Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Fungal growth during rice tapé fermentation

Fungal growth was quantified during Indonesian rice tapé fermentation using an agar-film technique following sample homogenization for 1 min at 25000 rev/min. After 72 h fermentation, mould hyphal length was 0·68 km/g, yeast hyphal length 2·1 km/g and the numbers of mould chlamydospores and single yeast cells were 14 times 10⁵/g and 5·1 times 10⁷/g respectively. The estimated fungal biomass in rice tapé after 72 h was 25 mg/g dry weight with 62% of this being mould hyphae, 24% mould chlamydospores, 13% yeast hyphae and 1% yeast cells.     

Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

The effect of repetitive haemorrhage on plasma cortisol, beta-endorphin and N-terminal pro-opiomelanocortin in conscious sheep

We measured the effect of repeated haemorrhagic stress, performed on four consecutive days in conscious adult sheep, on the plasma concentrations of cortisol and ACTH-related peptides to determine whether the pituitary-adrenal response was altered by stress repetition. Peptides from the C-terminus of the ACTH pro-hormone was measured by beta-endorphin RIA. Glycopeptides derived from the N-terminus of the ACTH pro-hormone were measured by tau 3-MSH RIA. The immunoreactive tau 3-MSH in sheep plasma was found to have an apparent molecular weight of approximately 10,000 by gel chromatography through Sephadex G-75, which is similar to the size of the major circulating form of pro-tau-MSH found in human and rat plasma. Daily haemorrhage consistently elevated plasma concentrations of cortisol and pro-tau-MSH. There was no significant difference in the daily responses of either cortisol or pro-tau-MSH when considered individually. However, there was a significant change over the four days in the relationship between the cortisol and pro-tau-MSH responses, as judged by analysis of variance of the difference in daily z-scores of cortisol and pro-tau-MSH. This trend indicated a relative increase in the secretion of pro-tau-MSH from the pituitary compared to the cortisol response, and suggested that repeated exposure to stressful stimuli may alter the pituitary-adrenal-axis.     

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.     

Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells

We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture.     

Mouse IgD half molecules with shortened IgD heavy chain result from alterations within C delta locus

An unusually small (51 KD) IgD myeloma protein was isolated from secretions of TEPC 1017 generation (gen) 24. The delta-chain mRNA and the delta-chain gene in this tumor were compared with those of TEPC 1017 of earlier generations. The gen 24 protein contained one normal-sized kappa-type light chain (21 KD) and one unusually short delta-heavy chain (30 KD). The delta-heavy chain was 15 KD shorter than that of TEPC 1017 of earlier generations, owing to a delta-mRNA (1.15 kb) which was 600 bp shorter than that of TEPC 1017 of earlier generations. TEPC 1017 is a tetraploid tumor, and the gen 24 appears to contain at least two different deletions on different chromosomes. The short mRNA was produced from one of these altered delta-chain genes which had a productive VDJ rearrangement but which had lost the C delta 3 domain and perhaps the C delta H domain as well. Despite these genetic insults, RNA splicing produced delta-mRNA with secreted termini and mRNA with membrane-binding termini. It is suggested that the mouse C delta gene has an unusual predilection for deletions because it normally lacks any vestige of C delta 2 and, during i.p. passage, it suffered further deletions or alterations.     

中华白蛉的自育性研究

现场及实验室结果表明,我国黄土高原的大多数中华白蛉(Phlebotomus chinensis)应属自育性品系,它通常在羽化后、吸血前经交配能依靠腹节内脂肪体发育卵泡,一般在产卵后始行吸血。其生理性状是:胃内无血、腹节内有大量块状或条状脂肪体。羽化24小时后,附腺内有暗色颗粒,卵巢内有发育的卵泡。在25℃士1℃下它的生活史分快、慢两型。快型从卵至成虫仅需44—50夭,慢型需要以四龄幼虫滞育,其长短随滞育期而定,最长的滞育期达301天。观察了白蛉幼虫在饥饿状态下对自育性的影响。此外,还比较了吸血白蛉与自育性白蛉的妊卵数。吸血白蛉的妊卵数约较自育性的高出1/5。这种自育性品系的中华白蛉在自然居群约内占92%、主要栖于洞穴内为野栖种类。本文对自育性中华白蛉的生态及其防制策略作了分析和讨论。