Activity Of B1 Research Articles

Reprimo, a New Candidate Mediator of the p53-mediated Cell Cycle Arrest at the G2 Phase

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G(2) arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2.cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G(2) arrest of the cell cycle.

Isoforms of Bone Alkaline Phosphatase: Characterization and Origin in Human Trabecular and Cortical Bone

Alkaline phosphatase (ALP) is a glycoprotein and functions as an ectoenzyme attached to the cell membrane by a hydrophobic glycosyl-phosphatidylinositol (GPI) anchor. Three bone ALP (BALP) isoforms in human serum were separated and quantitated by high-performance liquid chromatography. B/I, a minor fraction, is composed on average of bone (70%) and intestinal (30%) ALP, and two major isoforms, B1 and B2. Treatment with GPI-specific phospholipase C (GPI-PLC) did not influence the activities or retention times for B1 and B2, indicating that the biochemical differences between B1 and B2 are likely to be due to different glycosylation patterns. The B/I fraction in serum, on average 4% of total ALP, was found to be composed of B1 and B2 isoforms, each with an intact hydrophobic GPI cell membrane anchor. We investigated the origin of these three BALP isoforms and osteocalcin in human femora from five healthy individuals (four males), mean age 51 years, obtained from a tissue bank. Bone was sampled from three sites: cortical bone, trabecular bone from the diaphysis, and trabecular bone from the greater trochanter. Trabecular bone, from both sites, had higher BALP activities compared with cortical bone. Conversely, the osteocalcin content of cortical bone was more than 3-fold greater than that of trabecular bone. Cortical bone had approximately 2-fold higher activity of B1 compared with B2, whereas trabecular bone had approximately 2-fold higher activity of B2 compared with B1. We observed a previously undescribed BALP isoform (B1x) in all bone samples. B1x was also observed in sera from some patients (60%) with severe renal insufficiency and on chronic dialysis therapy (n = 20). The isoforms of BALP may provide information relating to bone metabolism within specific bone compartments.

P3B99 - The mutagenic activity of aflatoxin B1 in rat bone marrow

The effects of several carotenoids of vitamin A and of 3-methylcholanthrene have been tested on the initiation of hepatocarcinogenesis by aflatoxin B1, using the sequential protocol of Solt and Farber. AFB1-induced DNA single-strand breaks and AFB1-metabolism were also assessed. The P4501A inducer carotenoids (canthaxanthin, astaxanthin, β-apo-8′-carotenal) and 3-methylcholanthrene reduce the carcinogenicity of AFB1, divert AFB1-metabolism into the less genotoxic aflatoxin M1 and reduce AFB1-induced DNA single-strand breaks: we conclude that these carotenoids exert their protective effect through the deviation of AFB1 metabolism towards detoxification pathways. β-Carotene decreased AFB1 carcinogenicity but did not alter its metabolism, probably acting by other mechanisms.

Chloroquine stabilizes pancreatic lysosomes and improves survival of mice with diet-induced acute pancreatitis.

Activation of digestive zymogens by lysosomal enzymes has been suggested as a triggering event in acute pancreatitis (AP). chloroquine (CQ), a weak base that accumulates in the lysosomes and increases their pH, can inhibit the activity of lysosomal enzymes. In the present study, we examined the effect of CQ on choline-deficient, ethionine-supplemented (CDE) diet-induced AP. CQ-diphosphate (15-50 mg.kg-1) or vehicle was given intraperitoneally at 0, 24, and 48 h to female CD1 mice that were fed with either normal diet or CDE diet. For mortality studies, animals were observed for 168 h. Serum and pancreas samples were collected from animals sacrificed 56 h after the start of the CDE diet. Treatment with CQ at 50 mg.kg-1 significantly (p < 0.05) improved the survival of mice with CDE diet-induced AP. In the normal pancreas, CQ decreased the specific activity of lysosomal enzymes cathepsin B1, beta-hexosaminidase, beta-glucuronidase, and acid phosphatase. In the pancreas with AP, CQ did not modify the activity of cathepsin B1, whereas it increased the latency of all enzymes. In conclusion, our results confirm the beneficial effect of CQ on survival of mice with CDE diet-induced AP and suggest that this effect of CQ may be due to its stabilizing action on lysosomes.

Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation.

