Polymorphic restriction sites in the horse (β-globin gene cluster

Horse DNA samples digested with PstI and probed with the rabbit beta 1 globin gene show three phenotypes determined by one fragment of variable length (about 5.1 or 3.3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0.66 for the 3.3-kb fragment and 0.34 for the 5.1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7.5 and 3.8 kb) have been observed. Allelic frequencies of the 7.5- and 3.8-kb fragments are 0.24 and 0.76 respectively. The two polymorphic sites are non-randomly associated.     

Direct sequencing and bidirectional allelle specific polymerase chain reaction of the bovine β-casein B variant

Summary. We have used the polymerase chain reaction (PCR) to amplify exon VII of the bovine β-casein gene. The mutations responsible for the B variant were identified by direct sequencing of the amplification products. A bidirectional allele-specific PCR method (BAS-PCR) has been developed using oligonucleotides overlapping the mutation site at their 3' ends. This new procedure allows a rapid and reliable discrimination between the B and non-B alleles of β-casein.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

Rapid β-lactoglobulin genotyping of cattle using the polymerase chain reaction

A polymerase chain reaction (PCR) assay has been developed for genotyping beta-lactoglobulin A and B variants in dairy cattle. Either blood or semen samples can be used as a source of DNA. The method is accurate, faster than Southern blot analyses and should prove a useful tool in breeding programmes.     

Enhancement of γ-Aminobutyric Acid Binding by the Anxiolytic β-Carbolines ZK 93423 and ZK 91296

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.     

Chronic Morphine Administration Increases β-Adrenergic Receptor Kinase (βARK) Levels in the Rat Locus Coeruleus

Abstract: Based on the established role of β-adrenergic receptor kinase (βARK) and β-arrestin in the desensitization of several G protein-coupled receptors, we investigated the effect of chronic morphine administration on βARK and β-arrestin levels in selected brain areas. Levels of βARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of βARK, i.e., βARK1 and βARK2. It was found that chronic morphine treatment produced an ∼35% increase in levels of βARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of βARK2 immunoreactivity in any of the brain regions studied. Levels of β-arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by ∼20%) in the locus coeruleus. It is proposed that chronic morphine regulation of βARK1 and β-arrestin levels may contribute to opioid-receptor tolerance that is known to occur in this brain region.     

Phosphorylation-Dependent Immunoreactivity of Neurofilaments Increases During Axonal Maturation and β,β''-Iminodipropionitrile Intoxication

The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with beta,beta'-iminodipropionitrile (IDPN). A maturational increase in the relative binding of phosphorylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phosphorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal transport of NF proteins.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Poly(β-hydroxyalkanoate) biosynthesis and accumulation by different Rhizobium species

Abstract Synthesis and accumulation of poly(β-hydroxy-alkanoate) in Rhizobium leguminosarum , R. leguminosarum biovar. trifolii, Rhizobium 'galega', Rhizobium 'hedysarum' and R. meliloti were studied.
Poly(β-hydroxybutyrate) is accumulated up to 55% of the cell dry weight. At 30–50% air saturation R. meliloti accumulates 50% of its biomass (5.5 g·1⁻¹ dry weight) as PHB after 90 h of batch fermentation. At 90% air saturation maximum accumulation (37.5%) of PHB occurs after 70 h cultivation.
R. meliloti strain 41 is able to synthesize the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) at concentrations up to 58% of the biomass dry weight, containing up to 22 mol%β-hydroxyvalerate. Different concentrations of both copolymer and hydroxyvalerate were obtained when the microorganism was grown, in batch culture, in the presence of propionate or valerate and with glucose, sucrose or succinate as main carbon source.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

The Dietary Excitotoxins β-N-Methylamino-l-Alanine and β-N-Oxalylamino-l-Alanine Induce Necrotic- and Apoptotic-Like Death of Rat Cerebellar Granule Cells

Abstract: The neurotoxic properties of the dietary excitotoxins β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. β- N -Methylamino- l -alanine and β- N -oxalylamino- l -alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of β- N -methylamino- l -alanine were blocked by d (−)-2-amino-5-phosphonopentanoic acid, and those of β- N -oxalylamino- l -alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K⁺ (30 m M )- to a low K⁺ (10 m M )-containing medium. In cerebellar granule cells exposed to β- N -methylamino- l -alanine or β- N -oxalylamino- l -alanine (3 m M ), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine induce death over 12–72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Pronounced differences in the frequency of TaqI βA-inhibin alleles between sheep breeds with different reproductive performance

A total of 419 individuals of four breeds with differing fecundity (Rhoenschaf, Merinolandschaf, East Friesian Milksheep, Romanov) and several wild sheep of the Ovis musimon, O. orientalis, O. vignei and O. ammon groups were screened for genetic variation at the βA-inhibin (INHBA) locus with up to 11 enzymes. The four breeds differed significantly (P< 0.001) in TaqI allele frequencies. The frequency of the TaqI A allele coincided with the average litter size in each breed.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

A β-lactamase produced by a thermophilic Bacillus

Abstract A β-lactamase was purified from a thermophilic Bacillus strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the β-lactamase produced by Bacillus licheniformis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain.     

β-Glucanase expression by Ruminococcus flavefaciens FD-1

Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and ¹⁴C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β- d -cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and ¹⁴C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Separation of plasmid-mediated extended spectrum β-lactamases by fast protein liquid chromatography (FPLC system)

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.     

Novel carbenicillin-hydrolyzing β-lactamase (CARB-5) from Acinetobacter calcoaceticus var. anitratus

A strain of Acinetobacter calcoaceticus var. anitratus highly resistant to ticarcillin but susceptible to ticarcillin in combination with clavulanic acid (2 mg/l) was found to produce a constitutive beta-lactamase. This enzyme was periplasmic with a characteristic substrate profile of a carbenicillin-hydrolyzing enzyme. Enzyme inhibition was detected with antiserum (anti-CARB-3), pCMB, cloxacillin, clavulanic acid and sulbactam. This novel enzyme with a molecular mass of 28,000 resembles other plasmid-mediated carbenicillinases (CARB) but differs in its apparent isoelectric point estimated as 6.3 and has been designated CARB-5 on this basis.