Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found in WS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.     

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Inhibition of progesterone secretion and decrease of ovarian blood flow by isoxazole in pregnant rats

Pregnant rats were treated on day 11 of pregnancy with 0.62, 1.25, 2.5 or 5.0 mg of isoxazole per 100 g of body weight (bw) (Experiment I) or with 2.5 mg of isoxazole per 100 g of bw (Experiment II). In Experiment I animals were anaesthetized with pentobarbital at 48 hr. In experiment II animals were similarly anaesthetized at 0, 12, 24, or 48 hr following administration of isoxazole. Blood was collected separately from the ovary or from the uterus via a thin cannula which was inserted into the utero-ovarian vein. Blood was also collected from the vena cava inferior. The concentrations of progesterone (P), 17 beta-oestradiol (E2), and corticosterone were measured in blood samples by RIA. All of the animals exhibited reduced rates of ovarian venous blood flow and rates of P secretion when pregnancy was terminated by isoxazole (2.5 mg/100 g bw or greater). The concentration of P was found to be correspondingly reduced by 90% in ovarian venous blood and by 50% in peripheral blood, relative to the controls. After isoxazole had been administered, the rate of P secretion and also the concentration of P in peripheral blood continued to decrease rapidly and to remain at low levels for at least 48 hr. By contrast, the concentration of corticosterone in peripheral blood decreased during the first 12 hr but then it returned to the control levels by 48 hr. The ovarian secretion rate of E2 and the concentration of E2 in peripheral blood remained unchanged during the same 48 hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes

The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

A potential animal model for studying CF heterozygote advantage: genetic variation in theophylline-inducible colonic chloride currents among inbred strains of mice.

We have used Ussing chambers to measure chloride secretion by colonic segments (mucosa, muscularis, and serosa) from various inbred strains of mice. We found lower theophylline-induced Cl- secretion in the DBA/2J than in the C57BL/6J strain. Their F1 showed significantly higher levels of Cl- secretion than did the C57BL/6J parental strain while colonic segments from five recombinant inbred B x D lines ranged between the C57BL/6J and F1 values. No major component of the variation appeared to be associated with alleles of the met oncogene region of chromosome 6 or the H-2 region of chromosome 17.