Assignment of the fatty acid synthase (FASN) gene to bovine chromosome 19 (19q22) by in situ hybridization and confirmation by somatic cell hybrid mapping.
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Animals↗
Biomedical subjects
R Roy
Publications and source records attributed to R Roy.
Salmonella Give infection in 2 dairy herds.
Salmonella Give infection was investigated in 2 adjacent dairy herds because of the impact of milk contamination on public health. Once the status of the infection in a herd was established by fecal analyses, consecutive fecal testings, in conjunction with preventive measures, appeared to be consistent and cost-effective tools for the monitoring and control of salmonellosis in dairy herds.
Animals↗
Differentiation of tuberculous from pyogenic brain abscesses with in vivo proton MR spectroscopy and magnetization transfer MR imaging.
BACKGROUND AND PURPOSE: MR imaging features are nonspecific with respect to the causative organism for patients with brain abscesses. On the basis of the hypothesis that the biochemical environment depends on the infecting organism and might be different in tuberculous compared with pyogenic brain abscesses, this study attempted to determine whether pyogenic brain abscesses can be differentiated from tuberculous brain abscesses by use of magnetization transfer (MT) MR imaging and in vivo proton MR spectroscopy. METHODS: Twenty-seven patients with a total of 33 pyogenic brain abscesses and three patients with a total of 12 tuberculous abscesses were evaluated with in vivo MR spectroscopy and MT MR imaging. The diagnosis in all cases was based on the culture of the causative organisms and histopathology whenever done as a part of clinical management. RESULTS: All 27 patients with pyogenic brain abscesses had lipid and lactate levels of 1.3 ppm and amino acid levels of 0.9 ppm with or without the presence of succinate, acetate, alanine, and glycine, while the three patients with tuberculous abscesses showed only such lipid and lactate levels. The MT ratio from the wall of the pyogenic abscesses was significantly higher (P <.001) than that from the tuberculous abscess wall. CONCLUSION: It might be possible to differentiate tuberculous abscesses from pyogenic abscesses by using MT MR imaging and in vivo MR spectroscopy, which could be of value in influencing the management of such cases.
Adolescent↗
Leprosy bacillus--possibly the first chemoautotrophic human pathogen cultivated in vitro and characterised.
Leprosy bacillus (LB) and leprosy derived in vitro culture forms, the chemoautotrophic nocardioform (CAN) bacteria, showed an extremely close homology and identity with each other as regards a chemoautotrophic nutritional pattern, a nocardioform morphology, a weak acid-fastness coupled with Gram and Gomori's stain positivity, an exclusive mycolate and lipid profile, a phenolic glycolipid (PGL-I) and a highly sequestrated DNA characteristic, namely, a unique small size, a low G+C % mole, an exceptionally high gamma and UV radiation resistance, and a high thermal resistance. LB/CAN bacteria (CANb) gave positive signals for 36 kDa protein PCR, as well as, for 65 kDa epitope, and hybridisation with two or more probes and also by RFLP-analysis. Both LB/and CAN bacteria exhibited bacillary multiplication in the mouse footpads (MFP), nerve infiltration and evidences for local pathogenicity associated with pronounced systemic invasion. A highly reproducible mutilation model could be established which enabled a successful application of the postulates of Koch. The proof of their total identity was their anergic reactions in LL cases counterpoised against Mitsuda type strong nodular responses, mirroring the reactions of leprosy bacilli in TT cases, in accordance with the dictum of XIth International Leprosy Congress (1978). Thus, the chemoautotrophic nutritional requirements of LB, entirely unsuspected for a medically important pathogenic bacterium, having dimorphic (both bacillary and mycelial) characters with spores, mycelia and granules and unique pathogenicity of multilation manifested through the virulence factor, the enzyme collagenase, made LB or M leprae the highly enigmatic bacterium for so long.
Animals↗
Synthesis of persialylated beta-cyclodextrins.
