Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus

In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume–rhizobia symbiosis by modifying nodule development and metabolic processes.     

Possible Mechanism of Action of β-Lactam-Enhancing Factor on Methicillin-Resistant Staphylococcus aureus

We have recently found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of β-lactams on methicillin-resistant strains of staphylococcal species such as methicillin-resistant Staphylococcus aureus (MRSA), but shows only weak effects on methicillin-susceptible S. aureus and bacterial strains other than staphylococci. Factor T alone did not strongly inhibit cell metabolism and bacterial growth unless an excess amount was added. When Factor T was added to the culture medium beforehand, the growth of MRSA cells was rapidly suppressed just after addition of oxacillin (MPIPC). However, the growth of the cells was inhibited gradually when these two reagents were added in reverse order. For full expression of the enhancing effect, it seemed necessary for cells of MRSA strains to be incubated with Factor T for at least 2–3 hr. When the cells were washed after being sensitized by incubating them for 5 hr with Factor T, it took approximately 1 hr for the cells to recover their resistance to MPIPC. Factor T reduced the amount of penicillin-binding protein-2′ (PBP 2′), and thus sensitized the MRSA strains to β-lactams.     

Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase

Recently, a low molecular weight factor, which specifically stimulates sperm adenylate cyclase, was found in porcine seminal plasma (Okamura, N., and Sugita, Y. (1983) J. Biol. Chem. 258, 13056-13062). The purified factor was analyzed by 1H NMR, 13C NMR, infrared spectroscopy, and elementary analysis and identified as sodium bicarbonate. The effects of sodium bicarbonate both on adenylate cyclase activity in porcine spermatozoa and on sperm motility have been studied. Sperm adenylate cyclase was found to be specifically activated by bicarbonate over the physiological concentration range. In contrast, the adenylate cyclase activity in other tissues was not affected. The same concentration range of bicarbonate which resulted in activation of adenylate cyclase also stimulated sperm motility. The motility and enzyme activity of spermatozoa in all species so far tested (human, bovine, rat, mouse, and dog) were found to be similarly sensitive to bicarbonate concentration. These results show that the bicarbonate-sensitive adenylate cyclase system regulates sperm motility and suggest that this system is common to all mammals.     

Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation.

C57BL/6(B6)-jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6-jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6- +/+ mice into B6-jsd/jsd testes or tubules from B6-jsd/jsd mice into testes of (WB x C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6-jsd/jsd testes. In contrast, in B6-jsd/jsd tubules transplanted into WBB6F1-W/Wv testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6-jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.     

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.     

Effect of cyclic RGD peptide on cell adhesion and tumor metastasis.

Several kinds of cyclic peptides containing an L-arginine-glycine-L-aspartic acid RGD sequence were synthesized by the liquid phase method, and we investigated their effects on the attachment of mouse B16 melanoma cells onto fibronectin-coated well. Cyclo (GRGDSPA) inhibited the cell attachment at a 20-fold lower concentration than the linear form. The cell adhesion was inhibited by the synthetic peptides with the following relative order of activity: cyclo (GRGDSPA) much greater than cyclo (GRGD) greater than cyclo (RGDS), cyclo (GRGDSP) greater than cyclo (GRGDS) greater than cyclo (RGDSP), cyclo (RGDSPA). Cyclo (GRGDSPA) was more effective at inhibiting cell attachment to vitronectin than it was at competing with fibronectin attachment, as reported in the case of GRGDSP. Moreover, cyclo (GRGDSPA) significantly reduced the formation of colonies in mice injected with B16-FE7 melanoma cells.     

Coordination structure and chemical reactivity of hemoprotein-butyl peroxide complex.

ESR and optical spectra ascribed to be the hemoprotein-butylperoxide complex were detected for the frozen aqueous solution containing whale met-Mb and t- or n-butylhydroperoxide at pH 10. The observed ESR and optical parameters of the complex were characteristic to those of six coordinate ferric low-spin complexes, having the butylperoxide anion at the axial position of heme. pH dependent ESR measurements demonstrated the formation of the complex in the biological pH regions (7.0). Time dependent ESR and optical measurements indicated that the complex may be one of the intermediate species in the processes of heme degradation reaction induced by butylperoxide.     

Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines.

Hepatocyte growth factor (HGF) has potent mitogenic activity for mature hepatocytes and various normal epithelial cells. We now have evidence that HGF at 1-10 ng/ml, strongly inhibits the growth of HepG2 hepatocellular carcinoma cells, B6/F1 melanoma cells and KB squamous carcinoma cells. These tumor cells express high affinity receptors for HGF with a Kd of 25-28 pM, similar to findings with hepatocytes. HGF at 1-100 ng/ml had no significant cytolytic effect on tumor cells. Therefore, the anti-proliferative effect of HGF on tumor cells seems to be cytostatic, not cytolytic. As HGF apparently has bidirectional effects on cell growth, the possibility that it can serve as an anti-tumor agent merits attention.     

The activating effects of bicarbonate on sperm motility and respiration at ejaculation

Mature porcine sperm preserved in the cauda epididymis are quiescent. At ejaculation, they are mixed with the seminal vesicle fluid containing HCO3- and are rapidly activated. The role of HCO3- on the sperm activation process at ejaculation was studied in vitro. HCO3- quickly increased the motility, respiration rate and cAMP content of the porcine epididymal sperm. The extent of activation was proportional to the pCO2 in the medium. The activating effect of HCO3- on the motility was observed even in the absence of fructose as well as in the presence of KCN. 8-Bromoadenosine 3',5'-cyclic monophosphate and theophylline showed similar activating effects to that of HCO3-. However, HCO3(-)-free seminal plasma, Ca2+, amino acids, intermediates of the Krebs cycle, substrates of respiration and increases in the intracellular pH, extracellular pH or ionic strength of the medium had no effect. Fructose sustained the active state of the sperm and gradually increased both the motility and respiration rate when the dose of HCO3- was low. The anion channel blocker enhanced the activating effect of HCO3-. These results suggest that, upon ejaculation, HCO3- is a unique activator in vivo which makes the quiescent sperm motile via the HCO3(-)-adenylate cyclase-cAMP system, to which an endogenous HCO3- derived from metabolic CO2 may be related.