Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon.

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.     

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-convertingenzyme (ACE) to study the involvement of its glycanic chainsin its physicochemical and catalytic properties. The effectsof endoglycosidases F₂ and H, and of N-glycanase were assessedby ACE mobility in SDS-PAGE. N-Glycanase only was completelyeffective with or without previous denaturation, leading toa shift in ACE M_r from 172 to 135 kDa; endoglycosidase F₂ producedthe same shift but only without previous denaturation. DeglycosylatedACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine,and an identical Stokes radius as measured by size-exclusionhigh performance liquid chromatography. Neuraminidase had noeffect on ACE Stokes radius but slightly decreased its kcatwhich could be related to variations in ionization of the activesite. The isoelectric point of ACE, as, determined by isoelectricfocusing, increased from 4.5–4.8 to 5.0–5.3 aftereither endoglycosidase F₂ or neuraminidase digestion, but stillwith microheterogeneities which thus did not seem to be relatedto ACE glycans. Deglycosylated ACE did not bind onto agaroselectinsin contrast to native ACE which bound strongly to concanavalinA showing interactions involving oligomannosidic or biantennaryand sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin,an inhibitor of N-glycosylation, did not modify ACE secretionby endothelial cells. Thus, ACE glycans have no drastic effectson structural and biological properties of the protein, butthey may have a functional role on intracellular targeting ofboth secreted and membrane-bound ACE isoforms, also for theprotection of the soluble plasma form against hepatic lectinsand the maintenance of its hydrosolubility. converting enzyme (peptidyldipeptidase EC.3.4.15.1) endothelium glycosidases lectins     

Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α-demethylases (CYP51) from fungi and mammals

Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.     

Activation of Coupling Factor 1 from Euglena gracilis Chloroplasts : Conditions for Optimal Activation and Their Possible Physiological Significance

The recently described method for the activation of the Ca²⁺-ATPase of coupling factor 1 from chloroplasts (CF₁) of Euglena gracilis by low pH occurs optimally in high concentrations of NaCl, and is unaffected by the acid used to lower the pH to 4.5. Activation is inhibited by light, and this effect can be reversed by the presence of NADP⁺, ADP + inorganic phosphate, or an uncoupler. There appears to be no difference between the activities in the soluble and the particulate phases, and they seem to represent the same enzyme. The response of the activation process to light and to effectors of electron transport and phosphorylation indicates a possible physiological role for the acid activation of Euglena CF₁.     

Detailed cell cycle analysis in human lymphocytes; Application to γ-irradiated cells

Summary A method based on BrdU incorporation for analyzing in detail the kinetics of the cell cycle is described. The S phase has been subdivided into five subphases, each recognizable by their BrdU incorporation pattern at metaphase. The method can be useful for the study of abnormal cell cycles, and may have particular application in mutagenesis studies concerning the various subphases of the S phase, without using synchronization techniques. An application of the method is described, showing that -irradiation, during the course of the S phase, leads to a lack of cells which were in early S phase at the time of irradiation. This finding can be related either to a higher lethality at this stage of the cell cycle or to a delay in completion of DNA replication after irradiation.Hoider of a C.E.C. scholarship     

Qualitative dynamics of three species predator-prey systems

Summary A qualitative analysis of some two and three species predator-prey models is achieved by application of the method of averaging in conjunction with a Lyapunov function constructed from the appropriate Volterra-Lotka model. We calculate the limit cycle solution for a two-species model with a Holling type functional response of the predator to its prey by means of a time-scaled transformation. The existence of a bifurcation of steady states for a community of three species is discussed and the periodic solution around one of the unstable steady states is calculated to the lowest approximation. Several comments are made regarding the behavior of these systems under changes of some control parameters.This work was supported in parts by USERDA, Contract number E(11-1)-3001.     

Molecular and functional anomalies in two new mutant glucose-phosphate-isomerase variants with enzyme deficiency and chronic hemolysis

Summary Two new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients' parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal.GPI Paris was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI Enfants malades exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate»glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate»fructose-6-phosphate).No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other.Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.     

New developments in our knowledge of the chemistry of renin.

Although the role of renin in hypertension continues to be incompletely defined, recent progress in the chemistry of renin has been considerable. Extensive purifications of hog kidney renin and the renin-like mouse submaxillary gland enzyme have been achieved. Various inhibitory peptides based on tetradecapeptide renin substrate have been useful in renin kinetic studies and in renin affinity chromatography. Classification of renin as an acid protease results from its marked inhibition by pepstatin and from the discovery that free carboxyl at the active site is essential for activity in human and hog kidney and mouse submaxillary gland enzymes. The presence of pseudorenin in all tissues has limited the use of model peptides as renin substrates in plasma and crude tissue extracts, since the proteolytic properties of the two enzymes are nearly identical. The existence of renin in multiple, chromatographically separable forms has been known. More recently inactive forms have been found in plasma, amniotic fluid, and hog and rabbit kidneys. Prolonged storage or treatment with acid, trypsin, or pepsin causes activation; in some instances the conversion is from a higher than normal molecular weight. The implications of these findings with respect to the renin-angiotensin system need much further investigation.