M-phase promoting factor or maturation promoting factor, a key regulator of the G2-->M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. We have previously shown that Xenopus cyclin B1 has five sites of Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism is still unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala to abolish phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be enhanced by mutation of these residues to Glu to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Here we show that biological activity can be restored to cyclin B1Ala by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity was detected in association with cyclin B1Ala targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Furthermore, given the similar in vitro substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase-cyclin complexes.

Haltere Afferents Provide Direct, Electrotonic Input to a Steering Motor Neuron in the Blowfly,Calliphora

The first basalar muscle (b1) is one of 17 small muscles in flies that control changes in wing stroke kinematics during steering maneuvers. The b1 is unique, however, in that it fires a single phase-locked spike during each wingbeat cycle. The phaselocked firing of the b1's motor neuron (mnb1) is thought to result from wingbeat-synchronous mechanosensory input, such as that originating from the campaniform sensilla at the base of the halteres. Halteres are sophisticated equilibrium organs of flies that function to detect angular rotations of the body during flight. We have developed a new preparation to determine whether the campaniform sensilla at the base of the halteres are responsible for the phasic activity of b1. Using intracellular recording and mechanical stimulation, we have found one identified haltere campaniform field (dF2) that provides strong synaptic input to the mnb1. This haltere to mnb1 connection consists of a fast and a slow component. The fast component is monosynaptic, mediated by an electrical synapse, and thus can follow haltere stimulation at high frequencies. The slow component is possibly polysynaptic, mediated by a chemical synapse, and fatigues at high stimulus frequencies. Thus, the fast monosynaptic electrical pathway between haltere afferents and mnb1 may be responsible in part for the phase-locked firing of b1 during flight.

Supported transition-metal catalysts for the CC coupling reaction between ethene and diazoalkanes

The preparation of a series of immobilized transition-metal catalysts are reported. The catalysts were obtained by chemisorption of either rhodium(I) or iridium(I) complexes [MX(C 2H 4) 2] n (M  Rh, Ir; X  Cl, OAc, acac, f 3-acac, f 6-acac) on SiO 2 or MgO supports. The oxides were also activated by SiCl 4 or TiCl 4 to give support materials in which the acidic nature of the surface is substantially increased. The activity of the immobilized catalysts was tested, particularly in the reaction of ethene with diphenyldiazomethane which yields a mixture of 1.1-diphenylpropene ( 8) and 1.1-diphenylcyclopropane ( 9). It was found that the most active and most selective (highest ratio 8:9 ) catalyst B1 was formed support material B (SiO 2 activated by SiCl 4) and [RhCl(C 2H 4) 2] 2 ( 1) and that both the activity and selectivity of B1 was comparable with that of complex 1 in solution. In contrast, the supported catalysts A2, D2 and A3, D3 obtained from [Rh(OAc)(C 2H 4) 2] 2 ( 2) and [Rh(acac)(C 2H 4) 2] ( 3) were less active than compounds 2 and 3 in solution. The immobilized catalysts A6, A7, D7 and E7, which were generated from the chloro- and acetatoiridium(I) complexes [IrCl(C 2H 4) 2] 2 ( 6) and [Ir(OAc)(C 2H 4) 2] 2 ( 7), possessed a lower activity than the rhodium counterparts. With diazoalkanes other than Ph 2CN 2, the activity of the supported catalyst B1 was partly higher and partly lower than that of complex 1 in the homogeneous phase.

Aflatoxin B1 induced lacI mutation in liver and kidney of transgenic mice C57BL/6N: effect of phorone.

Transgenic C57BL/6N mice containing a lambda shuttle vector carrying a lacI target and an alpha-lacZ reporter gene have been used to study the modulating effect of phorone, a glutathione-depleting agent, on the mutagenic activity of aflatoxin B1 (AFB1) in vivo. Animals were treated with AFB1 (8 mg/kg) for four consecutive days and the animals sacrificed 21 days after the last treatment. Treatment with AFB1 alone did not result in a significant increase in mutation frequency in the liver and kidney. When the animals were treated with phorone 4 h prior to treatment with AFB1 a significant increase in mutation frequency was observed in the liver (4-fold) and kidney (1.5-fold). Phorone treatment did not increase the AFB1-induced mutation frequency in the lung and intestine. DNA sequence analyses of 30 independent clones isolated from the liver of AFB1-treated animals showed that G:C --> T:A transversion (60%) was the predominant mutational event. Mutations within the lacI gene could not be detected in seven of 30 mutants. The mutations were randomly distributed throughout the coding sequences of the lacI gene and no hotspots for the mutations were observed. However, codons 86 and 928 appeared to be major sites for mutation. The study shows that the transgenic mouse in vivo mutagenesis model can be used to study the influence of effect-modifying compounds on the mutagenic activity of known carcinogens.

