Two independent, light-sensing two-component systems in a filamentous cyanobacterium.

Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3' end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P(r) and P(fr) forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (lambda(max) = 685 and 735 nm for P(r) and P(fr), respectively) is indicative for a noncovalent incorporation of the chromophore (lambda(max) of P(r), P(fr) of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246-->Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P(fr) formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P(fr) formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Effects of pyridoxine deficiency and dl-p-chlorophenylalanine administration to rats on 5-hydroxytryptamine and noradrenaline concentrations in brain and 5-hydroxytryptamine concentration in blood

Pyridoxine deficiency in post-weanling rats caused a marked decrease in body weight and a small but significant decrease in brain weight. Although the concentration of circulating 5-hydroxytryptamine was markedly decreased, the concentrations of 5-hydroxytryptamine and noradrenaline in the brain were not affected. p-Chlorophenylalanine, an inhibitor of 5-hydroxytryptamine synthesis, decreased the 5-hydroxytryptamine content of brain to very low values in both the deficient and control animals, whereas the noradrenaline contents were not appreciably affected. The concentration of 5-hydroxytryptamine in blood, the origin of which is primarily gastrointestinal, was decreased only in the controls but not in the deficient animals after p-chlorophenylalanine treatment. These results suggest that whereas l-tryptophan hydroxylase (EC 1.14.3.2) is rate-limiting in the brain as has been reported by others, the pyridoxal 5'-phosphate-dependent enzyme 5-hydroxytryptophan decarboxylase (EC 4.1.1.28) may be more important in the gastrointestinal tract in the regulation of 5-hydroxytryptamine synthesis.     

Regional distribution of pyridoxal-5′-phosphate in developing and mature brains and its depletion in pyridoxine deficiency

A study was made of (a) the distribution of the coenzyme pyridoxal-5-phosphate (PLP) in four discrete regions of developing and mature rat brains and (b) the effect of dietary pyridoxine deficiency on the distribution. There was an increase in PLP concentration of all the regions from infancy to adulthood. Highest concentration of PLP was found in the medulla and pons in both infants and adults. Pyridoxine deficiency resulted in a more marked reduction of PLP in all regions of the neonatal brain as compared with those in the mature brain. This is consistent with the vulnerability of the developing brain to nutritional stresses.     

A New Appraisal of the Prokaryotic Origin of Eukaryotic Phytochromes

The evolutionary origin of the phytochromes of eukaryotes is controversial. Three cyanobacterial proteins have been described as ``phytochrome-like' and have been suggested to be potential ancestors of these essential photoreceptors: Cph1 from Synechocystis PCC 6803, showing homology to phytochromes along its entire length and known to attach a chromophore; and PlpA from Synechocystis PCC 6803 and RcaE from Fremyella diplosiphon, both showing homology to phytochromes most strongly only in the C-terminal region and not known to bind a chromophore. We have reexamined the evolution of the photoreceptors using for PCR amplification a highly conserved region encoding the chromophore-binding domain in both Cph1 and phytochromes of plants and have identified genes for phytochrome-like proteins (PLP) in 11 very diverse cyanobacteria. The predicted gene products contain either a Cys, Arg, Ile, or Leu residue at the putative chromophore binding site. In 10 of the strains examined only a single gene was found, but in Calothrix PCC 7601 two genes (cphA and cphB) were identified. Phylogenetic analysis revealed that genes encoding PLP are homologues that share a common ancestor with the phytochromes of eukaryotes and diverged before the latter. In contrast, the putative sensory/regulatory proteins, including PlpA and RcaE, that lack a part of the chromophore lyase domain essential for chromophore attachment on the apophytochrome, are only distantly related to phytochromes. The Ppr protein of the anoxygenic photosynthetic bacterium Rhodospirillum centenum and the bacterial phytochrome-like proteins (BphP) of Deinococcus radiodurans and Pseudomonas aeruginosa fall within the cluster of cyanobacterial phytochromes. Received: 9 December 1999 / Accepted: 10 May 2000     

Multiple organ dysfunction syndrome.

The multiple organ dysfunction syndrome (MODS), though newly described, has manifested itself in intensive care unit (ICU) patients for several decades. As the name implies, it is a syndrome in which more than one organ system fails. Failure of these multiple organ systems may or may not be related to the initial injury or disease process for which the patient was admitted to the ICU. MODS is the leading cause of morbidity and mortality in current ICU practice. While the pathophysiology of MODS is not completely known, much evidence indicates that, during the initial injury which precipitates ICU admission, a chain of events is initiated which results in activation of several endogenous metabolic pathways. These pathways release compounds which, in and of themselves, are usually cytoprotective. However, an over exuberant activation of these endogenous systems results in an inflammatory response which can lead to development of failure in distant organs. As these organs fail, they activate and propagate the systemic inflammatory response. No therapy has proven entirely efficacious at modulating this inflammatory response and the incidence and severity of MODS. In current ICU practice, treatment is focused on prevention and treating individual organ dysfunction as it develops. With increased understanding of the pathophysiology of MODS therapy will come newer modalities which inhibit or interfere with the propagation of the endogenous systemic inflammatory response. These newer therapies hold great promise and already some are undergoing clinical investigation.