Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35----A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.     

Dependence on exercise intensity of changes in electrolyte secretion from the skin sampled by a simple method

Secreta from the palm and forearm was sampled for 1-min periods by a new technique, using a glass cylinder. Subjects exercised for 10-min periods at successive intensities of 40%, 50% and 65% VO2max with a leg ergometer operated in the supine position. Changes in the concentrations (values) of Na+, K+ and Cl- in their secreta during exercise were investigated. Significant positive correlations were found between the values of any two electrolytes in samples from the palm or the forearm, but the correlations between values for any one of the three electrolytes from the two sites were not significant. Values for concentrations of the electrolytes were significantly higher in samples from the palm than in those from the forearm at rest, 10 min after the beginning of exercise and at the end of exercise. No significant correlation was found between values for electrolytes in samples from the palm and the exercise intensity, but values for Na+ in samples from the forearm increased stepwise with increase in exercise intensity, and similar tendencies were observed for values of K+ and Cl-. The values for the three electrolytes in samples from the forearm, but not the palm, were significantly correlated with values for blood lactate, the percentage of VO2max and the heart rate. These results suggest that the present technique is suitable for successive samplings of secreta from the forearm, and that values for the electrolytes in samples are useful indices of exercise intensity.     

Sodium-gradient-driven, high-affinity, uphill transport of succinate in human placental brush-border membrane vesicles.

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.     

Nucleotide sequence of the LuxC gene and the upstream DNA from the bioluminescent system of Vibrio harveyi.

The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. The luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency (1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA, B and D genes. The low G/C content of the luxC gene and upstream DNA (38-39%) compared to that found in the other lux genes of V. harveyi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend for more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.     

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Further technical considerations of the sleeve microanastomosis

In sleeve anastomoses, stenoses at the suture site have been the main concern. Mechanical dilatation is one way to prevent the stenosis, as suggested by Lauritzen. In the present study, 50 vessels (femoral and carotid) and 10 veins were used for sleeve anastomoses and the same numbers of vessels were used for conventional anastomoses (as control) to evaluate the effect of mechanical dilatation using resin corrosion cast (Mercox) because the Mercox cast facilitates three-dimensional stereoscopic views. Gradual dilatations around the suture sites were observed in seven carotid arteries, and three of seven resulted into aneurysm formation due to weakening of the inner vascular wall in the sleeve anastomosis. No dilatation or aneurysm was observed in the femoral arteries. Newly proliferating capillaries formed on the endothelial surfaces of the inner vascular walls around the suture sites after 4 weeks in the sleeve anastomoses. Operative time and endothelial trauma were markedly reduced with sleeve anastomoses. The gradual dilatation and aneurysm formation in the carotid arteries show that sleeve anastomoses should be used carefully for high-pressure arteries in clinical practice if mechanical dilatation is performed.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.