The synthesis of homogeneous hepta-antennated C-6 branched sialosyl cyclomaltoheptose derivatives (persialylated beta-cyclodextrins) has been performed in good to excellent yields, and the compounds have been fully characterized. The thioacetate N-acetylneuraminic acid derivative 6 was selectively de-S-acetylated and coupled by nucleophilic displacement in a one-pot reaction to the heptakis(chloroacetamido) beta-CDs 2 and 5, yielding multivalent sialosides 8 and 9, respectively. The thiourea-linked sialyl-CD 10 was obtained by reaction of the 4-isothiocyanatophenyl N-acetylneuraminic acid derivative 7 with the per-tert-butoxycarbonylamino beta-CD derivative 2 after suitable deprotection of the amino function.
Carbohydrate Conformation↗
Meso aryl heptaphyrins: the first 30pi aromatic expanded porphyrins with an inverted structure.
[reaction: see text] Synthesis of new meso aryl 30pi heptaphyrins 2, 3, and 4 is achieved. Spectroscopic studies reveal that 2, 3, and 4 are aromatic and possess an inverted structure.
Magnetic Resonance Spectroscopy↗
Cytokine and cytotoxic molecule gene expression determined in peripheral blood mononuclear cells in the diagnosis of acute renal rejection.
BACKGROUND: Prevention of acute rejection is the most prevalent measure used to reduce the long-term risk of chronic allograft rejection. Until now, biopsy was the only useful diagnostic tool for monitoring allograft acute rejection, but invasiveness of this procedure limits its use. The aim of this study was to investigate the implication of peripheral blood immune markers as a predictive diagnostic tool preceding biopsy in acute renal allograft rejection determination. METHODS: Of the 61 patients studied, 13 had no rejection episodes, 8 had a proven acute rejection, and 40 were excluded for graft dysfunction causes. Mitogen-induced peripheral blood mononuclear cells were tested for interleukin- (IL) 2, IL-4, IL-5, IL-6, IL-10, IL-15, Interferon-gamma, Perforin, Granzyme B, and Fas L using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). An up-regulated mRNA expression value was calculated in which a patient's sample was deemed positive if its differential expression value was equal or higher than the mean differential expression value calculated from the nonrejecting patients. RESULTS: IL-4, IL-5, IL-6, Interferon-gamma, Perforin, and Granzyme B mRNA levels were associated with acute rejection. When at least two of these cytokine markers were up-regulated in a given patient, 75% of the rejecting recipients were identified against 15% of the nonrejecting patients. CONCLUSIONS: We have shown that acute rejection episodes in renal transplant recipients were associated with an increase in mRNA expression of cytokines in mitogen-induced peripheral blood mononuclear cells. The evaluation of pro-inflammatory cytokines and cytotoxic molecules prove useful in the clinical identification of acutely rejecting transplant recipients and in the justification of concomitant antirejection therapy before histological diagnosis confirmation.
Biomarkers↗
Decreased LH pulsatility during initiation of gonadotropin superovulation treatment in the cow: evidence for negative feedback other than estradiol and progesterone.
LH pulse secretion is suppressed during superovulation of cattle. The objective of this study was to determine how soon after initiation of superovulation treatments this suppressive effect occurs, and to test the hypothesis that decreased LH pulsatility is not related to changes in circulating estradiol or progesterone. Heifers (n = 7/group) were injected with eCG (FOLLIGON: a single injection of 2,500 IU) or twice daily injections of decreasing doses of FOLLTROPIN-V (total equivalent of 280 mg of NIH-FSH-P1) or F.S.H.-P (total equivalent of 28 mg of Armour standard) or saline (time controls), starting on Day 10 (Day 0 = estrus). Blood samples were taken every 10 min for 12 h intervals on the day prior to first injection, at 8 to 20 h and 32 to 44 h after initiation of gonadotropin treatment, and also during prostaglandin (PG)-induced luteolysis. A simple method based on robust statistics and on graphical representations of time series was developed to characterize LH pulses. There was a significant interaction between time and treatment for mean LH, estradiol and progesterone when control and treated groups were analyzed together, and no interaction when only the gonadotropin groups were analyzed together. When compared to pretreatment values, pulse frequency of LH was significantly reduced (P<0.05) in each treatment group, 8 to 20 h and 32 to 44 h following initiation of gonadotropin treatment. Mean LH concentrations were also reduced 32 to 44 h following initiation of treatments (P<0.05). Mean estradiol concentrations increased two to threefold at 8 to 20 h following initiation of superovulation treatments (P<0.05). Progesterone concentrations also increased by 20 or 44 h. There was no significant correlation between estradiol or progesterone and LH pulse frequency, amplitude and mean concentrations at any time in control or superovulated animals. This study demonstrates that superovulation treatment in the cow causes a rapid decrease in pulsatile release of LH and suggests that this effect is not mediated through the negative feedback actions of estradiol and progesterone.
