Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

辣椒素引起脊髓P物质释放及其对血压的影响

为进一步研究脊髓 P 物质(SP)在调节心血管活动中的作用,在大鼠脊髓蛛网膜下腔注射(ith)辣淑素(cap),以刺激脊髓 SP 能神经末梢释放 SP,结果引起血浆去甲肾上腺素(NA)和肾上腺素(AD)含量增高,及具有剂量依赖性的动脉血压上升,心率升高。ith 具有高度特异性的 SP 受体拮抗剂或 SP 抗血清均可阻断 cap 引起的升压效应,免疫组化测定也观察到注入的cap 剂量越大,脊髓胸段 SP 样免疫阳性反应物的致密度越低,这些观察结果支持 cap 可以引起脊髓内 SP 的释放的说法。在第一颈段(C_1)横断脊髓后 ith cap 所引起的升压效应与完整动物 ith cap 的升压效应无显著差异。以上结果提示脊髓 SP 能神经末梢释放的 SP 可以通过交感肾上腺髓质系统引起心血管兴奋效应,SP 可能是引起交感节前神经元兴奋的神经递质。     

Cystatins from bovine brain: Purification,some properties,and action on substance P degrading activity

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. K_i values for the complexes of papain and the inhibitors were estimated to be 2.8×10^–10 M for HMM-cystatin and 1.3×10^–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin high molecular mass inhibitor - LMM-cystatin low molecular mass inhibitor - SP substance P - SPM synaptosomal plasma membranes - p-CMB 4-chloromercuribenzoic acid - BK bradykinin - Bz-Arg-Nap N-benzoyl-dl-arginine--naphthylamide - Arg-Nap dl-arginine--naphthylamide - P-Pxy-Hb hemoglobin initially coupled with pyridoxal-5-phosphate     

Occurrence of rye (Secale cereale) 350-family DNA sequences inAgropyron and otherTriticeae

Evidence is presented that in the R and P genomes (Secale cereale andAgropyron cristatum, respectively) of theTriticeae there exist closely related 350-family DNA sequences in the terminal heterochromatin. This observation is compared to the relationships between these two genomes derived from a comparison of theNor and5 S DNA loci as well as the available data on morphological characters, chromosome pairing, and isozyme studies. It is concluded that the R and P genomes are not closely related and that the common presence of very similar 350-family DNA sequences reflects the parallel amplification of this family of DNA sequences.     

Expression from symbiotic promoters ofRhizobium meliloti inAzotobacter vinelandii andAzospirillum brasilense

InRhizobium meliloti, the promoter P1 of thenif HDK operon, and also the promoter P2, have earlier been shown to be active in the bacteria present in alfalfa root nodules, but not in the bacteria grown aerobically in culture. Here we have looked at the expression from P1 and P2 in two non-symbiotic nitrogen-fixing bacteria,Azotobacter vinelandii andAzospirillum brasilense, using constructions in which the promoters are fused upstream of theβ-galactosidase gene. The promoter P1, but not P2, is active inA. vinelandii, while neither P1 nor P2 is active inAzospirillum brasilense.     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Rice cultivar evaluation for phosphorus use efficiency

Phosphorus deficiency is one of the most growth-limiting factors in acid soils in various parts of the world. The objective of this study was to screen 25 rice cultivars (Oryza sativa L.) at low, medium, and high levels of soil P. Number of tillers, root length, plant height, root dry weight and shoot dry weight were related to tissue P concentrations, P uptake and P-use efficiency. Shoot weight was found to be the plant parameter most sensitive to P deficiency. Significant cultivar differences in P use efficiency were found. Phosphorus use efficiency was higher in roots than shoots and decreased with increasing levels of soil P. Positive correlations were found among growth parameters such as plant height, tillers, root and shoot weight, and P content of roots and shoots. These results indicate selection of rice cultivars for satisfactory performance under low P availability can be carried out using shoot and root dry weight as criteria.