The meiotic chromosomes of man

Summary Information was obtained on the chromosome number, and the behavior of autosomes as well as of the sex chromosomes in meiosis in human male germ cells derived from 25 Japanese patients, 4 to 79 years in age, who were hospitalized mostly due to epididymitis, prostate cancer, undescended testes or infertility.In 16 out of the 25 specimens, the chromosome numbers, 46 in 2n and 23 in n, were consistently established together with an XY sex-determining mechanism based on spermatogonial and spermatocyte divisions. No reliable counts were obtained from the remaining 9 cases, because of that they provided no cells for precise investigation.The X and Y chromosomes during the leptotene stage were observed as two separate heteropycnotic bodies lying along the inner wall of the nucleus, while at pachytene they formed a sex-vesicle after homologous pairing. At the diplotene, diakinesis and first metaphase the X and the Y appeared as an isopycnotic bivalent showing an end-to-end association, though there were some cells in which they remained as two separate entities free from contact. Evidence was presented that the X and the Y seemed to associate with each other at the distal end of the short arm of each element.One or sometimes two smallest autosomal bivalents tended to show rather precociously a chiasma-terminalization at the first metaphase.The metaphase chromosomes of the second spermatocytes were evident by the haploid number as well as by their widely diverged chromatids with a characteristic spiral configuration.The testicular materials under study contained in most cases polyploid cells with a considerable frequency in spermatogonia as well as in first and second spermatocytes. Giant sperm heads were observed not infrequently, mostly being abnormal in shape. No significant correlation was obtained between the frequency of polyploid cells and the age of patients so far studied.Contribution No. 679 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. — It is our pleasure to dedicate this paper to Professor Dr. Hans Bauer, Max-Planck-Institut für Meeresbiologie, Tübingen, in honor of his sixtieth birthday.     

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

Cytochrome P-450 and chromosome damage by cyclophosphamide in LEC strain rats predisposed to hereditary hepatitis and liver cancer

LEC strain rats predisposed to hereditary hepatitis and liver cancer were examined for hepatic drug-metabolizing ability and the inducibility of chromosome damage by cyclophosphamide (CP) in somatic cells. Whereas the hepatic cytochrome P-450 contents and the activities of cytochrome P-450-catalyzed monooxygenases were lower in females than in males of both LEC and control LEA strains, male LEC rats exhibited significantly reduced cytochrome P-450 contents and monooxygenase activities compared with male LEA rats. When exposed to CP, a promutagen/procarcinogen requiring P-450-dependent metabolic activation, the frequencies of chromosome aberrations and sister-chromatid exchanges (SCEs) in bone marrow cells tended to be lower in females than in males of each strain and lower in LEC than in LEA rats of the same sex. In particular, the CP-induced SCEs were substantially lower in LEC rats. However, no such sex and strain differences were found in the SCE frequencies in regenerating hepatocytes of partially hepatectomized rats exposed to CP.     

Effects of the Cortex on Light- and Kinetin-Induced Accumulation of Betacyanin in the Epidermal Tissue of Phytolacca americana

Accumulation of betacyanin in the peeled green epidermis fromthe stem of P. americana was induced by incubating the epidermisin Murashige and Skoog's medium, under light, and was promotedby the presence of kinetin. However, in the epidermal tissuewith cortex attached, the accumulation of betacyanin was inhibited. (Received March 27, 1989; Accepted January 24, 1990)     

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.