Emergence of nalidixic acid resistant Vibrio cholerae O-1 in Karachi.
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Drug Resistance, Microbial↗
Biomedical subjects
A Mahmood
Publications and source records attributed to A Mahmood.
IgG binding and expression of its receptor in rat intestine during postnatal development.
The binding of 125I labelled IgG to the microvillus membranes (MVM) has been studied during postnatal development of rat intestine. The levels of mRNA encoding IgG receptor were also analyzed by liquid hybridization under these conditions. The IgG binding to MVM reached maximum levels by day 12 and showed a gradual decline upon weaning. The FcRn mRNA was markedly low in adult rats and was maximum during second week of postnatal development. Administration of cortisone or thyroxine to suckling rats, induced precocious decline of both IgG binding and the receptor expression. However, insulin administration did not affect the receptor expression. Scatchard analysis of IgG binding to MVM in cortisone injected pups revealed that the observed inhibition in IgG binding was a consequence of a decrease, both in the affinity constant (-Ka) as well as in the number of receptor sites (n) while thyroxine administration caused a reduction in the number of receptor sites from 2.29 in control to 1.14 nmoles/mg protein in thyroxine injected pups. These observations indicate that expression of IgG receptor during postnatal development is a hormone regulated process.
Animals↗
Ethanol effects on lipid peroxidation and glutathione-mediated defense in rat small intestine: role of dietary fats.
The effect of ethanol feeding for 5 weeks on lipid peroxidation status of small intestine was studied in rats maintained on either a rat pellet (RP) or a semisynthetic diet containing coconut oil (CCO), corn oil (CO), or fish oil (FO). Highest rate of iron/ascorbate-induced lipid peroxidation was observed in intestinal mucosa of FO-fed rats, which was further elevated (p < 0.05) upon ethanol administration. Purified brush borders from all the ethanol-treated dietary groups were more susceptible to iron-induced lipid peroxidation. Level of nonprotein thiols was increased by ethanol feeding to rats given CO or FO. FO-fed rats exhibited increased activities of glutathione reductase (GR), glutathione-S-transferase (GST), and catalase (Cat). Glutathione peroxidase (GPx) was the lowest in the CCO group. Ethanol-treated FO group exhibited increased GST and GPx activities compared to controls, whereas in rats fed the RP or CO diet, ethanol feeding significantly decreased GST activity. GR and Cat activities were not affected under these conditions. Thus, ethanol exposes the small intestinal mucosa to oxidative stress. The effects were more pronounced in rats fed n-3 fatty acid-rich (FO) diet. The corresponding rise in GPx and GST levels may reflect the adaptive changes in intestine.
Animals↗
Lysine-directed conjugation of ethidium homodimer to B72.3 antibody: retention of immunoreactivity but altered tumor targeting.
Ethidium homodimer (EHD) was conjugated to B72.3 monoclonal antibody using a method whereby 85-90% of the conjugated EHD remains available for DNA intercalation. Antibody was thiopropionylated by reaction with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction of pyridyldithio groups with dithiothreitol. EHD was maleimido-functionalized with succinimidyl-4-(N-maleimidoethyl)cyclohexane-1-carboxylate and treated with thiopropionylated antibody to obtain a conjugate containing approximately 3.4 EHD per antibody molecule. For biologic studies, 14C-labeled EHD was synthesized by reductive amination and conjugated as above. In vitro the conjugate maintained chemical integrity and immunoreactivity, while in vivo its targeting of LS174T tumors was reduced compared with that of iodinated antibody. A decrease in isoelectric point of the immunoconjugate was also observed.
Adenocarcinoma↗
Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.
