Doxorubicin application. Sense bcl-2, and anti-sense bcl-XL gene fragments were separately inserted into HepG2 cells via stable transfection. The expression profile of the Bcl-2 family proteins was determined by Western blot analysis. Chemosensitivity of the transfected cells was measured by Trypan blue exclusion assay and XTT reduction assay during drug application. In the absence of Bax protein, HepG2 cells with elevated Bcl-2 protein levels did not exhibit any significant increase in chemosensitivity towards the drugs. Transfected cells with reduced Bcl-XL levels became more sensitive to the drugs, and a significant difference in IC50 values was observed. The chemosensitivity of HepG2 cells to Taxol and Doxorubicin was not affected by Bcl-2 levels, while reduction of Bcl-XL levels rendered the cells more sensitive to the drugs. This suggests that the Bcl-2 protein alone could not protect HepG2 cells from drug-induced apoptosis, and that the Bcl-XL protein may be a target for gene therapy in hepatoblastoma treatment.     

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis

The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three approximately 2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln(421) and Phe(422) within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH(2)-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis.     

Solution structure of an N-capping peptide from the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The RNA genome of HDV encodes two proteins, the small delta antigen and the large delta antigen, which differ only with the latter having an additional 19 amino acids at the C-terminus. Previously, we have shown that dAg24-50, a synthetic peptide corresponding to residues 24-50 of the N-terminal leucine-repeat region of hepatitis delta antigen, binds to the viral RNA and forms an alpha-helical conformation in TFE-containing solution. However, it exhibited low alpha-helicity (less than 5%) in the absence of TFE. In order to obtain biologically active delta antigen peptides with higher structural stability in solution, an N-capping 21-residue polypeptide corresponding to residues 24-38 of hepatitis delta antigen (dAg(Cap24-38am)) was synthesized and, surprisingly, its solution structure was found to be a stable alpha-helix (64%) by circular dichroism and 1H NMR techniques. Moreover, the structure of the capping box shows the characteristic L-shaped bend perpendicular to the helix axis. This structural knowledge provides a molecular basis for understanding the role of the N-terminal leucine-repeat region of hepatitis delta antigen and has a significant potential for the development of diagnostic and therapeutic methods for HDV.     

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.     

A target within the target: probing cruzain's P1' site to define structural determinants for the Chagas' disease protease

BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.