The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster

Summary This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibire ^ts1. Uptake of horseradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19°C, but was blocked at 29°C. After 10 min at 29°C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19°C after 10 min at 29°C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19°C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesiclestubulessmall yolk spheresmature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Constitution of Mitochondrial and Chloroplast Genomes in Male Sterile Tobacco Obtained by Protoplast Fusion of Nicotiana tabacum and N. debneyi

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of malesterile tobacco plants obtained by fusion of Nicotiana tabacumprotoplasts and X-irradiated N. debneyi protoplasts were analyzed.Digestion of cpDNA isolated from ten male sterile lines withfour restriction endonucleases (EcoRI, XhoI, SmaI and HindIII)indicated that these lines possessed either one or the otherparental chloroplast genome. Neither mixture of two types ofcpDNA nor unique restriction fragments were detected in anyof the cases examined. The genetic constitution of chloroplastgenomes identified by restriction analysis of cpDNA showed goodagreement with that based on isoelectric focusing of the largesubunit of the Fraction I protein. The mtDNA from five fusion-derivedmale sterile plants showed banding patterns quite differentfrom each other and from the parental plants. Each plant exhibitednew restriction fragments not found in the parental species.These findings indicate that recombinational events in the mitochondrialgenomes take place rather frequently in the mixed cytoplasmsafter protoplast fusion, whereas the mixed chloroplasts becomesegregated to homogeneity. (Received June 19, 1987; Accepted October 5, 1987)     

Growth and turnover of microbial biomass during the decomposition of organic matter(Polygonum cuspidatum) in vitro

Air-dried fresh and dead specimens ofPolygonum cuspidatum were incubated for 250 days in the laboratory, and the growth and turnover of microbial biomass-C in the organic matter were studied. The biomass-C in the fresh leaf and fresh stem attained maximum levels on day 14 and day 7, respectively, and then settled down to stable levels. In the dead leaf and dead stem, increase in biomass-C ceased by day 4 and the biomass-C levels did not change thereafter. The turnover time of the biomass-C was estimated from the amount of biomass-C and the release rate of CO₂-C. The turnover was rapid in the early period of incubation. Then the turnover time became longer and after incubation for 70 days the values approached those in natural soils (longer than 16 days). During the incubation period, nitrogen was not mineralized in any organic matter. In the dead leaf and dead stem, asymbiotic nitrogen fixation activity increased after incubation for about 40 days and disappeared by the end of the incubation period, whereas nitrogen fixation was hardly detected in the fresh leaf and fresh stem.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [³H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.