Possible interference of lactose-fermenting marine vibrios in coliform -D-galactosidase assays

C.M. DAVIES, S.C. APTE AND S.M. PETERSON. 1995. An investigation into possible interferences in β-D-galactosidase-based assays for coliform bacteria in marine waters was carried out. A rapid instrumental fluorescence assay for β-D-galactosidase activity, using 4-methylumbelliferyl-β-D-galactosidase as a substrate, was used to investigate activities of this enzyme in non-coliform bacterial isolates from coastal waters. Only 2% of isolates showed slight enzyme activity after a 1-h incubation period at 44.5βC. At a lower incubation temperature of 20βC, 51% and 94% of the isolates showed some enzyme activity within 6 h and 48 h, respectively. Fifty-nine out of 67 of these isolates were identified as Vibrio species. A lac⁺ strain of Vibrio vulnificus was found to produce β-D-galactosidase which caused significant false-positive reactions in the Colilert-Marine Water assay when present at concentrations of 10 cfu ml⁻¹ or greater. This interference could be overcome by addition of the vibriostatic agent O/129. The high fluorescence of this reagent, however, precluded the simultaneous determination of Escherichia coli in the Colilert test and also its use in instrumental fluorescence assays. It was concluded that in assays employing high temperatures and short incubation times, Vibrio species are unlikely to cause significant interferences.     

Extracellular enzyme activities associated with epiphytic microbiota on submerged stems of the reed Phragmites australis

Abstract Fluorogenic 4-methylumbelliferyl (MUF) compounds were used as analogue substrates for assay of extracellular enzyme activities associated with epiphytic microbiota at submerged Phragmites australis stem surfaces. Incubations at a range of MUF substrate concentrations indicated that saturation of enzyme activity was achieved at a MUF substrate concentration of about 200 μmol 1⁻¹. Later determinations at a single, saturation, concentration of MUF substrate were, therefore, carried out at about 200 μmol l⁻¹. Such determinations were undertaken using P. australis stems from eight gravel-pit ponds. The rate of enzymatic hydrolysis of MUF phosphate (analogue substrate for phosphatase activity) was > MUF β- d -glucopyranoside (β- d -glucosidase) > MUF β- d -galactopyranoside (β- d -galactosidase) > MUF sulphate (sulphatase) and MUF palmitate (lipase) on stems from all eight ponds. Thus the relative magnitude of the various components of total epiphyton extracellular enzyme activity might be a conservative feature.     

Characterization of β-mannanase and β-mannosidase secreted from the yeast Trichosporon cutaneum JCM 2947

Y. ODA AND K. TONOMURA. 1996. β-Mannanase and β-mannosidase were purified from the culture fluid of the yeast Trichosporon cutaneum JCM 2947 (= T. beigelii CBS 5790). The molecular weights of the two enzymes were estimated to be 49 900 and 114000 by SDS-PAGE and 4500 and 193 000 by gel filtration, respectively. β-Mannanase contained 43% molecular weight as carbohydrate. The K _m and V _max values of β-mannanase for konjac glucomannan were 2.7 (mg ml^-1) and 10.6 (U mg protein^-1), and those of β-mannosidase for p -nitrophenyl β-D-mannopyranoside were 0.25 (mmol l^-1) and 91.7 (U mg protein^-1). Maximal activities were observed between pH 4.0 and 6.5 at 50°C for β-mannanase and around 6.5 at 40°C for β-mannosidase.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

Evidence for a third allele at the β-lactoglobulin (β-Lg) locus of sheep milk and its occurrence in different breeds

Summary. Milk samples from 189 Merinoland Sheep, 145 Black Faced Mutton Sheep, 89 East Friesian Milk Sheep, 36 Rhön, 36 Pleven, 23 Tsigaja, 25 Black Razka and 86 Hungarian Merino x Pleven (F1) sheep were analysed by polyacrylamide gel electrophoresis under acid conditions and isoelectric focusing in ultrathin layer polyacrylamide gels with carrier ampholytes. Six different β-lactoglobulin (β-Lg) phenotypes (A, AB, B, AC, BC and C) were observed by both methods. The occurrence of three codominant alleles (β-Lg^A, β-Lg^B, β-Lg^c) at an autosomal locus (β-Lg) was supported by family and population data on genetic equilibrium. Differences in gene frequencies between the breeds were observed.     

