Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.     

Phytochemical and morphological support for the existence of two species inMonoclea (Hepaticae)

Recognition of two different species in the liverwort genusMonoclea Hook. (monotypic orderMonocleales), viz.M. forsteri Hook. in New Zealand andM. gottschei Lindb. in the New World, is supported by characteristics of the sporophyte, antheridial receptacle and secondary metabolites.M. gottschei produces the greatest variety of flavonoids and the largest amount of bisbibenzyls ever encountered in a liverwort. In contrast,M. forsteri is poor in secondary metabolites. Two allopatric subspecies are recognized inM. gottschei, based on characteristics of the antheridial receptacle: subsp.gottschei in Chile (Valdivian region, Juan Fernandez Is.) and subsp.elongata Gradst. & Mues, subsp. nova, in tropical America. The exclusive occurrence inMonoclea of glucuronide and galacturonide flavone glycosides and the fact that capsule dehiscence may take place before full elongation of the seta are new arguments in support of the placement ofMonocleales in theMarchantiidae. Publication Nr. 43 of the Arbeitskreis Chemie und Biologie der Moose, Universität des Saarlandes, Saarbrücken. This paper is dedicated to DrElla O. Campbell, Massey University, Department of Botany and Zoology, New Zealand on the occasion of her 80th birthday.     

Hormonal responses to analgesic nitrous oxide in man

Analgesic concentrations of nitrous oxide were administered to 6 healthy male subjects, and blood samples were assayed for prolactin, ACTH, follicle stimulating hormone, luteinising hormone, growth hormone, cortisol and thyroid hormones. Analgesic nitrous oxide (mean concentration = 48.8%) produced statistically significant elevation of prolactin and depression of cortisol whilst not producing statistically significant changes in the other hormones assayed. The increase in prolactin and decrease in cortisol levels are similar to the hormonal changes associated with administration of opioids in man. We have also confirmed the findings of other workers that cortisol levels may not always be correlated with ACTH levels.     

Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae

alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.     

Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.