Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.     

Oligodeoxyribonucleotides containing 1,3-propanediol as nucleoside substitute.

1,3-Propanediol was protected with one dimethoxytrityl residue and converted into the methoxy- and cyanoethoxyphosphoramidites 2a and 2b, respectively. Solid-phase oligonucleotide synthesis, employing the phosphoramidite 2a resulted in the dodecamers d(CGCGAATTCGCG) (6-9), in which dA or dT residues were replaced by 1,3-propanediol. These oligomers showed a high tendency to form hairpins. Their phosphodiester bonds between the 3'-position of a nucleoside and the propanediol moiety was not cleaved by snake venom phosphodiesterase.     

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30     

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus)

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.     

Biotransformation of 5βH-pregnan-3βol-20-one and cardenolides in cell suspension cultures of Nerium oleander L.

Summary In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5H-pregnan-3ol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5H-steroidaglycones at C-3. The respective glucosides of the 5H-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5H-steroidglucosides.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - EtOAc ethylacetate - MeOH methanol - MS Murashige & Skoog     

Aminopyridazine derivatives and TXA2-PGI2 balance

Experiments carried out under the conditions adopted showed the strong affinity of aminopyridazine derivatives for the eicosanoids TXA2 and PGI2. But this affinity depended on the chemical structure of the molecule: a small change in the radical grafted onto the pyridazine ring could completely modify the pharmacological activity of the molecule. Consequently it should be possible to control the properties of pyridazine derivatives according to pharmacological needs. Thus: --pyridazin-3-one derivatives were mainly active on TXA2 biosynthesis: 2-aminoalkyl 5-arylidene 6-methyl (4H) pyridazin-3-ones inhibited the TXA2-synthesizing activity of cardiac tissue whereas 3-amino 4,6-diaryl pyridazin-3-ones were specific inhibitors of the TXA2 synthetase in vitro, but these effects were weak. --pyridazine derivatives were devoid of any effect on the TXA2-synthesizing activity of cardiac tissue: they acted on either TXA2 synthetase or PGI2 synthetase according to the radicals grafted onto the pyridazine ring. --none of the compounds under study was active on the PGI2-synthesizing activity of cardiac tissue.     

Comparative effects of some 3-amino 4,6-diarylpyridazines on the biosynthesis in vitro of TXA2 and PGI2- and on the TXA2- and PGI2-synthesizing activities of cardiac tissue

Some 3-amino 4,6-diarylpyridazine derivatives were tested for their effects on TXA2 and PGI2 biosyntheses in vitro and on the TXA2- and PGI2-synthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2- and PGI2-synthesizing activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXA2 synthetase and PGI2 synthetase sources). TXB2 and 6-keto PGF1 alpha were determined by RIA. Among the compounds under study, 3-morpholino 4,6-diphenylpyridazine (III) was shown to inhibit specifically the TXA2 synthetase. Substitution of the morpholino group by a dimethylamino one (I) reinforced the inhibiting effects on TXA2 synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholino moieties by either a 3-hydrazino (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamino group (VI) abolished completely the effects of the molecule on TXA2 and PGI2 synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of I neutralized all its inhibitory effects both on TXA2 and PGI2 synthetases in vitro. None of the 3-amino 4,6-diarylpyridazine derivatives was active on either the TXA2- or PGI2-synthesizing activities of cardiac tissue.