Destruction of phagocytosis-suppressing activity of aflatoxin B1by ozone

The impact of ozone on the immunity-impairing activity of aflatoxin B1 (AFB1) was studied. Phagocytosis by rat peritoneal macrophages, which was found to be suppressed in the presence of AFB1, remained unimpaired when the applied AFB1 was pretreated with ozone (1.2 mg l-1) for 6 min at a flow rate of 40 ml min-1. Hence, application of ozone on AFB1-contaminated foodcrops seems to be a promising preventive measure against any adverse immunological disorder in consumers.

Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C.

Susceptibility of Cacopsylla pyri and C. pyricola (Homoptera: Psyllidae) to Avermectin B1 Applied Topically and as Residues on Pear Foliage

Effects of avermectin B1 were determined by topical application for Cacopsylla pyri (L.) adults in Europe and by exposure to treated pear foliage for C. pyricola (Foerster) adults and nymphs in North America. Data were obtained for mortality and egg production by survivors. With C. pyri , probit regressions for males and females were similar, summer adults were more susceptible than winter adults. Age of summer adults at treatment did not affect slope, and mortality associated with time between treatment and evaluation increased linearly up to 72 h. Avermectin B1 reduced average daily oviposition and total egg production in C. pyri . Oviposition rate was affected more in summer females than in winter females. Slopes for dose- and concentration-mortality data were similar for the two species with the two exposure techniques. The technique of exposing nymphs to treated pear leaves was also used to measure residual activity of avermectin B1 in pear foliage. Applications at 7.5 mg/liter produced Comparable mortalities for ≈1 wk longer than did half that concentration. Data derived from these tests can serve as baselines for detecting changes in susceptibility of psyllid populations, and techniques reported here could be used to detect resistant populations. Implications of these data to the development of a use strategy for avermectin B1 against psyllids attacking pear are presented.

ChemInform Abstract: Quantum Chemical and Vibronic Analysis of the 1 2B2 ↔ 1 2A2, 2 2B2 Transition in Benzyl‐h7 and Benzyl‐d7 Radicals.

The QCFF/PI+CI method and the CNDO/S+CI method in the floating atomic orbital basis set are used to study vibronic structure of the 1 2B2↔1 2A2, 2 2B2 transition in benzyl‐h7 and benzyl‐d7 radicals, arising from the activity of a1 and b1 in‐plane modes. It is shown that the 6a10 line has its intensity almost entirely due to the borrowing from the 1 2A2→1 2A2 transition via the 1 2A2–1 2A2 coupling. We demonstrate tha the 1 2A2 and 2 2B2 states are strongly mixed by ν8b and ν6b modes. The unperturbed 1 2A2 state lies ∼850 cm−1 below the unperturbed 2 2B2 state. The coupling depresses strongly the position of the 6b10 level mixing it markedly with the level arising from C–CH2 rock (ν18b in other assignments) fundamental. Three‐mode vibronic analysis of the absorption and emission spectrum produces vibronic structure that agrees well with the observed spectra. The adiabatic frequency of the ν14 (b1) mode in the 1 2B2 state is shown to be depressed markedly by vibronically induced mode mixing with ν8b mode du...

Translaminar and Residual Activity of Avermectin B1 Against Plutella xylostella (Lepidoptera: Plutellidae)

The translaminar activity of avermectin B1 (AVMB1; 1.8% wt/vol EC) against second instars of a susceptible laboratory strain of Plutella xylostella L. varied considerably with crop species; the LC50 for AVMB1 was about 145 and 250 times lower on Chinese cabbage (cv. Pe Tsai) than on brussels-sprouts (cv. Winter Harvest) and cabbage (cv. Langed IJK4), respectively. Addition of emulsified safflower oil appeared to have little effect on the translaminar activity of AVMB1 on brussels-sprouts. Under greenhouse conditions, AVMB1 appeared to have between 50 and 100 times greater residual activity than cypermethrin on cabbage (cv. April) when tested against fourth instars of the laboratory strain and a field (Thailand) strain resistant to a number of insecticides. In the residue experiment, 96-h mortality data also suggested that the field strain was markedly less sensitive to cypermethrin and to a lesser extent to AVMB1 when compared with the laboratory strain. However, when mortality was assessed at eclosion (approximately 240 h), there was generally less difference between the two strains, particularly for AVMB1. Addition of emulsified safflower oil or Sunspray 6E oil extended the residual activity of AVMB1 against both strains.

