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Biomedical subjects

M Kirk

Publications and source records attributed to M Kirk.

At least 55 records · Page 3Linked to original sources
PubMed1998

Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines.

There is substantial variation in the growth inhibition of different human breast cancer cell lines by the isoflavones genistein and biochanin A. ZR-75-1 and BT-20 cells are > or = 2- to 4-fold less sensitive to these isoflavones than are MCF-7 cells, whereas T47D cells have a sensitivity similar to that of MCF-7 cells. To determine whether these differences are related to isoflavone metabolism by these cancer cells, each of the cell lines was incubated with [4-(14)C]genistein and [4-(14)C]biochanin A. Metabolites in the cell culture media were identified by radio-HPLC electrospray ionization mass spectrometry. One metabolite of genistein (genistein 7-sulfate) and 2 metabolites of biochanin A (genistein and genistein 7-sulfate) were detected by radio-HPLC. Further analysis by mass spectrometry identified 3 other metabolites, a hydroxylated methylated form of each isoflavone and a biochanin A sulfate. IC50 (the concentration at which the growth rate was halved) values of the breast cancer cell lines did not correlate well with production of genistein 7-sulfate from genistein or with biochanin A sulfate, genistein 7-sulfate, or genistein from biochanin A. However, IC50 values correlated with the production of the hydroxylated and methylated forms of the isoflavones. Only T47D cells produced these metabolites in this study, and only T47D cells had IC50 values similar to those of MCF-7 cells, which also produced the hydroxylated and methylated metabolites. These data suggest that the hydroxylated and methylated metabolites may be the active forms of genistein in human breast cancer cells and emphasize the importance of isoflavone metabolism in the mechanism of action of isoflavones.

Anticarcinogenic Agents↗
PubMed1998

Structure-activity study of the lantibiotic mutacin II from Streptococcus mutans T8 by a gene replacement strategy.

Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the mutA gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: delta N1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except delta N1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the "hinge" region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (delta N1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.

Amino Acid Sequence↗
PubMed1998

HPLC-mass spectrometry analysis of isoflavones.

The current interest in the role of dietary isoflavonoids, particularly the soy isoflavone genistein, in lowering the risk of several chronic diseases, has led to the need for rapid, sensitive and precise assays for isoflavones and their metabolites in food matrices and in various physiological fluids and tissues. HPLC has the advantage over GC-based methods in that all the conjugated and unconjugated isoflavonoids and their metabolites can be separated and analyzed without the need for derivatization. An important advance in mass spectrometry has been the introduction of effective interfaces between the HPLC and the mass spectrometer, namely the electrospray ionization (ESI) and the heated nebulizer-atmospheric pressure chemical ionization (HN-APCI) interfaces. Because of the isoflavonoid concentrations in fluids such as bile or urine, preliminary extraction, so essential for GC-MS and many other analytical methods, is not necessary. This immediately overcomes the thorny issue of finding an effective solid-phase extraction procedure. Using reversed-phase HPLC-ESI-MS, it is possible to obtain a mass/intensity map of all isoflavonoid metabolites in a single 20 min analysis. Analysis of isoflavonoid conjugates in serum/plasma samples requires initial extraction, but the conjugates can be measured intact either by capillary reversed-phase HPLC-ESI-MS or on regular reversed-phase columns by HPLC-HN-APCI-MS. In both cases, specificity is obtained by causing the parent isoflavonoid molecular ions to undergo collision-induced dissociation to form specific daughter ions in a triple quadrupole MS instrument. When it is only necessary to measure the total isoflavonoids and their metabolites in blood, hydrolysis can be performed directly in serum/plasma samples and isoflavonoids recovered by ether or ethyl acetate solvent extraction. The isoflavone aglucones can be analyzed by HPLC-MS under isocratic solvent conditions, thereby drastically shortening analysis time and opening up prospects for automation. Therefore, HPLC-MS is a technique that is broadly applicable to the major issues in phytoestrogen research.

Animals↗
PubMed1997

Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol.