Animals↗
Dynamics of activated escape and its observation in a semiconductor laser
We report a direct experimental observation and provide a theory of the distribution of trajectories along which a fluctuating system moves over a potential barrier in escape from a metastable state. The experimental results are obtained for a semiconductor laser with optical feedback. The distribution of paths displays a distinct peak, which shows how the escaping system is most likely to move. We argue that the specific features of this distribution may give an insight into the nature of dropout events in lasers.
Journal Article↗
Synthesis of "sugar-rods" with phytohemagglutinin cross-linking properties by using the palladium-catalyzed Sonogashira reaction
A palladium-catalyzed Sonogashira reaction has been applied for the syntheses of divalent "sugar-rods" which exhibited excellent lectin cross-linking properties. The procedure, which involves a tetrakis(triphenylphosphine)palladium-catalyzed cross-coupling reaction between an alkyne and a halogen-bearing sp2-carbon in DMF at 60 degrees C, is very efficient and the dimeric or heterobifunctional "sugar-rods" (8-13, 15-17) were isolated in 65-100% yields. Dimers 8a and 15a were both shown to form insoluble cross-linked lattices when mixed with the tetrameric plant lectin from Canavalia ensiformis (Concavalin A, Con A). Moreover, the relative inhibitory properties of the synthetic dimannosides were determined by means of the hemagglutination of rabbit erythrocytes, whereby dimer 15a was shown to be 20-fold more potent than monomeric methyl alpha-D-mannopyranoside.
Journal Article↗
Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect".
Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal alpha-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, DeltaH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, DeltaH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (TDeltaS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.
Calorimetry↗
Hexadecane mineralization and denitrification in two diesel fuel-contaminated soils.
The effect of nitrate, ammonium and urea on the mineralization of [(14)C]hexadecane (C(16)H(34)) and on denitrification was evaluated in two soils contaminated with diesel fuel. In soil A, addition of N fertilizers did not stimulate or inhibit background hexadecane mineralization (4.3 mg C(16)H(34) kg(-1) day(-1)). In soil B, only NaNO(3) stimulated hexadecane mineralization (0.91 mg C(16)H(34) kg(-1) day(-1)) compared to soil not supplemented with any nitrogen nutrient (0.17 mg C(16)H(34) kg(-1) day(-1)). Hexadecane mineralization was not stimulated in this soil by NH(4)NO(3) (0.13 mg C(16)H(34) kg(-1) day(-1)), but the addition of NH(4)Cl or urea suppressed hexadecane mineralization (0.015 mg C(16)H(34) kg(-1) day(-1)). Addition of 2 kPa C(2)H(2) did not inhibit the mineralization process in either soil. Denitrification occurred in both soils studied when supplemented with NaNO(3) and NH(4)NO(3), but was not detected with other N sources. Denitrification started after a longer lag in soil A (10 days) than in soil B (4 days). In soil A microcosms supplemented with NaNO(3) or NH(4)NO(3), rates of denitrification were 20.6 and 13.6 mg NO(3)(-) kg(-1) day(-1), respectively, and in soil B, they were 18.5 and 12.5 mg NO(3)(-) kg(-1) day(-1), respectively. We conclude that denitrification may lead to a substantial loss of nitrate, making it unavailable to the mineralizing bacterial population. Nitrous oxide was an important end-product accounting for 30-100% of total denitrification. These results indicate the need for preliminary treatability studies before implementing full-scale treatment processes incorporating commercial fertilizers.
Journal Article↗
Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT).
Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N(2)OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations, with an IC(50) for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N(2)OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N(2)OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N(2)OR was much more sensitive to TNT than NaR+NiR+NOR.
Journal Article↗
Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG).
N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.
Amino Acid Sequence↗
Acquired alkylating drug resistance of a human ovarian carcinoma cell line is unaffected by altered levels of pro- and anti-apoptotic proteins.
