Comparative study of urinary reproductive hormones in great apes

Urinary estrone conjugates (E₁C), pregnanediol-3-glucuronide (PdG), and follicle-stimulating hormone (FSH) were determined by enzyme immunoassays (EIAs) during the normal menstrual cycle in the orangutan, gorilla, chimpanzee, and bonobo. Furthermore, the data were compared to those levels in the human and long-tailed macaque. The results showed a typical preovulatory E₁C surge and postovulatory increase in PdG in all species. The pattern of E₁C during the menstrual cycle in the great apes more closely resembled the human than do the long-tailed macaque. A major difference of E₁C pattern between these species appeared in the luteal phase. In the great apes and the human, E₁C exhibited two peaks, the first peak detected at approximately mid cycle and the second peak detected during the luteal phase. On the other hand, in the long-tailed macaque, increase of E₁C in the luteal phase was small or nonexistent. The gorilla, chimpanzee, and bonobo exhibited similar PdG trends. The orangutan excreted one tenth less PdG than these species during the luteal phase. The long-tailed macaque also excreted low levels of PdG. The patterns of FSH in orangutan, chimpanzee, bonobo and long-tailed macaque showed a marked mid-cycle rise and an early follicular phase rise, similar to those in the human. Comparing similar taxa, a large difference was found in FSH of gorilla; there were three peaks during the menstrual cycle. Thus, there is considerable species variation in the excretion of these hormones during the menstrual cycle and comparative studies could be approached with a single method. The methods and baseline data presented here provide the basis for a practical approach to evaluation and monitoring of ovarian events in the female great apes. Electronic Publication     

Genistein enhances the cisplatin-induced inhibition of cell growth and apoptosis in human malignant melanoma cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy-refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 microM) did not induce apoptosis by itself but did enhance cisplatin-induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti-apoptotic proteins, bcl-2 and bcl-xL, and one pro-apoptotic protein, apoptotic protease activating factor-1 (Apaf-1), were examined. Genistein alone or cisplatin alone generally did not alter bcl-2 expression or bcl-xL expression, but slightly increased Apaf-1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl-2 and bcl-xL protein and increased Apaf-1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C-terminal domain

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperature-sensitive DBD mutation (cts62), some cysteine-replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease-resistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.     

Genetic diversity of fragmented populations of<Emphasis Type="Italic"> Polygala reinii</Emphasis> (Polygalaceae), a perennial herb endemic to Japan

Genetic variation at 10 allozyme loci was analyzed in 14 populations of Polygala reinii (Polygalaceae), a perennial herb endemic to central Honshu, Japan, with a fragmented geographical distribution. The levels of genetic variation within species (P=80.0, A=3.10, H_E=0.303) and within populations (P=42.1, A=1.61, H_E=0.163) were considerably higher than the mean for other endemic plants or short-lived perennial herbs. Genetic differentiation among populations was also high (G_ST=0.404). The genetic distance phenogram tended to show a clustering of the populations reflecting the fragmentation of the species range. A principal component analysis revealed the same tendency, as well as three groupings of populations in the Tokai district, on the Kii Peninsula and in the northern Kinki district. A negative correlation was obtained between the levels of gene flow and geographical distance among the populations (r=–0.745, P<0.0001). These results indicated limited gene flow among populations in P. reinii, presumably due to the geographical isolation accompanying the fragmented distribution. On the other hand, the geographical differentiation between the Japan Sea and Pacific Ocean sides was found in P. reinii, suggesting the influence of postglacial migration on the establishment of the genetic structure of this species.     

Molecular Basis of Anti-Apoptotic Effect of Immunophilin Ligands on Hydrogen Peroxide–Induced Apoptosis in Human Glioma Cells

To clarify the molecular basis of the cytoprotective properties of immunophilin ligands (IPLs), the anti-apoptotic effects of IPLs were determined in human glioma U251 cells. GPI1046 and V10367, non-immunosuppressive IPLs (NI-IPLs), as well as FK506, an immunosuppressive IPL (I-IPL), had cytoprotective effects against hydrogen peroxide (H20O)-induced apoptotic cell death in U251 cells. H2O2 increased both the ratio of bax/bcl-2 and the p53 mRNA expression. However, pre-treatment with FK506 and V10367 significantly prevented any increase in this ratio or p53 mRNA expression. GPI1046 also reduced the ratio of bax/bcl-2 to the normal level. In addition, H2O2 significantly increased activities of all three caspases, caspase-3, caspase-8, and caspase-9, in comparison with non-H2O2 controls. However, FK506 prevented the increase of these caspase activities. On the other hand, it is well-known that glutathione (GSH) and neurotrophic factor (NTF) is related to the induction of apoptosis in neuronal cells. In U251 cells, FK506, GPI1046 and V10367 had GSH-activating and NTF-activating effects. Thus, the immunosuppressive effect is not essential for the cytoprotective properties of IPLs, and IPLs have multiple beneficial properties such as the anti-apoptotic effect, GSH-activating effect, and NTF-activating effect, although the anti-apoptotic effect of NI-IPLs is independent of the regulation of apoptotic activators such as caspase-3.     

Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.     

Regulation of biological activity of laminin-5 by proteolytic processing of gamma2 chain

Laminin-5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino-terminal region of the gamma2 chain as well as at a carboxyl-terminal region of the alpha3 chain. To clarify the biological effect of the gamma2 chain processing, we prepared a human recombinant LN5 with the 150-kDa, non-processed gamma2 chain (GAA-LN5) and natural LN5 with the 105-kDa, processed gamma2 chain (Nat-LN5). Comparison of their biological activities demonstrated that GAA-LN5 had an about five-times higher cell adhesion activity but an about two-times lower cell migration activity than Nat-LN5. This implies that the proteolytic processing of LN5 gamma2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non-processed gamma2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed gamma2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the gamma2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.