Peroxynitrite (ONOO-), formed from the rapid reaction of superoxide (O2-.) with NO, is known to generate stable compounds capable of donating NO on reaction with thiols and molecules containing hydroxy groups. Using glycerol as a model compound for the reactions of ONOO- with biomolecules containing hydroxy groups, we separated the products and identified them by HPLC/MS. It was shown that both glyceryl mononitrate and glyceryl mononitrite were formed and released NO on incubation with copper and l-cysteine. The compounds were stable over a period of 4h when shielded from light and kept on ice. Slow spontaneous decomposition occurred in the buffer used for the bioassay, but this was not sufficient to explain the vasorelaxing properties of these NO donors. It is concluded that the stable organic nitrate and nitrite have the capacity to be metabolized by vascular tissues, resulting in vasorelaxation.

Animals↗
PubMed1997

A prolonged outbreak of Campylobacter infection at a training facility.

Campylobacter outbreaks are rarely detected despite Campylobacter being the most common food-borne illness notified to public health authorities. We report a prolonged outbreak of Campylobacter occurring over a three month period at a training facility. Seventy-eight cases were detected, 16 of which were confirmed Campylobacter infections. In seven affected groups of people using the facility, the attack rate ranged between 19% and 67%. An investigation of one sporting group showed that illness was associated with consumption of cucumber served at a self-serve salad bar. Six people attending the facility in other weeks also reported illness after eating only at the salad bar. Transmission of Campylobacter ceased after changes were instituted to food preparation and storage in the facility kitchen.

Adolescent↗
PubMed1997

Phase I dose-escalation pharmacokinetics of AZT-P-ddI (IVX-E-59) in patients with human immunodeficiency virus.

3'-Azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxy-5'-inosinic acid (AZT-P-ddI, IVX-E-59, Scriptene) is a heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (zidovudine or AZT) and one molecule of 2',3'-dideoxyinosine (didanosine or ddI) linked through their 5' positions by a phosphate bond. AZT-P-ddI exhibits enhanced antiviral activity and selectivity in vitro compared with AZT and ddI alone. The pharmacokinetics of AZT-P-ddI were studied in 12 patients with human immunodeficiency virus (HIV) who had CD4+ cell counts higher than 200 cells/mm3. Isotopic preparations of (14C)-AZT-P-(3H)-ddI were administered intravenously (50 mg and 100 mg) to eight patients; 1 month later these patients were crossed over to oral administration (100 mg and 200 mg). A second group of patients (n = 4) received only a 450-mg oral dose of AZT-P-ddI. Plasma levels of unchanged AZT-P-ddI after intravenous infusion declined rapidly and were undetectable 0.75 hours after the end of infusion, whereas the parent compound was not detected after oral administration, indicative of a very rapid metabolism. The parent entity was enzymatically cleaved in vivo yielding the two constituent drugs AZT and ddI, which were subsequently subjected to their respective pharmacokinetic and metabolic processes. The beta-glucuronide derivative of AZT (GAZT) represented the major metabolite of AZT, but there were no detectable levels of the toxic metabolite 3'-amino-3'-deoxythymidine (AMT). A major and previously unrecognized in vivo metabolite of ddI, referred as ddI-M, was detected in plasma and urine. Analysis by high-field proton nuclear magnetic resonance and mass spectrometry led to the identification of ddI-M as being R(-)-dihydro-5-(hydroxymethyl)-2(3H)-furanone. The formation of AZT and ddI metabolites was increased after oral administration of AZT-P-ddI compared with the intravenous infusion, with an area under the concentration-time curve (AUC) ratio of metabolite to AZT and metabolite to ddI being 7.7 and 5.7 (oral) and 3.8 and 1.1 (intravenous), respectively. The newly identified ddI-M exhibited sustained plasma levels for extended time periods with an apparent elimination half-life (t1/2) of approximately 10 hours after oral administration of AZT-P-ddI. Recovery of radioactivity associated with 3H and 14C in urine was essentially complete within 48 hours after oral and intravenous administration of AZT-P-ddI. The oral bioavailability of AZT (64.7-67.3%) and ddI (33.6-42.9%) and the other pharmacokinetic parameters were consistent with previous data reported with each nucleoside analog alone or in combination therapy.

Administration, Oral↗
PubMed1997

Influence of moderate physical exercise on insulin-mediated and non-insulin-mediated glucose uptake in healthy subjects.

