Central and peripheral changes in catecholamine biosynthesis and turnover in rats after a short period of ozone exposure

We investigated in rat the effects of ozone exposure (0.7 ppm) for 5 h on the catecholamine biosynthesis and turnover in sympathetic efferents and various brain areas. For this purpose, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was assessed in superior cervical ganglia and in two major noradrenergic cell groups, A2 and A6 (locus coeruleus). Tyrosine hydroxylase activity was estimated in vivo by measuring the accumulation of l-dihydroxyphenylalanine after pharmacological blockade of L-aromatic acid decarboxylases by NSD-1015 (100 mg/kg i.p.). The catecholamine turnover rate was measured after inhibition of tyrosine hydroxylase by alpha-methyl-para-tyrosine (AMPT, 250 mg/kg, i.p., 2.5 h) in peripheral sympathetic target organ (heart and lungs) as well as in some brain catecholamine terminal areas (cerebral cortex, hypothalamus and striatum). Ozone caused differential effects according to the structure. Catecholamine biosynthesis was stimulated in superior cervical ganglia (+44%, P < 0.05) and caudal A2 subset (+126%, P < 0.01), whereas catecholamine turnover was increased in heart (+183%, P < 0.01) and cortex (+22%, P < 0.05). On the other hand, catecholamine turnover was inhibited in lungs (-53%, P < 0.05) and striatum (-24%, P < 0.05). A brief exposure to ozone, at a concentration chosen to mimic pollution level encountered in urban areas, can modulate catecholamine biosynthesis and utilization rate in the sympathetic and central neurones.     

Control of centromere localization of the MEI-S332 cohesion protection protein

In mitosis and meiosis, cohesion is maintained at the centromere until sister-chromatid separation. Drosophila MEI-S332 is essential for centromeric cohesion in meiosis and contributes to, though is not absolutely required for, cohesion in mitosis. It localizes specifically to centromeres in prometaphase and delocalizes at the metaphase-anaphase transition. In mei-S332 mutants, centromeric sister-chromatid cohesion is lost at anaphase I, giving meiosis II missegregation. MEI-S332 is the founding member of a family of proteins important for chromosome segregation. One likely activity of these proteins is to protect the cohesin subunit Rec8 from cleavage at the metaphase I-anaphase I transition. Although the family members do not show high sequence identity, there are two short stretches of homology, and mutations in conserved residues affect protein function. Here we analyze the cis- and trans-acting factors required for MEI-S332 localization. We find a striking correlation between domains necessary for MEI-S332 centromere localization and conserved regions within the protein family. Drosophila MEI-S332 expressed in human cells localizes to mitotic centromeres, further highlighting this functional conservation. MEI-S332 can localize independently of cohesin, assembling even onto unreplicated chromatids. However, the separase pathway that regulates cohesin dissociation is needed for MEI-S332 delocalization at anaphase.     

Thirty years after fibronectin discovery: role in malignant transformation and in ageing

1973 was the year of a major discovery: two proteins were shown to disappear from cell surface when cells were transformed by tumor viruses. Later on a large number of glycoproteins were recognized as identical and coined as fibronectin. In spite of 30 years of studies, interest in fibronectin remains vivid. Thanks to its study, understanding of extracellular matrix organisation, cell-matrix interactions and signalling through integrins, formation of fibronectin based fibrillar matrix made great progress. Furthermore importance of matricryptic sites emerged as well as matricryptins. Such peptides show properties that original proteins do not possess. Some of these peptides are used as therapeutic agents. On the contrary others appear to be involved in vicious circles underlying age-dependent decline of tissue function. This fascinating molecule and the parent molecules will certainly remain of interest for some time.     

Absence of accessory subunit in the DNA polymerase gamma purified from yeast mitochondria

In yeast and animals, replication of the mitochondrial genome is carried out by the DNA polymerase gamma. In mammals this polymerase is composed of a catalytic and an accessory subunit. Yeast DNA polymerase gamma was purified over 6600-fold from mitochondria. The catalytic polypeptide of this enzyme was identified as a 135-kDa protein by a photochemical crosslinking procedure and its native molecular weight was estimated between 120 and 140 kDa by gel filtration and glycerol gradient sedimentation. These results indicate that yeast DNA polymerase gamma contains only one subunit and thus has a different quaternary structure from its counterpart in animals.     

Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.     

Comparative analysis of two slc11 (Nramp) loci in Takifugu rubripes

To study the evolution of the solute carrier family 11 (slc11; formerly Nramp) protein, we isolated and characterized two paralogs from the pufferfish Takifugu rubripes (Fugu). These teleost genes, designated Fugu slc11a-a and Fugu slc11a-b, comprise open reading frames of 1743 nucleotides (581 amino acids) and 1662 nt (554 aa), respectively. The proteins are 81% similar, and both exhibit signature features of the slc11 family of proteins including 12 transmembrane domains, a conserved transport motif and a glycosylated loop. Both Fugu paralogs are more Slc11a2-like based on sequence homology and phylogenetic studies. Analysis of gene environment placed both in the proximity of multiple loci syntenic to human chromosome 12q13, that is, within a SLC11A2 gene environment. However, Fugu slc11a-a also gave one match with chromosome 2q35, where human SLC11A1 resides. Functional diversification was suggested by differences in tissue distribution and subcellular localization. Fugu slc11a-a exhibits a restricted expression profile and a complex subcellular localization, including LAMP1 positive late endosomes/lysosomes in transiently transfected mouse macrophages. Fugu slc11a-b is expressed ubiquitously and localizes solely to late endosomes/lysosomes. This comparative analysis extends our understanding of the evolution and function of this important family of divalent cation transporters. [Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ496547/8/9 and AJ496550.]     

Targets of fibroblast growth factor 1 (FGF-1) and FGF-2 signaling involved in the invasive and tumorigenic behavior of carcinoma cells

Fibroblast growth factor (FGF)-1 and -2 have potent biological activities implicated in malignant tumor development. Their autocrine and nonautocrine activity in tumor progression of carcinoma was investigated in the NBT-II cell system. Cells were manipulated to either produce and be autocrine for FGF-1 or -2 or to only produce but not respond to these factors. The autocrine cells are highly invasive and tumorigenic and the determination of specific targets of FGF/fibroblast growth factor receptor (FGFR) signaling was assessed. In vitro studies showed that nonautocrine cells behave like epithelial parental cells, whereas autocrine cells have a mesenchymal phenotype correlated with the overexpression of urokinase plasminogen activator receptor (uPAR), the internalization of E-cadherin, and the redistribution of beta-catenin from the cell surface to the cytoplasm and nucleus. uPAR was defined as an early target, whereas E-cadherin and the leukocyte common antigen-related protein-tyrosine phosphatase (LAR-PTP) were later targets of FGF signaling, with FGFR1 activation more efficient than FGFR2 at modulating these targets. Behavior of autocrine cells was consistent with a decrease of tumor-suppressive activities of both E-cadherin and LAR-PTP. These molecular analyses show that the potential of these two growth factors in tumor progression is highly dependent on specific FGFR signaling and highlights its importance as a target for antitumor therapy.     

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3′ end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3′ end of 18S rRNA and formation of active 40S ribosomal subunits.     

Structures of Escherichia coli DNA mismatch repair enzyme MutS in complex with different mismatches: a common recognition mode for diverse substrates

We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by ~60° upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.