Comparative study on cell envelope-associated proteinases in natural isolates of mesophilic lactobacilli

Lactobacilli isolated from different natural sources were screened for the presence of cell envelope-associated proteinases (Prt⁺ strains). Among them 17 of 75 tested isolates were Prt⁺. All Prt⁺ strains were producers of a serine-type proteinase, since their proteolytic activity was inhibited by phenylmethylsulfonyl fluoride. Most of the natural isolates of mesophilic lactobacilli degraded only β-casein such as Lactobacillus paracasei subsp. paracasei strains BGLI17 and BGLI18 and Lact. rhamnosus BGEN1. Only Lact. divergens BG742 cleaved all three, α-, β- and κ-caseins, even in the presence of Ca²⁺ ions. Total DNA isolated from Lact. paracasei subsp. paracasei strains BGLI17 and BGLI18 hybridized to the lactococcal proteinase gene probes originated from Lactococcus lactis subsp. cremoris Wg2. Hybridization could not be linked to the plasmid DNA, and pulse-field gel electrophoresis analysis suggested that the proteinase genes of these two strains are most probably chromosomally located.     

Sensitivity of terpene emissions to drought and fertilization in terpene-storing Pinus halepensis and non-storing Quercus ilex

We studied the effects of water stress, fertilization and time course on foliar volatile terpene emission rates by Quercus ilex and Pinus halepensis in a garden experiment. The terpenes mostly emitted by both species were α -pinene, β -pinene, β -myrcene and Δ³-carene. P. halepensis emission rates (average 31.45 μg g⁻¹ DM h⁻¹) were similar to those of Q. ilex (average 31.71 μg g⁻¹ DM h⁻¹). The effects of drought (reduction to one-third of full watering) and fertilization (250 kg N ha⁻¹, 250 kg P ha⁻¹, or both) were different depending on the species: the drought treatment significantly increased the terpene emissions from Q. ilex by 33%, and the fertilization treatments reduced the terpene emissions from P. halepensis by 38%. Terpene emission rates increased with time course in parallel to raising summer temperatures in P. halepensis and Q. ilex , whose emission rates were temperature related (r = 0.42 and r = 0.68, respectively) and light related (r = 0.32 and r = 0.57, respectively). There was a positive relationship for P. halepensis , and a negative relationship for Q. ilex, between emission rates and relative water contents. No relationship was found between emission rates and N or P foliar concentrations. The results of this study show complex species-specific responses with stronger and faster short-term responses in terpene-non-storing than in storing species and indicate that terpene emissions may significantly change in the warmer, drier and more fertilized conditions predicted for the next decades in the Mediterranean region.     

Hormonal regulation of the European sea bass reproductive cycle: an individualized female approach

Fifteen tagged female sea bass Dicentrarchus labrax were sampled weekly from September to April and plasma vitellogenin (VTG), testosterone (T), 17β-estradiol (E₂), and two potential maturation inducing steroids (MISs): 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) assayed. An oocyte sample was obtained via intraovarian cannulation at each sampling time from every female and the stage of development of the most advanced clutch of oocytes determined and related to VTG and hormone plasma levels for each female. The mean number of ovulations per female was 1·75+0·25 when those females that did not present ovulations were excluded and up to 4 ovulations detected in some females. The highest plasma levels of T ( c. 6 ng ml^-1) were observed during postvitellogenesis and the beginning of maturation while maximum plasma levels of E2 (>5 ng ml^-1) were obtained during late vitellogenesis. VTG plasma levels increased throughout vitellogenesis peaking ( c. 2·5 mg ml^-1) at postvittelogenesis. For the first time significant changes of plasma progestogens were detected in European sea bass during the sexual cycle. The highest plasma level of 17,20βP ( c. 1·1 ng ml^-1) was observed during postvitellogenesis while the highest level of 20βS ( c. 1·4 ng ml^-1) coincided with final maturation. These results suggest that 17,20βP and 20βS play a role in the early and final maturation, respectively, in the European sea bass.     