PURPOSE: To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA. MATERIALS AND METHODS: A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in DNA has been synthesized. Upon depurination of plasmid DNA and reaction with FARP a stable oxime bond is formed between FARP and the generated open chain aldehydes. RESULTS: By independently quantitating the generated abasic sites, it is shown that most of the generated abasic sites under acidic conditions become fluorescently labelled. The limit of sensitivity with the fluorometer used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled over a wide range of FARP:base pair ratios following different extent of depurination, and fluorescent loadings of 1 FARP:20000 base pairs up to 1 FARP:10 base pairs are demonstrated. The heavily labelled samples display significant fluorescence quenching due to the proximity of abasic sites labelled with FARP, that undergo fluorescence energy transfer. Treatment of heavily labelled DNA samples with nuclease P1 results in an increase in fluorescence due to the release of the fluorescent labels in the solution. The relative increase in fluorescence is a quantitative measure of the proximity of labelled abasic sites. Furthermore it is shown that if only 1% of DNA contains abasic sites generated in close proximity (within 10-20 base pairs or less of each other) the resulting quenching is significant enough to detect, even if the rest of the DNA contains isolated abasic sites. CONCLUSIONS: The present approach constitutes a novel fluorescence-based method to detect abasic sites in nucleic acids and demonstrates the feasibility of detecting the presence of closely-spaced damage sites in DNA via fluorescence energy transfer. The technique also comprises a general and convenient method to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.
Aldehydes↗
Effect of chronic ethanol feeding on intestinal alkaline phosphatase activity in rats.
The effect of chronic ethanol feeding has been studied on intestinal alkaline phosphatase (IAP) activity. The enzyme was assayed using p-nitrophenyl phosphate (PNPP), phenylphosphate (PhP) and beta-glycerophosphate (beta GP) as the substrates. Feeding of ethanol for 10 days did not effect the enzyme activity but it was markedly elevated in animals fed ethanol for 20 or 30 days. However, ethanol administration to rats, for over 6 wk exhibited a decline in IAP activity, both in the soluble and membrane fractions of intestine. Kinetic studies revealed an enhancement in Vmax with no change in apparent Km after 30 days of ethanol feeding and a decrease in Vmax after 6 wk of ethanol ingestion. These results were confirmed by assaying the enzyme activity in non-denatured polyacrylamide gels using 5-bromo-4-chloro-3-indoly1 phosphate (BCIP) as the substrate. These findings suggest differential changes in IAP activity in response to ethanol feeding in rats.
Alcoholism↗
Effect of sodium on Saccharomyces cerevisiae invertase activity.
Effect of Na+ ions on yeast invertase activity has been studied as a function of pH. At acidic pH, Na+ (5-100 mM) had no effect but invertase activity was inhibited (38-44%) by Na+ ions (100 mM) with an increase in pH (6.8 and 8.0). Kinetic analysis revealed that invertase inhibition by Na+ ions was non-competitive and reversible in nature. Value of K(m) remained unaltered (33.3 mM) in presence of Na+ (20-100 mM) while Vmax decreased by 21-44% under these conditions. Value of Ki was of the order of 85 mM. Mechanism of observed inhibition of invertase activity as a consequence of Na+ ions interactions at the active site of the enzyme has been described.
Glycoside Hydrolases↗
Harmaline interactions with yeast invertase.
The effect of harmaline, a plant alkaloid has been studied on yeast invertase activity in the absence and presence of 50mM Na+ as a function of pH. Harmaline (1-3 mM) inhibited the invertase activity at pH 5.2, 6.8 and 8 both in the absence (44-92%) and (22-85%) of Na+ ions. Kinetic analysis revealed that harmaline is a non-competitive inhibitor of invertase, at pH 5.2 and 6.8 but at pH 8, it produced a mixed type of inhibition, Km increased by 450% and 175% and Vmax decreased by 82% and 63% in the absence and presence of 50mM Na ions respectively. The observed inhibition of invertase by harmaline was reversible in nature. These findings suggest that the presence of Na+ site is not a prerequisite for the inhibition of enzyme by harmaline.
Glycoside Hydrolases↗
A technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain.
