Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.     

Thymocytes express a mRNA that is identical to hypothalamic luteinizing hormone-releasing hormone mRNA

1. A luteinizing hormone-releasing hormone (LHRH)-like molecule produced by thymocytes is similar to hypothalamic LHRH in both bioactivity and antigenicity. 2. We determined whether this thymic LHRH is identical to or only homologous with hypothalamic LHRH by synthesizing and sequencing the cDNA of rat thymus LHRH. 3. The thymocyte and hypothalamic LHRH cDNAs are identical, indicating, that the amino acid sequences of LHRH produced in the hypothalamus and the immune system are also identical. 4. This is the first report showing conclusively that cell of the immune system transcribe the authentic mRNA for a hypothalamic releasing factor, LHRH.     

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Polymorphisms within the HLA-DR3 haplotypes

HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA-DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed.No reprints available     

Stridulation and hearing in the noctuid mothThecophora fovea (Tr.)

Summary MaleThecophora fovea (Tr.) (Noctuidae) sing continuously for several minutes by rubbing the 1. tarsal segment of the metathoracic leg against a stridulatory swelling on the hindwing. In Northern Yugoslavia (Slovenia) the males emerge in late October and start stridulating about a week later when the females emerge.The sounds are pulse trains consisting of 10–12 ms long sound pulses with main energy around 32 kHz and a PRR of 20 pulses/s. The mechanics of the sound producing apparatus was studied by activating the stridulatory swelling with short sound impulses. The impulse response of the swelling was recorded by laser vibrometry and amplitude spectra of the vibrations showed maximum velocities between 25 and 35 kHz. Hence, it seems likely that the stridulatory swelling is driven as a mechanical oscillator with a resonance frequency which determines the carrier frequency of the sounds.Audiograms of both males and females showed peak sensitivities at 25–30 kHz. The median threshold at the BF was 36 dB SPL. The peak intensity of the sound pulses was 83 dB SPL at 1 m, which should enable the moths to hear each other at distances of around 30 m. Therefore sound production inT. fovea might function in long distance calling. It is argued thatT. fovea can survive making such a noise in spite of being palatable to bats because it flies so late in the year that it is temporally isolated from bats.Abbreviations PRR pulse repetition rate - SPL sound pressure level - BF best frequency     

Studies on stimulus-response coupling in human neutrophils. II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.