Kinetics and thermodynamics of the structural transformations of thiamine in basic aqueous media. Part 3. Interpretation of the lability of the 2-proton via an intramolecular σ-adduct

The structural transformations of thiamine into its yellow form J– in very basic media (pH > 11) have been investigated by pH stopped-flow jump and temperature-jump techniques. This transformation via the deprotonation of the thiazolium 2-position to give the resonance-stabilized carbene A–H, hitherto considered as the biocatalyst of vitamin B1 metabolic activity, is questioned, since deprotonation of the thiamine 2-position is unlikely for pK < 15. Formation of the J– species occurs through the σ-adduct D resulting from intramolecular NH2 base-promoted nucleophilic addition at the thiazolium 2-position. This ring formation occurs with a second-order rate constant k14of 99 l mol–1 s–1. σ-Adduct D generates J– by a base-promoted reaction: k45 5.15 × 106 l mol–1 S–1. The deuterium isotope effect k45(H2O)/k45(D2O) 2.05 and ΔpK4 0.75 for this D ⇄ J– reaction imply the involvement of several proton transfers. It is surmised that this D ⇄ J– transformation takes place through the deprotonation of the 2-position of D into a carbanion intermediate D– which by prototropic ring-opening yields J–. This hypothesis accounts for the half-life of the 2-proton, known from n.m.r. measurements in neutral aqueous media. This tentative mechanism answers a long standing question on the role played by the amino pyrimidine moiety in the biocatalytic activity of thiamine. The existence of such a carbanion D– species as a candidate among the catalysts which monitor the metabolic activity of vitamin B1 is, therefore, proposed with caution.

Effect of Pressure‐Heat Treatments on Cathepsin B1 Activity

ABSTRACTThe substrate Nα‐benzoyl‐DL‐arginine‐2‐naphthylamide was used to measure the activity of crude cathepsin Bl in reaction mixtures that had been subjected to various combined pressure and heat treatments. Pressures of 100 KPa (atmospheric), 100 MPa and 150 MPa and temperatures in the range 30–30°C were used. High ressure appeared to protect the enzyme against heat inactivation. Maximum activity was observed at 60°C and 150 MPa and was 12 times that observed at 60°C and atmospheric pressure. An increase in activity of cathepsin B1 may account, at least in part, for the tenderization of meat by pressure‐heat treatments.

Inhibition of aflatoxin B1-hepatocarcinogenesis in rats by beta-naphthoflavone.

Effects of beta-naphthoflavone (beta NF) on the activity of hepatic microsomal aflatoxin B1 (AFB1)-4-hydroxylase - the cytochrome P-450-dependent enzyme system which catalyzes the metabolism of AFB1 to AFM1 - and on AFB1-induced in vivo hepatocarcinogenesis were investigated in weanling male Fischer rats. A single i.p. injection of beta NF in doses of 20 mg/kg and 150 mg/kg induced AFB1-4-hydroxylase 3- and 4-fold, respectively, 48 h post injection. Feeding of diet containing 0.01% beta NF for a period of 9-weeks induced AFB1-4-hydroxylase approximately 2-fold. AFB1, given by intubation in a dose of 25 micrograms five times/week for 8 weeks, produced 42 weeks later a 100% incidence of liver lesions (neoplastic foci, nodules or tumors), but feeding beta-NF in diet at a concentration of 0.015% for one week prior to and during the 8 weeks of AFB1 treatment inhibited AFB1 hepatocarcinogenesis by approximately 75%. These results are in accord with the suggestion that AFB1-4-hydroxylase induction may be associated with the inhibition of AFB1 carcinogenesis, possibly occurring as a consequence of accelerated detoxification of AFB1 via its conversion to AFM1.

Differential sensitivity of Ah-responsive mice to beta-naphthoflavone-induced metabolism and mutagenesis of benzo[a]pyrene and aflatoxin B1.