The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a marker for certain neurological diseases. In particular, the DAT is depleted in Parkinson's disease, and the extent of depletion correlates with the loss of dopamine. Herein we describe the design, synthesis, and biological evaluation of technepine, the first 99mTc-labeled SPECT imaging agent which targets the dopamine transporter in striatum. We have demonstrated that the DAT can accommodate a chelating unit attached to the 8-amine function of a tropane skeleton. Further, we have demonstrated for the first time that a molecule can be designed to carry the radionuclide 99mTc across the blood-brain barrier in sufficient quantity to obtain in vivo images of the striatum in monkeys. This advance will undoubtedly lead to the design of new receptor and transporter-mediated 99mTc agents which can label specific transporter and receptor targets in the central nervous system.
Animals↗
Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation.
The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.
Amidines↗
Maternal mortality in India. Preventable ill health occurs in Britain too.
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Female↗
Technepine: a high-affinity 99m-technetium probe to label the dopamine transporter in brain by SPECT imaging.
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Animals↗
Metastasis in meningioma.
Meningioma is a neoplastic growth originating from the leptomeninges. Although meningiomas are usually benign, malignant meningiomas with distant metastases occur infrequently. There is little precise information in the literature regarding the frequency of metastases in meningiomas; their incidence has been vaguely reported to be less than 1 per 1,000. Furthermore, most of the previous studies have also included haemangiopericytomas which most recent authorities do not consider meningiomas. In our experience with the management of 396 meningiomas over the past 18 years, 7 meningiomas were classified as malignant by defined histological criteria. After initially presenting as solitary intracranial neoplasms, three of the malignant meningiomas metastasized to extracranial tissues. Collectively, the metastases involved the vertebral bodies, liver, pelvis, long bones, and the spinal cord. This confers an incidence of metastasis of 0.76% when considering all the meningiomas, and an incidence of approximately 43% when considering only malignant meningioma; both percentages are significantly higher than reported previously. This high incidence of metastasis in the malignant meningioma indicates a worse prognosis than formerly assessed and also characterized the malignant meningioma as a primary central nervous system neoplasm with one of the highest rates of metastasis. In addition, when malignant meningioma is classified by following strict criteria, the risk of metastasis in the ensuing clinical course can be predicted with a higher reliability.
Aged↗
A new approach to labeling cells with technetium-99m, Part II. Evaluation of radiolabeled human-lymphocyte immune functions.
Technetium-99m-modified polylysine (MPL) is a mild and efficient method for cell labeling that is easily applicable to human lymphocytes. 99mTc-MPL uptake is maximal in 40 min at room temperature. Cell labeling efficiency (from 60 to 80%) increases with rising concentrations of cells and labeling agent. In vitro stability of 99mTc-MPL on lymphocytes incubated in serum at 37 degrees C is high. Microautoradiography indicates that 99mTc-MPL is equally distributed within the cells. The radiolabeling procedure does not alter expression of surface receptors involved in T lymphocyte effector functions, adhesion function, cytolytic activity or proliferative response to IL-2.
Antibodies, Monoclonal↗
A new approach to labeling cells with technetium-99m, Part I. Preparation of modified polylysine and in vitro cell labeling.
A method for labeling cells with technetium-99m via hydrophilic, polycationic poly D-lysine modified by N-acetyl homocysteine has been developed. The modified polylysine (MPL) is labeled with 99mTc in > 95% yield and is stable for > 12 h. Maximum cell labeling is achieved by a 1-h incubation at room temperature with isolated leukocytes, granulocytes and peripheral blood mononuclear cells attaining 60-75% 99mTc incorporation, and red blood cells 35%. Ninety-two percent of the label is retained by leukocytes after a 1-h incubation at room temperature in 50% serum. The cell uptake of 99mTc-MPL is affected by the presence of negatively charged species in the medium; the inhibitory effects of 5% serum or serum albumin can be reversed by increasing the concentration of 99mTc-MPL, while those of heparin are not.
Adult↗
The construction of novel mobilizable YAC plasmids and their behavior during trans-kingdom conjugation between bacteria and yeasts.