The relationship between physical and chemical conditions and low microbial diversity in the Blue Lagoon geothermal lake in Iceland

Abstract: The Blue Lagoon in Iceland is a shallow geothermal lake with average temperatures of 37°C, pH 7.5 and about 2.5% salinity. It was formed in 1976 from the effluents of the Svartsengi geothermal power plant and is saturated with silica which constantly precipitates in the lake. It has been colonized by a few types of specialized microorganisms which seem to proliferate in this unusual ecosystem. The average bacterial colony count in the lake was 1.3 × 10⁵ ml⁻¹ on plate count agar made with 50% Blue Lagoon fluid but 2.6 × 10⁶ ml⁻¹ when determined with the MPN method. A total of 99 isolates were purified and characterized by 54 phenotypic tests and then grouped using Numerical Taxonomy. At similarity values of 80%, one major cluster was formed containing 85% of the isolates. Four representative strains from this cluster were further characterized and all shown to be Gram-negative, obligately aerobic, non-motile rods. They were oxidase positive, catalase negative and grew optimally at 45°C and in 3.5% NaCl with doubling time of about 80 min.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin

Abstract 80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with ΔT_m-values ranging from 0.0–2.3°C. In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for β-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a ΔT_m-value of 5.7°C and produced γ-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate^TM, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.     

Differential gene expression in infarct scar and viable myocardium from rat heart following coronary ligation

Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na⁺-K⁺ATPase α-1 and −2, phospholamban (PLB), α-myosin heavy chain (α-MHC), ryanodine receptor (Rya) and Ca²⁺ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Giα2 and β-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na⁺-K⁺ ATPase-β, Na⁺-Ca²⁺ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the a-1 subunit of Na⁺-K⁺ATPase, α-3, β-isoforms, Rya, β-MHC, Giα2, Gs and Na⁺-Ca²⁺ exchanger were significantly elevated while expression of the a-2 subunit of Na⁺-K⁺ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2α mRNA was elevated in noninfarcted heart (145 ± 15%) and diminished in the infarct scar (66 ± 13%) vs controls. Expression of β-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 ± 31% and 183 ± 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Relationship of plasma steroids to germ cell development and the presence of protamine mRNA in rainbow trout during the induction of spermatogenesis with partially purified salmon gonadotropin

The gonadosomatic index (GSI) of pre–pubertal male rainbow trout, which had been injected biweekly with partially purified salmon gonadotropin (sG–G100, 50 μ mUg kg⁻¹ body weight), increased from 0.05 to 1.85 over 21 weeks from injection, while control GSI remained below 0.05. Plasma testosterone (T) increased from 2 to 11.34 ng ml⁻¹ by week 21 in injected fish, while control level remained below 1.5 ng ml⁻¹. In injected fish plasma 11–ketotestosterone (KT) and 17,20–dihydroxyprogesterone (17α20βP) levels increased from 20.2 to 41.9 and 8.9 to 219.7 ng ml⁻¹ respectively. Plasma T, 11–KT, and 17α20βP were all correlated with the GSI ( P <0–001) in injected fish. The most advanced stage of germ cells present in the control fish were spermatogonia. However, in injected fish spermatozoa were present by week 21. Eggs fertilized at this time with spermatozoa from injected fish achieved a 78% fertilization rate, whereas the testicular homogenate was incapable of fertilizing eggs.     

Amyloid β Protein (25–35) Stimulation of Phospholipase C in LA-N-2 Cells

Abstract: The amyloid β protein (25–35) stimulated appearance of ³H-inositol phosphates from [³H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid β protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid β protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC₅₀ values were 12 µ M for propranolol, 15 µ M for AP-3, and 25 n M for [Tyr⁴, d -Phe¹²]bombesin. Additional support comes from results of densensitization and resensitization experiments. Amyloid β protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans -1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid β peptide. In a similar manner, LA-N-2 cells previously treated with amyloid β protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid β protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid β protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

Solubilization of galactosyltransferase that synthesizes 1,4-beta-galactan side chains in pectic rhamnogalacturonan I

β -1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 m M 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred ¹⁴[C]galactose from UDP-¹⁴[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for ¹⁴[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The ¹⁴[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4- β - Endog alactanase could not digest the former product, whereas the latter product was digested to ¹⁴[C]galactobiose and ¹⁴[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4- β -linked.