Gene expression during CAM induction under salt stress in Mesembryanthemum: cDNA library and increased levels of mRNA for phosphoenolpyruvate carboxylase and pyruvate orthosphosphate dikinase

Mesembryanthemum crystallinum responds to high salinity in the soil by shifting the mode of carbon assimilation from the C3 mode to Crassulacean acid metabolism (CAM). Several enzymes of carbon metabolism have increased apparent activities in the CAM mode, including phosphoenolpyruvate carboxylase (PEPcase) and pyruvate orthophosphate dikinase (PPDK). We have identified cDNA clones for PEPcase and PPDK by immunological screening of a cDNA library constructed in the protein expression vector lambda gt11. The clones were characterized by immunoblotting and RNA blotting techniques. RNA blotting showed that during CAM induction the steady-state level of mRNAs for both PEP case and PPDK increased.Abbreviations IPTG isopropyl thiogalactoside - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PPDK pyruvate orthophosphate dikinase - Xgal-5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside     

Development and analysis of pectic screening media for use in the detection of pectinase mutants

Summary Two solid pectic media were devised for mutually exclusive detection of extracellular polygalacturonase and pectin lyase produced by fungi including the vascular parasite of tomato Verticillium albo-atrum. These media allowed detection of pectinase-defective mutants. Polygalacturonase detection medium contains non-methylated polygalacturonan (sodium polypectate) is buffered at pH 5.0 (Na citrate, 0.05 M) and is calcium-free. In contrast pectin lyase medium contains polymethylgalacturonan (pectin), is buffered at pH 8.0 (HEPES, 0.05 M) and contains calciumrich agar. When glucose was added to the media for selection of catabolite-resistant mutants, enzyme synthesis was still evident, whereas in comparable conditions in liquid culture production was almost completely repressed. This apparent discrepancy is discussed in terms of the influence of basal synthesis, colony biomass and accumulation of oligouronides which repress induced synthesis and activity.Abbreviations CR catabolite repression - CTAB cetyltrimethyl ammoniumbromide - GALA galacturonic acid - NAPP sodium polypectate - PG polygalacturonase - PL pectin lyase - TBA thiobarbituric acid - UGALA unsaturated galacturonic acid     

Cytoplasmic Poly(ADP-Ribose) Polymerase Associated with Free Messenger Ribonucleoprotein Particles in Rat Brain

Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (free mRNP particles) carrying messenger RNA has been characterized in rat brain. There were first-order kinetics for NAD with an apparent Km for NAD of 90.5 +/- 0.70 microM and Vmax of 19.7 +/- 2.8 pmol ADP-ribose incorporated min-1 mg protein-1. Five poly(ADP-ribose) protein acceptors were identified in the Mr 37,000-120,000 range. It is hypothesized that ADP-ribosylation of specific free mRNP proteins might play a role in the derepression and translation of the silent mRNAs of free mRNP particles.     

Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro

Astrocyte-enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10-90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10-90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.     

Loss of striatal mu1 opiate binding by substantia nigra lesions in the rat

Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. Mu1 binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and mu2 binding assays supported these observations. No change in delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.     

Effects on growth and sporulation of inactivation of a Bacillus subtilis gene (ctc) transcribed in vitro by minor vegetative cell RNA polymerases (E-sigma 37, E-sigma 32)

Summary The E-³⁷ gene ctc was inactivated by a site-specific insertion into the Bacillus subtilis chromosome. The resulting mutation inhibited sporulation by 95% at elevated temperatures (48° C). If the ctc ^- mutation is placed in a strain that carries a mutation in the closely linked but distinct spoVC gene, ctc now affects both growth and sporulation at elevated temperatures. Growth of the ctc ^- spoVC285 strain was transiently inhibited when exponentially growing cultures were shifted from 37° C to 48° C. A similar, but less pronounced growth lag, was also seen in a B. subtilis strain carrying only the spoVC-285 mutation. This finding suggests that both the ctc and spoVC products function in vegetatively growing B. subtilis.     

Correlations between carbon isotope ratio and microhabitat in desert plants

Summary Water is usually considered to be the key limiting factor for growth of desert plants, yet there is little information available of the water-use efficiency of species within a desert community. Leaf carbon isotope ratios, an indicator of long-term intercellular carbon dioxide concentrations and thus of water-use efficiency in C₃ plants, were measured on species occurring within a Sonoran Desert community, consisting of wash, transition and slope microhabitats. Along a soil moisture gradient from the relatively wetter wash to the relatively drier slope, leaf carbon isotope ratios increased in all species, indicating that water-use efficiency increased as soil water availability decreased. Leaf carbon isotope ratios of long-lived perennials were substantially more positive than in short-lived perennials, even though plants were growing adjacent to each other. Leaf carbon isotope ratio and leaf duration (evergreen versus deciduous) were not correlated with each other. The results are discussed in terms of how the efficiency of water use may affect community structure and composition.     