In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5 - 10-fold more resistant to nitrogen mustards (IC50 of 50 - 60 microM) and other DNA crosslinking agents, e.g., cisplatin, and also to DNA topoisomerase inhibitor etoposide (ETO) than A2780. CBL (125 microM) induced extensive apoptosis in A2780 associated with mitochondrial damage but not in A2780(100). No significant differences were observed between A2780 and A2780(100) cells in the basal levels, or the enhanced levels in some cases after CBL treatment, of DNA repair proteins involved in repair of alkyl base adducts or in repair of DNA crosslinks or double strand break repair. However, the basal levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were 4 - 8-fold higher in A2780(100) than in A2780 neither of which expressed Bcl-2. In contrast, the levels of pro-apoptotic Bax and Bak were 3 - 5-fold higher in the CBL-treated A2780 but not in A2780(100). ETO (5 microM) induced apoptosis in A2780 without altering the levels of Bax and Bak in these cells. At the same time, neither overexpression of Bcl-xL in A2780, nor its antisense expression in A2780(100), and nor overexpression of Bax in A2780(100), significantly affected drug sensitivity of either line. Our results suggest that a change in an early step in DNA damage processing which affects intracellular signaling, such as enhanced DNA double-strand break repair, could be the primary cause for development of resistance in A2780(100) cells to drugs which induce DNA crosslinks or double strand-breaks.
Antineoplastic Agents, Alkylating↗
The hamster heart: a paradox in itself.
Perfusion of all mammalian heart muscle except hamster with Ca(2+)-free Tyrode and thereafter reperfusion with normal Tyrode causes irreversible damage, the calcium paradox. Our study aims at deciphering the role of creatine kinase, high energy phosphates and Ca(2+)influx in the genesis of myocardial injury in the rat and comparing it with the hamster. Isolated hearts from hamster and rats were perfused in the Langendorff mode at 37 degrees C for 30 min with normal Tyrode, for 15 min with Ca(2+)-free Tyrode and thereafter for 30 min of reperfusion with normal Tyrode. The 'high energy phosphate compound' levels were monitored by(31)P-NMR, creatine kinase (CK) release was measured in the perfusate.(45)Ca influx was estimated in the papillary muscle. We observed that in the rat heart: (a) high energy phosphate levels declined significantly within 1 min of Ca(2+)reperfusion; (b) a massive release of CK occurred upon Ca(2+)reperfusion; (c) there was a significant increase of Ca(2+)influx. In the hamster heart, there was preservation of high energy phosphates, CK release was prevented completely and no rise in(45)Ca influx was observed upon Ca(2+)reperfusion. These results suggest that the hamster heart has a remarkable capacity for Ca(2+)homeostasis which protects the heart from Ca(2+)overload.
Adenosine Diphosphate↗
Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.
The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.
Aldehyde Oxidoreductases↗
Mechanism of thymocyte apoptosis induced by serum of tumor-bearing host: the molecular events involved and their inhibition by thymosin alpha-1.
The observations presented in this paper indicate that serum of Dalton's lymphoma (DL) bearing mice contained certain soluble factor(s) that augmented the induction of apoptosis in thymocytes in a time- and dose-dependent manner. DL-ascitic fluid and DL-conditioned medium could also induce apoptosis of thymocytes in vitro, though the magnitude of the same was consistently lower than that induced by serum of DL-bearing mice. It was observed that the interaction of FasL and TNFalpha with their respective receptors could trigger apoptosis in thymocytes. Elucidation of the signal transduction mechanism revealed involvement of protein tyrosine kinase, protein kinase C and ser/thr phosphatases with concomitant increase in the level of protein products of apoptosis associated genes p53, bax, bad, fas and fas ligand and cleavage of N-terminal 23 kDa fragment of Bcl-2 that exhibited Bax-like death effector properties. Further, we report, for the first time, the ability of thymosin alpha-1, an immunopotentiating thymic hormone, to antagonize apoptosis in thymocytes induced by factors present in serum of DL-bearing mice. The underlying mechanism of tumor serum induced apoptosis inhibition by thymosin alpha-1 was also analyzed. The signal transduction cascade evoked by thymosin alpha-1 involves activation of protein kinase C with a decrease in the level of protein products of proapoptotic genes like bax and bad and increase in the protein products of bcl-2 gene.
Animals↗