Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.     

A novel technique to overcome browning in tissue culture

Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed.     

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.     

Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.     

Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes

Mitochondrial DNA (mtDNA) from two breeds of cattle, viz., [Hariana (Bos indicus), Holstein (Bos taurus)] and Indian water buffalo (Bubalis bubalus), was analyzed using 13 restriction endonucleases which recognized an average of about 40 six-base sites. Polymorphism among cattle was detected with six of these enzymes. The two Holstein differed at six sites, whereas the Hariana breed (Bos indicus) did not show any site polymorphism. Surprisingly, the Hariana type differed by only one site from one of the Holstein types. The total size of buffalo mtDNA was estimated to be 16.4 kb. Polymorphism within the Murrah buffalo breed was observed with respect to aBglI site. Scarcely any of the restriction fragments of buffalo mtDNA matched those of cattle mtDNA.     

Gynecomastia in a mortician. A case report

The fine needle aspiration (FNA) observations in a case of gynecomastia occurring in a mortician are described. The FNA smear showed cytomorphologic features typical of a gynecomastia. Morticians use an embalming cream that contains estrogens or estrogenlike compounds; these substances may be absorbed percutaneously and cause the development of gynecomastia. The mortician should be made aware of the potential risk of such an occupational exposure and development of the "embalmer's curse. "Recognition by the clinicians and cytopathologists of such an occurrence and its implications can help with the proper management.     

Diffusion of phosphate to plant roots in soil

Summary A method is described for computing the theoretical distribution of solutes around a root with root hairs. The P concentration profile around the primary root of a rape seedling with root hairs, growing in a low-P soil, showed considerably greater depletions than predicted by this method, using the relationship between P in soil and in solution obtained from a desorption isotherm experiment. This suggests an enhanced release of soil P into solution in the rhizosphere.Soil Science Laboratory, Department of Agricultural Science, University of Oxford     

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.     

Flagellated coryneform bacteria from the intestine of earthworms

Summary Representative strains of distinctly flagellated, but doubtfully motile, coryneform bacteria were isolated from the intestine of Indian earthworms. Their morphological, cultural, physiological and nutritional characters are described, and their taxonomic position discussed.     

Adult human brain expresses four different molecular forms of neural cell adhesion molecules

Using antibodies to rat neural cell adhesion molecules (NCAM), we analyzed the NCAM of adult human brain. Various regions of the brain were analyzed quantitatively by Western blot. Grey matter showed four bands of NCAM with apparent molecular weights of 180,000, 170,000, 140,000 and 120,000. White matter showed one major band with an apparent M_r of 120,000 and a minor band of 180,000. Cerebellar grey matter contained mainly 170,000, 140,000 and 120,000, white cerebellar white matter had only 180,000 and 120,000 M₁ NCAMS. Spinal cord showed mainly 120,000 M_r NCAM. Deglycosylation using N-glycanase resulted in 170,000, 160,000, 130,000 and 110,000 M_r proteins, suggesting that the four forms of human NCAM are derived from individual polypeptides. The presence of 170,000 M₁ NCAM is unique to human brain.