To establish the relative importance of insulin sensitivity and glucose effectiveness during exercise using Bergman's minimal model, 12 nontrained healthy subjects were studied at rest and during 95 minutes of moderate exercise (50% maximum oxygen consumption [VO2max]). Each subject underwent two frequently sampled intravenous glucose tolerance tests (FSIGTs) for 90 minutes, at rest (FSIGTr) and during exercise (FSIGTe). Plasma glucose, insulin, and C-peptide were determined. Insulin sensitivity (S(I)), glucose effectiveness at basal insulin (S(G)), insulin action [X(t)], and first-phase (phi1) and second-phase (phi2) beta-cell responsiveness to glucose were estimated using both minimal models of glucose disposal (MMg) and insulin kinetics (MMi). Glucose effectiveness at zero insulin (GEZI), glucose tolerance index (K(G)), and the area under the insulin curve (AUC(0-90)) were also calculated. Intravenous glucose tolerance improved significantly during physical exercise. During exercise, S(I) (FSIGTr v FSIGTe: 8.5 +/- 1.0 v 25.5 +/- 7.2 x 10(-5) x min(-1) [pmol x L(-1)]-1, P < .01), S(G) (0.195 +/- 0.03 v 0.283 +/- 0.03 x 10(-1) x min(-1), P < .05), and GEZI (0.190 +/- 0.03 v 0.269 +/- 0.04 x 10(-1) x min(-1), P < .05) increased; however, no changes in phi1 and phi2 were found. Despite a significant decrease in the insulin response to glucose (AUC0-90, 21,000 +/- 2,008 v 14,340 +/- 2,596 pmol x L(-1) x min, P < .01), insulin action [X(t)] was significantly higher during the FSIGTe. These results show that physical exercise improves mainly insulin sensitivity, and to a lesser degree, glucose effectiveness. During exercise, the insulin response to glucose was lower than at rest, but beta-cell responsiveness to glucose did not change.

Adult↗
PubMed1997

Immunological effects of an arginine side chain contaminating synthetically prepared peptides.

The side chain, 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), is a protective group coupled to arginine to mask the omega-nitrogen, in order to protect the guanidino function during peptide synthesis by the 9-fluorenylmethoxycarbonyl (Fmoc) procedure (Walker, 1994). This group is removed at the completion of peptide synthesis; however, the cleavage process can be incomplete. We have found that animals injected with a mixed population of pure, i.e. unmodified, and Mtr-containing MBP peptides have an immunodominant humoral response to the Mtr-bearing peptide. This response is dependent on the characteristics of the MBP peptide involved. For two MBP peptides, the Mtr-containing peptide had increased binding to antibody over pure peptide. For two other peptides, only the Mtr-containing peptide bound antibody while the unmodified peptide did not. In a separate system involving a polyclonal response to an unrelated peptide from beta2-microglobulin (beta2 m), the dominance of the Mtr group was also evident. These results provide further evidence that a small side chain on a single amino acid in a peptide can markedly alter the immunogenicity and antigenicity of that peptide for antibody reactivity. This evidence emphasizes the need for a critical awareness of each component of peptide synthesis and its potential to alter the immunoreactivity of the final product.

Amino Acid Sequence↗
PubMed1997

Pearls, pitfalls, and updates in toxicology.

Pearls and pitfalls learned from our practical experiences caring for poisoned patients are presented. Clinical pearls include the following: using diagnostic tests to detect end-organ toxicity, applying physiologic principles to the management of hemodynamically unstable poisoned patients, and dealing with psychologic injuries from hazardous materials incidents. Recognizing serious complications from poisoning and adverse drug effects, including the serotonin syndrome, are offered as pitfalls. Pharmaceutical companies are rapidly developing and marketing new therapies. Therefore, updates on the evolving role of NAC as an antidote for acetaminophen poisoning, new psychotropic medications, and new antidotes were included in this article. These pearls, pitfalls, and updates are intended to provide practical information that is readily applicable to the clinical practice of emergency medicine.

Adverse Drug Reaction Reporting Systems↗
PubMed1997

Chemical warfare. Nerve agent poisoning.

The threat of civilian and military casualties from nerve agent exposure has become a greater concern over the past decade. After rapidly assessing that a nerve agent attack has occurred, emphasis must be placed on decontamination and protection of both rescuers and medical personnel from exposure. The medical system can become rapidly overwhelmed and strong emotional reactions can confuse the clinical picture. Initially, care should first be focused on supportive care, with emphasis toward aggressive airway maintenance and decontamination. Atropine should be titrated, with the goal of therapy being drying of secretions and the resolution of bronchoconstriction and bradycardia. Early administration of pralidoxime chloride maximizes antidotal efficacy. Benzodiazepines, in addition to atropine, should be administered if seizures develop. Early, aggressive medical therapy is the key to prevention of the morbidity and mortality associated with nerve agent poisoning.

