Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

Calcium in hippocampus following lidocaine induced seizures: an electron cytochemical study

The effect of lidocaine seizures on cellular accumulation of calcium was studied in hippocampal subfields CA1 and CA3 and the dentate gyrus of rats, using the combined oxalate-pyroantimonate method. The specificity of the reaction was ascertained by EGTA treatment and X-ray microanalysis. In control rats, calcium was visualized between myelin lamellae of axons, in synaptic vesicles and in some lysosomes. Two hours after onset of lidocaine seizures selective neuronal degenerations appeared in hippocampal subfields CA1 and CA3 but not in the dentate gyrus. Calcium deposits were present in numerous mitochondria of pyramidal cells and, occasionally, also of neuroglial cells. Many of these mitochondria exhibited ultrastructural alterations. Calcium uptake was most prominent in the CA3 sector but was also present in the CA1 subfield as well as the dentate gyrus. Intracellular calcium uptake, in consequence, is not the unique attribute of selectively vulnerable hippocampal neurons.     

Serum cortisol level changes in the maternal-fetoplacental unit between the 28th-40th weeks of pregnancy

Changes in the serum cortisol level of maternal venous, umbilical venous and umbilical arterial blood were studied separately between the 28th-36th weeks in cases of preterm deliveries (n = 74) and in the 40th week in cases of term delivery (n = 34). Results indicate that between the 28th-40th weeks of pregnancy the cortisol concentration increased only in the serum of the umbilical artery; the "umbilical arterial/umbilical venous concentration X100" quotient rose from a value of 86% measured at 28th-32nd weeks to 103% in the 40th week of pregnancy. Positive correlation was found between the cortisol concentration of the three samples. On the basis of these results the authors believe that fetal adreno-cortical activity increases before birth.     

Early increases in the frequency of DNA initiations and of phospholipid synthesis discontinuities after nutritional shift-up in Escherichia coli

Cultures of Escherichia coli (strains ML30 and K12 AB1157), synchronized by repeated phosphate starvation, were submitted to nutritional shifts-up at various cell ages. The progression of the replication forks was assessed by DNA-DNA hybridization of pulse-labelled chromosomal DNA with plasmid DNA probes containing specific chromosomal sequences. The rate of phospholipid synthesis and its cyclic discontinuities were measured by continuous and pulse labelling with palmitate. The DNA-DNA hybridization experiments showed that a shift-up induces a burst of initiation from the oriC region. These hybridization results, taken together with older data from the literature, suggest that most DNA initiations belonging to this burst are not followed by complete replication. Following a shift-up, the rate of phospholipid synthesis is maintained for 13-20 min, depending on cell age at shift-up, then doubles. The new steady-state rate of phospholipid synthesis is reached through a series of three doublings, while the cell mass doubles approximately twice. This discrepancy brings the rate of phospholipid synthesis per mass unit to its steady-state postshift value.     

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).