Cloning of rabbit uricase cDNA reveals a conserved carboxy-terminal tripeptide in three species

cDNA clones encoding uricase have been isolated from a rabbit liver cDNA library. The nucleotide sequences of the cDNAs have been determined and those of the rat uricase cDNA have been revised. In all three uricases, the carboxy-terminal tripeptides are Ser-Arg/Lys-Leu sequences, which have recently been suggested as an essential element of peroxisomal targetting signals for many but not all peroxisomal proteins.     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Alternative life-history styles of Japanese freshwater sculpins revisited

Synopsis Japanese freshwater sculpins consist of 6Cottus and 1Trachidermus species, with various types of life-cycles, namely, catadromous, amphidromous, lacustrine land-locked and fluvial land-locked. Among them,C. amblystomopsis andC. nozawae have been regarded as sibling species. Because of very similar morphological and ecological adult characteristics they were formerly classified as a single species.C. amblystomopsis mainly inhabits the lower courses of rivers and produces many small eggs from which pelagic larvae are formed. In contrast,C. nozawae lives in the middle or upper courses of rivers and deposits few, large eggs, from which well-developed benthic young emerge, well on the way to the definitive phenotype as fully formed juveniles. These two species have distinctly different life-cycles: amphidromous forC. amblystomopsis and fluvial forC. nozawae. A comparison of the early ontogeny shows that, amongCottus species, the small-egg, amphidromousC. amblystomopsis is altricial, while the large-egg, fluvialC. nozawae is precocial. Assuming thatC. nozawae has been derived phylogenetically fromC. amblystomopsis or its ancestral relatives, it is reasonable to consider that the fluvial land-locked life-history style ofC. nozawae has evolved from the ancestral arnphidromous one through adapting to the upstream habitat by an increase in egg size and consequent truncation of the larval period.     

Experiments inducing prospective polar body nuclei to participate in embryogenesis of the sawfly Athalia rosae (Hymenoptera)

Summary Mature eggs dissected from ovaries of unmated females of Athalia rosae (Hymenoptera: Tenthredinidae), if placed on a filter-paper soaked with distilled water, are activated and develop to haploid males. Occasionally, however, diploid females develop from these artificially activated eggs. Treatment of mature unfertilized eggs dissected from diploid females with ice-cold temperatures immediately before activation and with a high temperature (36° C) upon and immediately after activation resulted in the production of diploid males, diploid females, triploid females and gynandromorphs at high frequency. The same treatment of mature unfertilized eggs dissected from triploid females resulted in the production of only triploid survivors. These results, together with the results on the segregation of a marker mutation, yellow fatbody (yfb), appear to indicate that meiotic divisions were complete in the treated eggs, and that all four nuclei became potentially capable of participating in development with or without automictic fusion.Studies on the sawfly, Athalia rosae (Insecta, Hymenoptera, Tenthredinidae), part V     

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.     

Possible regulation of autologous mixed lymphocyte reaction by OKM1+ NK cells

T cells are stimulated by autologous non-T cells and interleukin 2 (IL-2) is produced in the conventional autologous mixed lymphocyte reaction (AMLR) in young healthy controls. The role of cells with natural killer (NK) cell markers (OKM1+ cells or Leu 7+ cells) in the AMLR was studied. There were significant inverse correlations between the percentage of input OKM1+ cells minus monocyte (OKM1+ NK cells) and either AMLR proliferation (gamma = -0.9, P less than 0.001) or IL-2 production (gamma = -0.75, P less than 0.01) in the AMLR cultures after 7 days measured at 7 days. A statistically significant correlation was observed between the percentage of input Leu 7+ cells and AMLR proliferation (gamma = -0.64, P less than 0.05), but not IL-2 production. These results suggest that the AMLR is controlled by OKM1+ NK, perhaps acting through IL-2 regulation.     

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.     

Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.     

Application of 39K NMR Spectroscopy to Potassium Transport in Mung Bean Root Tips

The intracellular K⁺ concentration and its change in mung bean[Vigna mungo (L.) Hepper] root tips were investigated non-invasivelywith ³⁹K nuclear magnetic resonance spectroscopy using a membraneimpermeable shift reagent, dysprosium (III) tripolyphosphate[Dy(PPP_i)^7–₂]. The K⁺ resonance was shifted to highermagnetic field in proportion to the concentration of the shiftreagent. In addition to a reference capillary peak for measuringthe K⁺ concentration, two well-resolved peaks (intra- and extracellularK⁺ resonances) were observed in the 39K NMR spectra of mungbean root tips. The intracellular K⁺ concentration was determinedto be 41 mM, which was similar to the value obtained by flamephotometry. When 20 mM KCl was added to the external medium,the intensity of the intracellular K⁺ resonance gradually increasedand the net K⁺ uptake rate was calculated to be 4.1 micromolesper gram fresh weight per hour. After removal of KCl from theperfusion medium, the intracellular K⁺ concentration considerablydecreased. With ³¹P NMR method, 2.5 mM Dy(PPP_j)^7–1₂ and20 mM KCl had little effect on the ATP level in the cells. Wehave indicated that the ³⁹K NMR method can be used to determinethe K⁺ levels and net fluxes of the K⁺ transport in perfusedroot tips successively. (Received April 6, 1988; Accepted September 29, 1988)     

Individual growth and smoltification of juvenile masu salmon, Oncorhynchus masou Brevoort, under rearing conditions

In a reared population of individually marked juvenile masu salmon, individual growth was monitored from the first autumn in 1983 to the following spring. The potential smolts were not significantly greater in mean fork length and body weight than the potential parr in late August of the first year, but they then grew faster until March of the second year. As a result, the potential smolts formed the upper mode of the bimodal length distribution after February. Especially in autumn (October and November) the specific growth rates of potential smolts were significantly greater than those of parr, and the bimodality in growth rate distribution was more distinct for males than females. These suggest that there are two groups having different growth rates in autumn of the first year and that sufficient growth in this period may play an important role in smoltification in the following spring.