Ig isotypes produced by EBV-transformed B cells as a function of age and tissue distribution

EBV can transform human B cells giving rise to lymphoblastoid cell lines that produce and secrete Ig. Herein B cells from various tissues of newborns and adults were transformed by EBV and their Ig products were analyzed with isotype-specific mAb. Although IgG- and IgA-bearing B cells were present in the newborn, EBV transformed IgM-producing cells almost exclusively in both newborn blood and breast milk. IgM-secreting cells were derived from IgM+ B cells and IgM- pre-B cells present in neonatal blood, but only from IgM+ cells in adult blood. Whereas in adults most EBV-transformed cells produced IgM, producers of IgG and of IgA were present in frequencies that varied according to the tissue source. Precursors of IgG-producing cells were relatively abundant in blood, spleen, and tonsil, and relatively infrequent in bone marrow and appendix. EBV-inducible IgA producers were relatively concentrated in the appendix and to a lesser extent in tonsils and blood. Differences in the subclass composition of EBV-transformed populations of IgG- and IgA-producers were also observed for the various adult lymphoid tissues. IgG1-producing cells predominated in most tissues, and precursors of IgG2 were largely confined to the circulation. Whereas IgA1-producing cells were predominant in all tissues, a marked enrichment in IgA2-producers was observed in the appendix. These results indicate a remarkable heterogeneity in the isotype distribution pattern of EBV-transformable B cells that is determined both by developmental age and tissue localization. We propose that EBV selectively transforms primed B cells, the isotype commitment of which varies according to tissue origin and age.     

BP-3 alloantigen. A cell surface glycoprotein that marks early B lineage cells and mature myeloid lineage cells in mice

To explore the cell surface molecules expressed on pre-B cells we have produced a panel of alloantibodies against transformed pre-B cells from BALB/c mice by immunizing a wild mouse, Mus spretus. One of these antibodies, BP-3, recognized glycoproteins of Mr 38,000 to 48,000 on pre-B cells transformed either by the Abelson murine leukemia virus or an erb B oncogene construct. Removal of N-linked oligosaccharides from the BP-3 Ag revealed a single core protein of Mr 32,000. The Ag was expressed by bone marrow cells in all but one (A/J) of the inbred mouse strains tested and in wild mice of biochemical groups Mus-1 and Mus-2. Analysis of the tissue distribution revealed expression of the BP-3 reactive molecule on normal pre-B and B cells in the bone marrow, 35% of B cells in the circulation, 30% of the B cells in the spleen, and less than or equal to 20% of B cells in lymph nodes, peritoneal cavity, and Peyer's patches. The subpopulation of BP-3+ B cells in bone marrow and peripheral tissues displayed an immature phenotype (IgM IgD +/- ). Examination of a panel of transformed B lineage cells confirmed the early stage-specific expression of the BP-3 alloantigen. In addition, a myeloid cell line and normal myeloid cells were found to express the BP-3 alloantigen. In contrast to B lineage cells, the level of BP-3 expression increased as a function of myeloid cell differentiation. Myeloid cells in the bone marrow expressed relatively little Ag, whereas circulating neutrophils and peritoneal macrophages expressed relatively high levels of the BP-3 alloantigen with Mr 38,000, 41,000, and 46,000. The data suggest that this variably glycosylated cell surface protein could play different roles in the differentiation of B lineage and myeloid lineage cells. The BP-3 alloantigen appears to be a useful marker for virgin B cells that have recently migrated from the bone marrow to the periphery.     

CR2 ligands modulate human B cell activation

A considerable body of evidence from this and other laboratories indicates that complement receptor type 2 (CR2) modulates B cell activation and growth. In the present studies we have examined the effects of three different types of CR2 ligands, i.e., monomeric, aggregated, and latex-bound C3dg; mAb to different CR2 epitopes; and UV-inactivated, non-transforming EBV (EBVUV) for their actions on highly purified, high density resting tonsil B cells. Although none of these ligands induced B cells to enter the cell cycle or synergized with either anti-mu or low m.w. B cell growth factor in triggering B cell mitogenesis, aggregated C3dg, latex-bound C3dg, the OKB7 anti-CR2 mAb, and EBVUV-enhanced thymidine incorporation by phorbol ester-activated tonsil B cells. Such enhancement was not T cell or monocyte dependent. The major action of the CR2 ligands thus seems to be to enhance the transition of B cells activated by certain stimuli from the G1 to the S phase of the cell cycle. In contrast to the action of aggregated and latex-bound C3dg, monomeric C3dg was inhibitory for phorbol ester and aggregated C3dg-induced B cell activation. The HB-5 anti-CR2 mAb, which reacts with a different epitope on CR2 from that of OKB7, did not synergize with PMA in B cell activation. These data provide additional evidence for a role for the CR2 in the control of B cell growth and provide a useful model for studying the CR2-mediated signals that affect the growth of B cells.