Trans-kingdom conjugation is an easy and efficient method for gene transfer from prokaryotes to eukaryotes since it does not require DNA extraction and purification. We constructed novel mobilizable plasmids pAY-YAC-B and pAY-YAC-E. The origin of conjugal transfer (oriT) was inserted at two different positions, pAY-YAC-B contains oriT region in between two telomeres whereas pAY-YAC-E has oriT at the cloning site of pYAC4. By conjugation, both plasmids were successfully transferred from E. coli to S. cerevisiae and S. kluyveri yeasts with the aid of helper plasmid pRH220 which harbors mob and tra genes. The plasmids were transferred more efficiently in S. cerevisiae compared to S. kluyveri. The analyses by restriction enzyme digestion and Southern hybridization indicated that both plasmids maintained their original structure and size in transconjugant yeasts, therefore, reflecting the faithful nicking and subsequent resealing of plasmids during conjugation. The comparison between conjugative transfer and transformation has also been performed and discussed.
Chromosomes, Artificial, Yeast↗
Clinical significance of a polymerase chain reaction assay for the detection of Mycobacterium tuberculosis.
Various investigators have reported rapid detection of Mycobacterium tuberculosis (MTB) in clinical samples by polymerase chain reaction (PCR). To improve the specificity and efficiency of PCR, the authors adopted a variety of conditions, then analyzed sputum specimens from 217 patients clinically suspected of having MTB. Sputum samples were sonicated to obtain MTB DNA. The DNA was subjected to PCR using primer sets from the region of 650-900 in MTB. The PCR product was detected by direct gel electrophoresis and Southern blot hybridization using digoxigenin-labeled MTB-specific probe. The results of smears, cultures, and PCR were concordant in 93% (202) of the 217 specimens and discordant in 7% (15). Fifteen of the discordant specimens, all from patients who had received antituberculosis medications for days to months, were PCR positive. Of these, 11 were culture negative and 3 were smear negative. Only one specimen was false negative on PCR. Our results indicate that PCR is the method of choice when clinical suspicion is high, but smears or cultures are negative. When smears are positive, PCR permits rapid distinction between MTB and other mycobacterial infections. Because PCR can detect nonviable MTB DNA, positive PCR should be interpreted in conjunction with clinical information.
Bacteriological Techniques↗
Dietary fat effects on brush border membrane composition and enzyme activities in rat intestine.
The effect of dietary fats on the chemical composition and enzyme activities has been studied in intestinal brush border membranes (BBM) or rats. Animals were given commercial rat pellet diet (RP) or semisynthetic diet rich in either saturated [coconut oil (CCO))] or polyunsaturated [n-6, corn oil (CO) or n-3, fish oil (FO)] fat at the 10% level for 5 weeks. The membrane cholesterol/phospholipid ratio was augmented in CO- or RP-fed rats. There was an increase in level of saturated fatty acids in BBM from CCO- or FO-fed animals. n-3 polyunsaturated fatty acid content was raised in FO-fed rats, while the proportion of linoleic acid and arachidonic acid was enhanced in animals given a CO diet. Membrane fluidity was in the order of CCO < RP = CO < FO. The membrane hexose content was high (p < 0.05) in the CCO group. Hexosamines were elevated (p < 0.05) in CCO- or FO-fed rat brush borders. Membrane fucose was unaltered, while sialic acid content was elevated in CO- (p < 0.05) and FO- (p < 0.01) fed vs. CCO-fed rats. Lectin binding to brush borders corroborated these findings. The activities of alkaline phosphatase, sucrase and lactase were augmented (p < 0.001) in CCO-fed animals. Leucine-aminopeptidase and sucrase activities were depressed by FO feeding. The activities of PNP-beta-glycosidases were the highest in FO-fed rats. These results indicate that dietary fat quality markedly affects microvillus membrane lipid composition, glycosylation and enzyme functions in rat intestine.
Alkaline Phosphatase↗