Effects of the administration to C57BL/6ha (Ah-responsive) mice of a low (10 mg/kg) and a high dose (150 mg/kg) of beta-naphthoflavone (BNF) on the hepatic microsome-mediated mutagenesis and metabolism of benzo[a]pyrene (BP) and aflatoxin B1 (AFB1) were studied. Hepatic microsome-mediated mutagenesis of benzo[a]pyrene was not enhanced by the low dose (10 mg/kg) but at the high dose (150 mg/kg) the mutagenic activation was enhanced several fold relative to control (corn oil-treated). Mutagenic activity of aflatoxin B1 was however depressed by both the low and the high doses of beta NF. These results are consistent with the effects of beta NF administration on hepatic microsome-mediated metabolism of BP to its phenolic products and on the metabolism of aflatoxin B1 to aflatoxin M1 catalyzed by aflatoxin B1-4-hydroxylase. Relative to control, pretreatment of the mice with 10 mg/kg beta NF did not induce aryl hydrocarbon hydroxylase activity (a measure of BP metabolism), however, the same pretreatment induced the metabolism of AFB1 to AFM1 by 2.7 to 4.7-fold. Microsomal preparations from 150 mg/kg beta NF-pretreated mice showed a 3-fold induction of aryl hydrocarbon hydroxylase activity and a 6.8-fold induction of AFB1-4-hydroxylase activity. These results suggest that two different enzyme systems are involved in the metabolism of BP and the metabolism of AFB1 to AFM1.

Intracellular protease changes in acute experimental hypersensitivity pneumonitis.

Proteases are among the potentially toxic agents that may be produced by lung cells during nonspecific or specific immunologic inflammation. We studied levels of selected intracellular lysosomal proteases in an established rabbit model of acute hypersensitivity pneumonitis to determine if demonstrable changes in these proteases occurred. Cathepsins B1, B2, C and D, dipeptidylaminopeptidase II and elastolytic enzyme in lung homogenates and subcellular fractions were quantitated using specific substrates, and reported as total activity (per lung) and as specific activity (activity per mg protein). Rabbits undergoing acute inflammation had significantly increased total activity for cathepsins B1, B2, C and D in lysosomal fractions compared to controls, with increased total activity of cathepsins B1, C and D in cytoplasmic fractions as well. Specific activity was significantly increased for cathepsins B1, B2 and C in the lysosomal compartment and of cathepsin C in the cytoplasmic fraction. The intracellular lung proteases exhibited a variety of changes during systemic immunization, inhalation of aerosolized antigen and recovery from inflammation. The cellular origin of these enzymes and whether their changes contribute to injury, aid in recovery, or are simply epiphenomena remain to be determined.

Dinoflagellate neurotoxins related to saxitoxin: Structure and latent activity of toxins B1 and B2 1

Toxins B1 and B2 from cultured dinoflagellates of the genus Protogonyaulax are shown to be the carbamoyl-N-sulfo derivatives of saxitoxin and neosaxitoxin, the structures confirmed by synthesis from the corresponding decarbamoyl toxins.

Effect of adjuvant arthritis on collagenase and certain lysosomal enzymes in relation to the catabolism of collagen.

The activity of collagenase, cathepsin B1, cathepsin D and Hyaluronidase was determined in skin, bone, liver, kidney, spleen and serum of adjuvant induced arthritic rats during the acute and chronic phase of the disease. Collagenase was assayed directly in tissue extract by a solution method using radioactive labelled substrate. The activity of collagenase, cathepsin B1 and D was found to increase significantly at both phases of the disease. The activity of hyaluronidase decreased significantly in liver, kidney and spleen of arthritic rats, while in skin, bone and serum no significant change was observed. The results are discussed with respect to catabolism of collagen in adjuvant induced arthritis. Prednisolone and L-thyroxine were administered to arthritic rats and the activity of collagenase, cathepsin B1, cathepsin D and hyaluronidase was determined in the treated groups during the acute and chronic phase of the disease. Prednisolone was found to suppress the development of arthritis which, in turn, decreased the increased activity of collagenase and lysosomal enzymes cathepsin B1 and D in tissues and serum of arthritic rats. L-Thyroxine was found to slowly diminish the development of inflammation and its beneficial action was found in mesenchymal tissues and skin of arthritic rats but not in bone.