Plasmid Involvement in Parathion Hydrolysis by Pseudomonas diminuta

An organism identified as Pseudomonas diminuta was found to hydrolyze parathion. Cells grown for 48 h contained 3,400 U of parathion hydrolase activity per liter of broth. Expression of enzymatic activity was lost at a high frequency (9 to 12%) after treatment with mitomycin C. Hydrolase-negative derivatives were missing a plasmid present in the wild-type organism. The molecular mass of this plasmid (pCS1), as determined by electron microscopy, was about 44 × 10⁶ daltons.     

A collagen-like glycoprotein from elastin-rich tissues

Extracts of bovine aorta and nuchal ligament contain several large glycoproteins. The major glycoprotein species has been isolated and has been shown to be collagenase sensitive with an apparent molecular weight of 140,000 daltons. The protein exists in disulphide-bonded aggregates, contains hydroxyproline and hydroxylysine in 1:1 ratio and is unlike any of the known collagen types in amino acid analysis. Its presencein ligament extracts indicates that it is not derived from basement membranes. The evidence suggests that this protein is not derived from the microfibrillar components of the elastic tissues.     

Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides

In Rhodobacter sphaeroides, many of the structural genes encoding enzymes of the Calvin cycle are duplicated and grouped within two separate clusters. In this study, the nucleotide sequence of a 5627-base pair region of DNA that contains the form I Calvin cycle gene cluster has been determined. The five open reading frames are arranged in the order, fbpA prkA cfxA rbcL rbcS and are tightly linked and oriented in the same direction. The results of insertional mutagenesis studies suggest the genes are organized within an operon. Consistent with this proposal, the cfxA gene has been tentatively identified as a gene encoding the Calvin cycle enzyme, aldolase. Measurement of the activities of various Calvin cycle enzymes in the insertion mutants showed that inactivation of genes within one CO2 fixation cluster affected expression of genes within the second cluster, revealing a complex regulatory network.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.

YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.     

Mate choice in lekking sage grouse revisited: the roles of vocal display, female site fidelity, and copying

In lekking sage grouse (Centrocercus urophasianus), femalesexhibit relatively unanimous mate choice for particular males,but a satisfactory explanation for this unanimity has been elusive.We present analyses of mating distributions from two leks over4 years that provide evidence for female choice based on differencesin vocal display performance of males, the locations at whichhens mated in the previous year, and the choices of other females(copying). The unanimity of female choice varied markedly amongleks and years in correlation with changes in the mean numbersof hens that mated at the same time and hence the opportunityto copy. The results confirm that hens assess phenotypic traitsof males directly but also indicate that the secondary tacticsof site fidelity and copying are often important componentsof female choice. The occurrence of these secondary tacticshas three implications: the variance in mating success amonglek males will be a poor predictor of the intensity of sexualselection on specific traits; female preferences may generatemore clustered dispersions of displaying males than predictedby hotspot settlement models; and direct assessment of malesby females may be difficult or costly, a conclusion that supportsadaptive models of sexual selection over a nonadaptive Fisherianprocess. [Behav Ecol 1991;2:165–180]     

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.     

Partial characterization of the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causative agent of chancroid, a genital ulcer disease.

The first preliminary structure of a surface lipooligosaccharide from Haemophilus ducreyi has been determined. The major oligosaccharide was released by mild acid hydrolysis and analyzed by liquid secondary ion and tandem mass spectrometry. The mass spectral data combined with composition and methylation analysis yielded the most probable structure; Gal1----4GlcNAc1----3Gal1----4Hep1----6Glc1----( Hep1----2Hep1----)3,4Hep1---- KDO, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or KDO) exists in an anhydro form. This anhydro species results from the elimination of a phosphate from C-4 of KDO during mild acid hydrolysis. The core heptose trisaccharide consists of L-glycero-D-manno-heptose, but analysis of the peracetylated sugars indicated that the 1,4-linked heptose is likely D-glycero-D-manno-heptose. The monoclonal antibody 3F11 generated against Neisseria gonorrhoeae also binds to this lipooligosaccharide and suggests that the terminal trisaccharide is Gal beta 1----4GlcNAc beta 1----3Gal beta 1----, an epitope found in the glycose moiety of the human erythrocyte glycosphingolipid lactoneotetraglycosylceramide. Mass spectrometric and composition analysis of the lipid A moiety shows that it is similar to the lipid A of Haemophilus influenzae strain I-69 Rd-/b+ proposed by Helander et al. (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Z?hringer, U. (1988) Eur. J. Biochem. 177, 483-492). Electrospray mass spectrometric analysis of the intact O-deacylated lipooligosaccharides gave an average Mr of 2710, and supported an overall structure consisting of the above nonasaccharide linked directly to a diphosphorylated lipid A moiety through the single KDO which is phosphorylated. This structure should provide a framework to investigate the roles of lipooligosaccharides in the host immunochemical response and pathology of H. ducreyi infection, a leading cause of genital ulcer disease.