Effects of temperature and pH on hemoglobin release from hydrostatic pressure-treated erythrocytes

The release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45 degrees C) and pH (5.5-8.8) at atmospheric pressure. Only in the case of high pressure (2,000 bar) did the release of hemoglobin increase significantly with decreasing temperature and pH. Maleimide spin label studies showed that the temperature and pH dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky.     

The addition of exogenous gangliosides to cultured human cells results in the cell type-specific expression of novel surface antigens by a biosynthetic process

After the observation that human mAb 32-27M reacts only with melanoma and astrocytoma cells cultured in the presence of fetal bovine serum, a novel pathway for the uptake of exogenous gangliosides, their further biosynthesis, and expression at the cell surface as novel Ag has been elucidated. The addition of fetal bovine serum to melanoma and astrocytoma cells growing in synthetic medium (insulin-transferrin-selenium) resulted in reactivity with Ab32-27M. As antibody 32-27M detects N-glycolylneuraminic acid (NeuGc)-containing gangliosides, the effect of adding a number of different gangliosides to melanoma and astrocytoma cells cultured in the synthetic medium was studied. Only the addition of NeuGc-GM3 resulted in the development of Ab32-27M reactivity. The identity of the antigenic structures developed after addition of fetal bovine serum or NeuGc-GM3 was determined by analysis of the gangliosides from both samples. The major component detected in melanoma cell lines was shown to be N-acetylneuraminic acid-NeuGc-GD3. Another, slower moving component, present in some melanomas and in astrocytomas may be N-acetylneuraminic acid-NeuGc-GD2. The cell type specificity for these processes can be most readily explained by postulating that all cells can take up exogenous gangliosides but only melanoma and astrocytoma cells have sufficiently high levels of GM3 alpha 2----8-sialyltransferase for the conversion of added NeuGc-GM3 to disialogangliosides to be effective. These results demonstrate a novel pathway for exogenous glycolipid processing that can lead to novel Ag expression but may also play a role in normal glycolipid metabolism and function.     

Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

Responses of non-spiking giant interneurons to substrate tilt in the crayfish, with special reference to multisensory control in the compensatory eyestalk movement system

In the brain of the intact crayfish, three pairs of non-spiking giant interneurons (G1, G2, G3; NGIs) scarcely responded to substrate tilt about the longitudinal axis of the body either in the dark or in the presence of an overhead light. However, when the statolith was removed, these NGIs responded with depolarizing and hyperpolarizing potentials respectively to upward movements of the ipsilateral legs (2nd–5th pereiopods) and upward movements of the contralateral legs produced by substrate tilt. The relationships between the polarity of the potential and the direction of movement in the contralateral legs were opposite to those in the ipsilateral legs. The amplitude of the responses was proportional to the frequency (0.5-0.05 Hz) and amplitude of tilting. When the legs were moved unilaterally, the NGIs responded with depolarizing and hyperpolarizing potentials to upward movements of the ipsilateral legs and to upward movements of the contralateral legs, respectively. When the legs were moved bilaterally in the same direction by upward or downward movement of the substrate, the NGIs scarcely responded to the leg movements. A hypothetical model is presented to account for the pathways of sensory inputs to the NGIs and the role of NGIs in compensatory oculomotor system.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Electronic structure of siloxene model compounds

The electronic structures of one-dimensional and two-dimensional siloxene (Si₆O₃H₆) model compounds have been examined theoretically, using the semiempirical tight-binding self-consistent field crystal orbital (SCF-CO) method. These compounds are formed by silicon-based chain and planar structures containing a regular array of oxygen atoms. Results show that the two-dimensional polysilane in which OH groups are substituted for H atoms possesses a relatively smaller direct gap than other siloxenes. It is assumed that the electronic structures of siloxenes are affected not only by the dimensionality of Si-Si -conjugational networks due to an array of oxygen atoms, but also by the diminishing of the electron population in the Si-Si bonding orbitals caused by oxygen atoms with large electronegativity.     

Induction of {alpha}-Amylase is Repressed by Uniconazole, an Inhibitor of the Biosynthesis of Gibberellin, in a Dwarf Mutant of Rice, Waito-C

A system for induction of synthesis of     

Histological studies on male sterility of hybrids between laboratory and wild mouse strains

In this study the cellular mechanisms of male sterility in F1 hybrids (BNF1) between BALB/c and wild-derived M.MUS-NJL (NJL) was investigated. Cell proliferation and differentiation in the sterile testis were examined by bromodeoxyuridine-labeling and use of germ cell stage-specific antibodies. In BNF1 testes, spermatogonia actively proliferated with a seminiferous epithelial cycle, and were retained in the basal layer of the tubules. However, preleptotene, leptotene and zygotene spermatocytes moved to the adluminal region. Immunohistological data with germ cell stage-specific antibodies indicated the presence of few, if any, pachytene spermatocytes in BNF1 testes. Thus, spermatogenesis seemed to be blocked at the zygotene stage. For examination of germ cell-Sertoli cell interactions, testes of aggregation chimeras between BNF1 and C3H/HeN were analyzed immunohistologically with C3H-specific antibody. Results showed that spermatogenesis of C3H-germ cells was normal, even when these cells in contact with BNF1-Sertoli cells. Differentiation of BNF1-germ cells progressed from zygotene to pachytene stage spermatocytes when these cells were surrounded by C3H-Sertoli cells, but never proceeded beyond the pachytene stage. These observations suggest that at least two different cellular factors may be involved in spermatogenesis, one acting in the germ cells and the other mediated by Sertoli cells. Furthermore, mating experiments revealed that the degree of spermatogenesis varied in different F1 hybrids, and that the major sterility factor was closely linked to the T -locus on chromosome 17.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.