Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.     

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.     

ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma-35S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines.     

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.     

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new species of Crepidostomum (Allocreadiidae: Digenea) from northeastern Finland,with comments on its possible origin

Crepidostomum wikgreni n. sp. is described from the gall-bladder and intestine of the whitefish Coregonus acronius in Lake Yli-Kitka in NE Finland. It is morphologically similar to Crepidostomum farionis, with which it occurs sympatrically and sometimes concurrently; but it differs in that the eggs are much larger, i.e. 96±6.5 m mean-length, as opposed to 71±4.7 m mean-length for C. farionis in the same host and locality.It is suggested that the new species has arisen from C. farionis after deglaciation and since c. 8,400 BP, at which time the waters of the Lake Kitka System were isolated from those in the rest of Finland and flowed eastwards into the White Sea Basin. The isolation of the White Sea Basin appears to have been maintained by watersheds running north-south close to the Fenno-Soviet border and east-west through central Karelia. It is further suggested that C. farionis currently occurring in Lake Yli-Kitka is a recent re-introduction brought about by the translocation of fish-stocks.     

Alterations in Retinal Tyrosine and Dopamine Levels in Rats Consuming Protein or Tyrosine-Supplemented Diets

The ad libitum ingestion of casein diets varying in protein content altered serum and retinal levels of tyrosine. The serum tyrosine level rose when protein ingestion was increased from 6 to 24% casein. In rats consuming high-protein diets (40% casein), no further increase in serum tyrosine level occurred, although the levels of other large neutral amino acids, which compete with tyrosine for retinal uptake, continued to rise. The activity of the liver enzyme tyrosine aminotransferase varied directly with the percentage of protein in the diet and may partially explain the failure of chronic high-protein feeding to increase serum tyrosine levels. The retinal tyrosine concentration was significantly correlated with the serum tyrosine level and with the serum tyrosine ratio at all levels of protein intake. Retinal 3,4-dihydroxyphenylalanine synthesis and dopamine (DA) level varied in parallel with the level of the precursor, tyrosine. Addition of pure L-tyrosine (1, 2, or 4%) to normal protein diets resulted in a stepwise increase in serum and retinal tyrosine levels and retinal DA turnover. Alterations of retinal tyrosine level as a result of change in amount of dietary protein or by its addition to the normal diet can influence retinal DA synthesis and release.     

Biotinylated human beta-endorphins as probes for the opioid receptor

The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human beta-endorphin to cross-link the mu and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.