Animals↗
PubMed1997

Superoxide dismutase catalyzes nitration of tyrosines by peroxynitrite in the rod and head domains of neurofilament-L.

Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO-), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in alpha-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.

Amino Acid Sequence↗
PubMed1997

Intestinal uptake and biliary excretion of the isoflavone genistein in rats.

The intestinal absorption, biliary excretion and metabolism of genistein, a potent and specific protein tyrosine kinase inhibitor that occurs naturally in soy foods, was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. 4-14C-Genistein, when infused into the duodenum, was rapidly absorbed from the intestine, taken up by the liver and excreted into the bile as its 7-O-beta-glucuronide conjugate. Cumulative recovery of 14C-radioactivity in the bile over a 4-h period was 70-75% of the dose. When genistein was infused into the portal vein, it was also taken up efficiently by the liver, conjugated with glucuronic acid and transported into bile. However, portal blood collected after duodenal infusions of genistein contained mostly genistein 7-O-beta-glucuronide, suggesting that in vivo glucuronidation occurred in the intestinal wall rather than the liver. This was confirmed using everted intestinal sac preparations. Reinfusion of genistein 7-O-beta-glucuronide into the duodenum or into the mid small intestine resulted in its reappearance in the bile, albeit more slowly than when genistein was infused. Over a 4-h collection period, the cumulative recovery of 14C-radioactivity in bile was 27 and 70-75% of the administered dose for duodenal and ileal infusions, respectively. These data indicate that genistein is highly bioavailable in rats and because of its enterohepatic circulation may accumulate within the gastrointestinal tract.

Animals↗
PubMed1997

Health effects of exposure to cyanobacteria (blue-green algae) during recreational water-related activities.

The aim of this study was to investigate effects on health of exposure to cyanobacteria as a result of recreational water activities. Participants, who were aged six years and over, were interviewed at water recreation sites in South Australia, New South Wales and Victoria on selected Sundays during January and February 1995. Telephone follow-up was conducted two and seven days later to record any subsequent diarrhoea, vomiting, flu-like symptoms, skin rashes, mouth ulcers, fevers and eye or ear irritations. On the Sundays of interview, water samples from the sites were collected for cyanobacterial cell counts and toxin analysis. There were 852 participants, of whom 75 did not have water contact on the day of interview and were considered unexposed. The 777 who had water contact were considered exposed. No significant differences in overall symptoms were found between the unexposed and exposed after two days. At seven days, there was a significant trend to increasing symptom occurrence with duration of exposure (P = 0.03). There was a significant trend to increasing symptom occurrence with increase in cell count (P = 0.04). Participants exposed to more than 5000 cells per mL for more than one hour had a significantly higher symptom occurrence rate than the unexposed. Symptoms were not correlated with the presence of hepatotoxins. These results suggest symptom occurrence was associated with duration of contact with water containing cyanobacteria, and with cyanobacterial cell density. The findings suggest that the current safety threshold for exposure of 20,000 cells per mL may be too high.

Adult↗
PubMed1997

Urinary phytoestrogen levels in young women from a multiethnic population.

Phytoestrogens include several classes of chemical compounds (i.e., isoflavones, coumestans, and lignans) which are structurally similar to endogenous estrogens. In biological systems, they have both estrogenic and antiestrogenic effects and may reduce the risk of developing certain types of hormonally related diseases. However, little information is available on population differences in exposure to phytoestrogens. To examine racial/ethnic differences in urinary phytoestrogen levels, 50 young women (ages 20-40 years) were randomly selected from participants in a previous epidemiological study in which 24-h urine specimens and a dietary assessment were obtained. Subjects were members of the Kaiser Permanente Medical Care Program of northern California. Selection was stratified on race/ethnicity. Urinary levels of seven phytoestrogens were measured using high-performance liquid chromatography-mass spectrometry. Substantial variation in phytoestrogen levels was observed and racial/ethnic differences are described. The highest levels of coumestrol and the lignans were observed in white women and the lowest levels in Latina and African American women. Genistein levels, however, were highest in Latina women; other isoflavone levels did not differ significantly by race/ethnicity.

Adult↗