N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-l-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.Abbreviations BMT S-adenosyl-l-methionine: bergaptol O-methyltransferase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Respiratory load compensation in neuromuscular disorders

First-breath ventilatory responses to graded elastic (delta E) and resistive (delta R) loads from 10 people with spinal muscular atrophy (SMA), 15 people with Duchenne muscular dystrophy (DMD), and 80 able-bodied people were compared. The SMA and DMD groups produced equal tidal volume, respiratory frequency, inspiratory duration (TI), expiratory duration, mean inspiratory airflow, and duty cycle responses to both delta E and delta R. Thus SMA (primarily a motoneuron disorder) and DMD (primarily a muscle disorder) have the same net effect on loaded breathing responses. The SMA and DMD groups failed to duplicate the normal group's short expirations during delta E, long inspirations during delta R, and thus, extended duty cycles during both delta E and delta R. The deficit in load compensation therefore was due to impaired regulation of respiratory timing (reflecting neural mechanisms) but not airflow defense (reflecting mechanical and neural mechanisms). One-fifth of the normal but none of the SMA or DMD subjects actively generated an "optimal" TI response (defined theoretically as TI greater than 160% control during large delta R and TI less than 75% control during large delta E). This lack of optimal responses, which is the same abnormality exhibited by quadriplegic people, suggests that SMA and DMD also impair human ability to discriminate between large delta R and delta E. These findings support the hypothesis that neuromuscular disorders can lead to disturbances in respiratory perception.     

Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing piIC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili.