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PubMed2004

Enzymatic and oxidative metabolites of lycopene.

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD+, KCI, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of 2H10 lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C18H24O2 or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z =272 and (2) 3,4-dehydro-5,6-dihydro-15-apo-lycopenal (C20H28O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-l-al) with lambdamax= 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-ene-5,8-lycopenal-furanoxide (C37H50O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C40H56O2) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z =568; (5) lycopene-5,8-furanoxide isomer (I) (C40H56O2) with lambdamax = 410 nm, 440 nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C40H56O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C40H54O2) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.

Animals↗
PubMed1987

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.

Animals↗
PubMed1999

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.

Benzenesulfonates↗
PubMed2001

Using an in vivo phagemid system to identify non-compatible loxP sequences.

The site-specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34-bp loxP site. The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another. We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence. As expected, homologous loxP sequences efficiently underwent Cre-mediated recombination. However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100%. Some of these loxP sequences have previously been reported to be non-compatible with one another. Our study also confirmed other heterologous loxP pairs that had previously been shown to be non-compatible, as well as defined additional combinations that could be used in designing new recombination vectors.

Attachment Sites, Microbiological↗
PubMed2003

Spectrum of molecular changes during hepatocarcinogenesis induced by DEN and other chemicals in Fisher 344 male rats [Mechanisms of Ageing and Development 123 (2002) 1665-1680].

UNLABELLED: Unlike other tissues such as breast, colon and renal cell carcinoma, it is not an easy task to single out any representative oncogene or tumor suppressor genes in the development of hepatocellular carcinoma (HCC), which play a pivotal role. To investigate putatively altered main pathways in HCC, F344 male rats were treated with a single injection of N-nitrosodiethylamine (DEN), followed by either twice/week injections of nodularin for 10 weeks or thioacetamide (TAA) in drinking water for 39 weeks. p53 expression was dramatic in both hepatocytes and mesenchymal cells after a single injection of DEN, however, PCR-SSCP assay could not detect any p53 mutation during the development of hepatocellular adenoma (HCA). The data indicate that wtp53 response was mostly for removal of damaged cells during the initiation of carcinogenesis. When treated with DEN-TAA, induction of gankyrin expression during hepatic fibrosis preceded the loss of pRB protein, accompanied with significant expressions of G1phase cyclins and CDKs. Moreover, p16(INK4A) exon 1 was hypermethylated during the development of poorly differentiated HCCs. These changes would result in complete inactivation of the pRB regulatory pathway during hepatocarcinogenesis. Induction of TGF-beta1 expression with loss of its receptor expression occurred rapidly in the altered hepatocytes by DEN-nodularin treatment. CONCLUSION: Therefore, escape from TGF-beta1 induced apoptosis and severe degradation of pRB protein during the early stage of carcinogenesis can perform a symphony to proliferate and to transform the altered hepatocytes to tumor cells. Inactivation of p16(INK4A) and p53 genes at the later stage of carcinogenesis would endow HCC with malignancy, which is highly resistant to any therapeutic trials.

Aging↗
PubMed1998

Markers of 'cell senescence'.

In the current literature cells that have finished their proliferative life span in vitro and have reached a terminal post-mitotic state are called senescent cells. This definition originated from the belief that the irreversible non-dividing state has a relationship with aging of the organism. Attempts have been made to find markers of the so-called senescent cell in order to detect their presence in vivo in donors of different ages. One marker which was supposed to demonstrate an increase of post-mitotic cells with aging is a marker of a long resting phase whether reversible or irreversible. Other markers suggest that the postmitotic cell does not increase with aging of the organism, that it is irrelevant for aging, that it is found in an increased number in pathology, and that the term senescent cell is a misnomer that should be used only in an operational manner.

Biomarkers↗
PubMed2003

Craniometaphyseal dysplasia: a case report and review of medical and surgical management.

Craniometaphyseal dysplasia (CMD) is a genetic syndrome involving cranial and tubular bone anomalies that commonly present at a young age, often with otolaryngologic manifestations. In this paper, we report a rare case of a sporadic form of the disease resulting in an early state of hypocalcemia with secondary hyperparathyroidism. A conductive hearing loss is also documented prior to 12 months of age. The clinical aspects of CMD will be covered along with its pathogenesis. The current concepts surrounding medical and surgical treatments will be reviewed, and the management of our patient will be discussed.

Camurati-Engelmann Syndrome↗
PubMed2002

Erratum to "characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris".

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

Amino Acid Sequence↗
PubMed2003

A longitudinal study of a natural lice infestation in growing cattle over two winter periods.

A group of 61 cattle which were naturally infested with lice was followed over two winter periods. Data were collected on the number of lice found at various body sites over this period. Summary statistics were produced and due to the repeated measured and hierarchical structure of the data, multi-level analysis was used to model the population dynamics of Bovicola bovis and assess the influence of the various hierarchical levels. A four level Poisson model was produced-level one, the individual number of lice at each parting; level two, the body site examined (shoulder, midline or rump); level three, the time of the inspection and level four, the animal. Seasonal fluctuation in lice numbers was modelled using a cosine function transformation of time. A seasonal pattern was seen in both years with lice counts higher in the first than the second year. The midline area was the most sensitive to detection of B. bovis. Variance contributed by the individual animal was less than that contributed by the body site examined and the time of the inspection. The model suggested that lice numbers within the shoulder and rump sites were near random following a Poisson distribution, but aggregation of lice occurred at the midline site with the distribution between animals following a Negative Binomial Pattern. The midline site was the most sensitive site for detecting B. bovis. Infestation numbers were higher in the first winter when cattle were younger. In the second winter, there was no difference in lice numbers between cattle exposed to infection for the first or second time.

Animals↗
PubMed2003

Planar gamma scintigraphy--points to consider when quantifying pulmonary dry powder aerosol deposition.

Methodological aspects of planar gamma scintigraphy used to quantify pulmonary aerosol deposition were investigated using an experimental dry powder formulation. Particles of micronized salbutamol sulphate were labelled with technetium-99m and admixed to an ordered mixture of unlabelled micronized salbutamol sulphate and larger carrier particles of lactose. The radioaerosol was administered to 24 healthy subjects, 12 in each of two consecutive, similarly designed studies. Pulmonary deposition was determined using two methods: repeated planar imaging, and pharmacokinetic assessments following charcoal co-administration to prevent gastrointestinal salbutamol absorption. After due consideration had been taken to ensure appropriate radiolabelling, image acquisition and processing procedures, a scintigraphic estimate of 26.2% (with 95% confidence interval of 24.2-28.4%) was obtained, which did not significantly differ from the pharmacokinetic estimate of 26.4% (24.4-28.7%). In summary, pre-study validation of the radiolabelling technique, quality control of radioaerosols produced during the study, correction for re-distribution of radiolabel from the lungs, selection of regions of interest, assessment of lung contours, correction for tissue attenuation of gamma rays and establishment of the actual recovery of radioactivity in the scintigraphic measurements could potentially affect the accuracy of the scintigraphic estimate of pulmonary deposition and, thus, should be carefully considered in the design or evaluation of any such study.

Administration, Inhalation↗
PubMed1999

Determination of roquefortine C in blue cheese using on-line column-switching liquid chromatography.

A method is described for the determination of roquefortine C in (blue) cheese. After liquid-liquid extraction with a mixture of hydrochloric acid and methanol, and filtration, an aliquot is analysed using column-switching reversed-phase liquid chromatography. The recovery of roquefortine C in Fetta cheese is about 85%, the calibration curve is linear from 10 to 2500 ng g(-1) (r2 = 0.998), and the detection limit is about 10 ng g(-1). In different batches of Danish Blue concentrations of 1000-2000 ng g(-1) of roquefortine C are found. As regards the stability of roquefortine C its half-life in diffuse daylight is ca. 50 min, while after irradiation with ultraviolet light, it is about 10 min.

Acetates↗
PubMed1999

Meropenem in elderly and renally impaired patients.

The safety profile of meropenem in the elderly (aged > 65 years, n = 843) and/or renally impaired (creatinine clearance < 51 ml/min, n = 436) was assessed by evaluating data from 26 phase III studies which compared the use of meropenem (0.5 or 1.0 g, i.v. every 8 h) with other antimicrobial agents in patients with bacterial infections. The overall pattern and frequency of adverse events following meropenem therapy in the elderly and/or renally impaired were similar to those in younger and/or non-renally impaired cohorts and to imipenem/cilastatin and injectable third generation cephalosporins. Both dosages of meropenem (0.5 and 1.0 g, i.v. every 8 h) were generally well tolerated. There was no clinically significant mean change in indicators of renal flux between baseline and the end of treatment in any patient sub-group. Importantly, meropenem-related seizures were rare (0.1%), even in patients with renal impairment. In summary, meropenem has an excellent safety profile and is therefore suitable for use in elderly and/or renally impaired patients.

