Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors

In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.     

A non-amyloidogenic function of BACE-2 in the secretory pathway

beta-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave beta-amyloid precursor protein (betaAPP) at the beta-secretase site and to be required for the generation of amyloid beta-peptide (Abeta), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of betaAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves betaAPP in the middle of the Abeta domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-alpha- and p3-like products and reduced production of Abeta species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of betaAPP at Asp1 of the Abeta domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of betaAPP differentially affect the generation of Abeta.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

Role of anti-beta-glucan antibody in host defense against fungi

We have recently detected an anti-beta-glucan antibody in normal human and normal mouse sera. The anti-beta-glucan antibody showed reactivity to pathogenic fungal Aspergillus and Candida cell wall glucan. Anti-beta-glucan antibody could bind whole Candida cells. It also enhanced the candidacidal activity of macrophages in vitro. The anti-beta-glucan antibody titer of DBA/2 mice intravenously administered either Candida or Aspergillus solubilized cell wall beta-glucan decreased remarkably dependent on dose. Moreover, in deep mycosis patients, the anti-beta-glucan antibody titer decreased, and this change correlated with clinical symptoms and other parameters such as C-reactive protein. It was suggested that the anti-beta-glucan antibody formed an antigen-antibody complex and participated in the immune response as a molecule recognizing pathogenic fungi.     

Chitinolytic activity in the autolysis of Aspergillus nidulans

Abstract Chitinolytic activity in filtrates of Aspergillus nidulans cultures was studied at the start of the autolysis (maximum dry weight of mycelium) and during autolysis in 24 different media. During the growth the chitinolytic activity was induced only by the presence of ascorbic acid or colloidal chitin in the medium. During autolysis an increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β - N -acetylgucosaminidase and endochitinase was detected. The possible induction of these enzymes during A. nidulans autolysis is established.     

Plasma levels of C18-, C19- and C21-steroids in captive and feral female sea bass

Morphometric analysis of the gonads of sea bass Dicentrarchus labrax revealed that captive fish matured 1 month later than feral fish, but levels of gonadal steroids were identical in both groups at the same stage of sexual development. 17β-oestradiol (E₂) (up to 3 ng ml-1) and testosterone (T) (up to 4 ng ml-1) were highest during the gametogenetic period while 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) (free and sulphated) were maximal during the spawning period. Free 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was very low and did not change (c. 0·5 ng ml⁻¹) while 17,20β-P-sulphate increased during the spawning period in both groups (up to 2 ng ml⁻¹). In contrast cortisol levels were higher in captive fish and increased during the spawning period (up to 100 ng ml⁻¹). These results suggest that captivity delays vitellogenesis and spawning in sea bass without affecting the final levels of the gonadal steroids and further indicates a role for cortisol in the latter period. The increased levels during the spawning period suggests a pheromonal role for 17,20β-P-sulphate and 17,20β,21-P-conjugates and the involvement of 17,20β,21-P in final ooccyte maturation.     

Pre-and Postnatal Developmental Changes of Adrenoceptor Subtypes in Rat Brain

beta-Adrenergic receptor subtypes, beta 1 and beta 2, were studied during pre- and postnatal development in the rat brain. [125I]Iodocyanopindolol (6-300 pmol/L) binding assays in the presence of 5-hydroxytryptamine (0.6-6 mumol/L) were used to measure exclusively beta-adrenergic receptors. In forebrain tissue, saturable and stereoselective binding was detected on gestational day 13. The amount of beta-adrenergic binding increased until postnatal day 23, when adult values were reached. The dissociation constants of [125I]iodocyanopindolol binding remained the same throughout development, as did the affinity of several beta-adrenergic and non-beta-adrenergic compounds. The proportion of the beta 2-adrenergic receptors was determined using the beta 1-selective antagonist ICI-89406 (7-150 nmol/L) and was found to change from 65% in prenatal forebrain tissue to 28% in adulthood. In cerebellum/medulla pons tissue, however, the proportion of beta 2-receptor binding (80%) remained unchanged during the whole developmental period.     

Characterization of a novel SHV β-lactamase variant that resembles the SHV-5 enzyme

Abstract An SHV type β-lactamase frequently found in enterobacteria isolated in Greek hospitals was analyzed. The enzyme (SHV-5a) conferred resistance to ceftazidime and aztreonam. The DNA sequence of the structural gene was determined. The deduced amino acid sequence showed that positions 70–73 were occupied by the active site tetrad Ser-Thr-Phe-Lys. As in SHV-5, Ser-238 and Lys-240 were present. However, one deletion (Gly-54) and three substitutions (Arg-140 for Ala, Asn-192 for Lys and Val-193 for Leu) differentiate SHV-5a β-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediated SHV-1 β-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they were functionally indistinguishable was provided by the similar MICs of β-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.     

Effect of auxin on cell wall glycanhydrolytic enzymes in epicotyls of Cicer arietinum

Cell wall glycanhydrolytic enzymes have been related to cell wall loosening and cell growth, although the mechanism of this relationship has not been clarified. Since auxins are plant hormones that stimulate growth in elongating organs, in the present work we studied the effect of auxin on cell wall glycanhydrolytic enzymes, which were extracted with LiCl. Our results show that incubation of sections of Cicer arietinum epicotyls with indoleacetic acid elicit some minor changes in electrophoretic patterns of cell wall proteins when compared with control sections. This indicates that there is no appearance of a specific polypeptide synthesized de novo in response to the hormone, although there are increases in the intensity of some of the polypeptides, which could indicate an enhancement of wall protein biosynthesis. Brief incubation with IAA led to a general increase in the specific activities of these different cell wall enzyme fractions separated by chromatography, with the exception of the α-fraction, with α-galactosidase activity. Longer incubation resulted in an increase in the amount of protein associated with some of the enzyme fractions. In particular, it induced a large increase in the amount of protein associated with the β111-galactosidase fraction that is involved in the autolytic process of cell walls of chick-pea epicotyls. Our results indicate that auxin-enhanced growth could be the result of the action of the hormone al the level of the cell wall glycanhydrolytic proteins that have been related to the wall-loosening process.     

NMR spectrometric assay for determining enzymatic hydrolysis of β-lactam antibiotics with bacteria in aqueous solution

Abstract An application of a nuclear magnetic resonance (NMR) spectrometer for the measurement of β-lactamase activity in clinical material containing bacteria is presented. By means of proton (¹H)-NMR, it was easy to measure quantitatively β-lactamase activity in human bacteriuria, without performing any such pretreatment as isolation of bacteria or extraction of crude enzymes and without preparing special reagents for the detection. This is the first report on the application of ¹H-NMR analysis of structural changes for determining hydrolysis of β-lactam antibiotics with β-lactamase-producing bacteria in aqueous solution.     

Dissemination of transferable CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli in Korea

AIMS: Among 365 Escherichia coli isolated in 2003, 31 cefotaxime-resistant isolates were obtained from clinical specimens taken from adults hospitalized in Busan, Korea. Six extended-spectrum beta-lactamase (ESBL)-producing isolates were investigated further to determine the mechanism of resistance. METHODS AND RESULTS: These isolates were analysed by antibiotic susceptibility testing, pI determination, plasmid profiles, transconjugation test, PCR-restriction fragment length polymorphism (RFLP), enterobacterial repetitive consensus (ERIC)-PCR and DNA sequencing. All six of these isolates were found to contain the CTX-M-type ESBL genes. Five clinical isolates and their transconjugants produced CTX-M-3. One clinical isolate (K17391) and its transconjugant (trcK17391) produced CTX-M-15. Five clinical isolates also produced another TEM-1. One clinical isolate (K12776) also contained another TEM-52. CTX-M-3 ESBL gene was responsible for the resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam. CTX-M-15 or TEM-52 was especially responsible for the resistance to ceftazidime. CONCLUSIONS: These results appear to represent the in vivo evolution of CTX-M-type beta-lactamase genes (bla(CTX-M-3) --> bla(CTX-M-15)) under the selective pressure of antimicrobial therapy (especially ceftazidime). PCR-RFLP is a reliable method to discriminate CTX-M-15 gene from CTX-M-3 gene. ERIC-PCR analysis revealed that dissemination of CTX-M-3 was not due to a clonal outbreak of a resistant strain but to the intra-species spread of resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of CTX-M-1 cluster ESBLs in Korea. A more comprehensive survey of these ESBL types from Korea is urgently needed because of the in vivo evolution of CTX-M-15 from CTX-M-3. The emergence of these CTX-M-type ESBLs suggests that diagnostic laboratories should screen for ESBLs with ceftazidime as well as cefotaxime; they should still perform clavulanate synergy tests on resistant isolates.     

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for beta- and beta'-cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.     

Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

Nucleotide sequences of the genes coding for the TEM-like β-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2)

Abstract Two bla _TEM-like genes were characterized that encoded IRT β-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with β-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 β-lactamase resembled the bla _TEM-1B gene, and that for IRT-2 resembled bla _TEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the bla _IRT-1 gene and C to A transversion in the bla _IRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to β-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic p I (p I =5.2) of these IRT enzymes compared to that of TEM-1 (p I =5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this β-lactamase to inhibition by p -chloromercuribenzoate.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Transgene activity following somatic transgenesis in Nile tilapia Oreochromis niloticus

A detailed study was made of the persistence and expression of a plasmid-enclosed reporter gene construct after intramuscular injection into the somatic muscle tissue of juvenile Nile tilapia Oreochromis niloticus and also the effect of injecting a potentially growth-promoting gene construct. The plasmid-enclosed DNA proved stable at the site of injection, lasting in some cases for up to 6 months, and was, at a very low frequency, detected in gonad tissue, indicating occasional substantial movement from the injected muscle site. It was observed that the reporter gene and regulatory sequences were also functional within the somatic cells. In a comparison of expression levels by direct somatic injection, the 1·6 kb tilapia β-actin regulatory sequence (tiβAP) resulted in c. three-fold higher β-galactosidase activity than the 4·7 kb carp β-actin regulatory sequence (cβAP) when spliced to the lacZ gene. The enhancer element near the end of first intron in the tiβAP, when co-injected with tiβAP/lacZ plasmid at a 3:1 ratio, drove significantly higher reporter activity in somatic cells than the tiβAP/lacZ sequence alone. The introduction of a growth-promoting construct, the Nile tilapia growth hormone gene driven by a tiβAP, yielded no detectable growth enhancement.     

Cyanogenic glycosides and their metabolic enzymes in barley, in relation to nitrogen levels

The possible role for cyanogenic glycosides as nitrogen storage compounds was studied in barley, Hordeum vulgare (cv. Golf), cultivated under different nitrogen regimes. Cyanogenic glycosides were absent in seeds and roots but were synthesized in seedlings where they accumulated at a level of about 150 nmol shoot⁻¹ in control plants and 110 nmol shoot⁻¹ in nitrogen-starved plants. An enzyme involved in the breakdown of cyanogenic glycosides, β-glucosidase (EC 3.2.1.-) exhibited high activity in seeds and was also detected in roots and shoots. The activity of β-cyanoalanine synthase (EC 4.4.1.9), which is involved in the metabolism of HCN, was low in seeds but very high in roots and shoots. There was no correlation between the activities of the two enzymes and the content of cyanogenic glycosides or nitrogen. The relative content of nitrogen in cyanogenic glycosides never exceeded 0.3% of total nitrogen, and the amount of cyanogenic glycosides decreased at a low rate even at a stage when nitrogen limitation inhibited growth.