Photoproduction of amino acids by mutant strains of N2-fixing cyanobacteria

Summary Mutant strains of the N₂-fixing cyanobacterium bacterium Anabaena variabilis resistant to 6-fluorotryptophan or to ethionine were isolated. Many of these strains liberated amino acids into their media in the absence of 6-fluorotryptophan and ethionine. Nitrogenase activity was higher in mutant strains than in the parent strain. Mutant strains were immobilised in calcium alginate and sustained photoproduction of amino acids has been demonstrated.Abbreviations ETH ethionine - FT 6-fluorotryptophan - Hepes 4-(2-hydroxyethyl)-1, piperazine ethanesulphonic acid - PEP phosphoenolpyruvate - DAHP 3-deoxy-d-arabinoheptulosonate 7-phosphate - chl a chlorophyll a     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene.

The complete 648 amino acid sequence of the human raf oncogene was deduced from the 2977 nucleotide sequence of a fetal liver cDNA. The cDNA has been used to obtain clones which extend the human c-raf-1 locus by an additional 18.9 kb at the 5' end and contain all the remaining coding exons.     

Cohort study of multiple brain lesions in sport divers: role of a patent foramen ovale.

OBJECTIVE: To investigate the role of a patient foramen ovale in the pathogenesis of multiple brain lesions acquired by sport divers in the absence of reported decompression symptoms. DESIGN: Prospective double blind cohort study. SETTING: Diving clubs around Heidelberg and departments of neuroradiology and neurology. SUBJECTS: 87 sport divers with a minimum of 160 scuba dives (dives with self contained underwater breathing apparatus). MAIN OUTCOME MEASURES: Presence of multiple brain lesions visualised by cranial magnetic resonance imaging and presence and size of patent foramen ovale as documented by echocontrast transcranial Doppler ultrasonography. RESULTS: 25 subjects were found to have a right-to-left shunt, 13 with a patent foramen ovale of high haemodynamic relevance. A total of 41 brain lesions were detected in 11 divers. There were seven brain lesions in seven divers without a right-to-left shunt and 34 lesions in four divers with a right-to-left shunt. Multiple brain lesions occurred exclusively in three divers with a large patent foramen ovale (P = 0.004). CONCLUSIONS: Multiple brain lesions in sport divers were associated with presence of a large patent foramen ovale. This association suggests paradoxical gas embolism as the pathological mechanism. A patent foramen ovale of high haemodynamic relevance seems to be an important risk factor for developing multiple brain lesions in sport divers.     

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ.

The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Expression of glutamine synthetase in mutant strains of the cyanobacterium Anabaena variabilis which liberate ammonia

Abstract The nucleoside analogue ganciclovir has clinical efficacy in the treatment of serious infections with human cytomegalovirus (CMV) in AIDS patients. The mechanism of action of the drug against CMV is different from that described for herpes simplex viruses (HSVs) as the crucial formation of the monophosphate derivative appears to be carried out by cellular rather than virus-coded enzymes. Adenovirus infections also induce the expression of cellular genes including kinase activity and a novel DNA polymerase and the results reported here show that these viruses are sensitive to ganciclovir. The 50% effective dose (ED₅₀) range for known serotypes and one clinical isolate was 4.5−33 μM. By comparison with the sensitivity of CMV in vitro and the known clinical response of infections with this virus to ganciclovir, our results suggest that this drug or its analogous may form the basis of chemotherapy for adenovirus infections.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-alpha signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38(mapk) and the p46(jnk) and p54(jnk) c-jun NH(2)-terminal kinase isoforms, but not p42(mapk/erk2) or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42(mapk/erk2) and Akt by TNF-alpha was also inhibited in the presence of NaCl. Because the activation of p42(mapk/erk2) and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-alpha in the presence of NaCl compared with stimulation with TNF-alpha alone or NaCl alone. Furthermore, pharmacological inhibition of p42(mapk/erk2) and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.