Aged↗
PubMed1998

Endothelial damage due to ischemia and reperfusion is prevented with SIN-1.

BACKGROUND: Acute ischemia followed by reperfusion results in direct endothelial damage characterized by cell swelling, increased permeability and loss of acetylcholine-mediated vasorelaxation. Ischemia followed by reperfusion in a New Zealand white rabbit hindlimb has been shown to result in loss of acetylcholine-induced relaxation of superficial femoral arteries. This loss of relaxation in response to acetylcholine is a reflection of the decreased nitric oxide availability that occurs with reperfusion injury. The purpose of this investigation was to evaluate the effect of SIN-1, a direct nitric oxide donor, on this endothelial injury. METHODS: New Zealand white rabbits underwent complete ischemia of the right hindlimb for 3 h followed by 2 h of reperfusion. Aliquots of 20 ml of either 0.88-mM SIN-1 or normal saline was infused via a lateral branch of the right common iliac artery during the first 20 min of reperfusion. Sham vessels were subjected to the 5-h operative intervention to control for anesthetic effect. Control vessels were harvested from rabbits not exposed to ischemia or reperfusion. Superficial femoral artery rings were evaluated in vitro for endothelial cell-mediated relaxation. Rings were contracted with potassium chloride and norepinephrine and then exposed to standardized incremental doses of acetylcholine to measure percent relaxation. Artery sections were sent for hematoxylin and eosin staining. RESULTS: No significant differences were seen in contraction caused by either potassium chloride or norepinephrine in all four experimental groups. Saline infused vessel rings relaxed a mean of 8.42 +/- 2.39% and 49.57 +/- 8.65% in response to acetylcholine doses of 3 x 10(-8) M and 1 x 10(-7) M, respectively. In contrast, SIN-1 infused vessels relaxed a mean of 57.82 +/- 2.65% and 100.23 +/- 1.53% to the same doses of acetylcholine. Control and sham arteries showed a similar relaxation response as compared with SIN-1 infused vessels. Differences in relaxation when comparing saline infused vessels with SIN-1 infused, sham and control arteries, were significantly different at each dose of acetylcholine from 3 x 10(-8) M to 1 x 10(-7) M (P < 0.05, ANOVA). Histologic examination of the vessels revealed no morphologic differences among the experimental groups. All vessels were structurally normal with an intact endothelium. CONCLUSION: In this model of rabbit hindlimb ischemia, preservation of endothelial cell-mediated vasorelaxation occurs with administration of intra-arterial SIN-1 during reperfusion. This preservation of endothelial function cannot be explained by histologic changes in the arterial wall or attributed to altered arterial contractility in response to potassium chloride or norepinephrine.

Animals↗
PubMed1996

Effects of the anthelmintics clorsulon, rafoxanide, mebendazole and arprinocid on Echinostoma caproni in ICR mice.

Female ICR mice, 5 to 6 weeks old, were exposed by stomach tube to 25 metacercarial cysts of Echinostoma caproni per mouse. At 14 days post-exposure, mice were fed by stomach tube clorsulon (1000 mg/kg, 500 mg/kg and 100 mg/ kg) or rafoxanide (50 mg/kg, 25 mg/kg and 5 mg/kg) dissolved in dimethylsulphoxide (DMSO) carrier and mebendazole (1000 mg/kg and 500 mg/ kg) or arprinocid (100 mg/kg and 50 mg/kg) suspended in a 2:1 polyethylene glycol (PEG)/DMSO carrier. All drugs were obtained from Merck Inc. (Rahway, New Jersey, USA) and only single dose regimes were used. Experimentally infected mice that served as controls received either DMSO or 2:1 PEG/DMSO carriers or were not given the carrier. Mice were necropsied 15, 16, 18 and 20 days postexposure to worms. Doses of 100 mg/kg of clorsulon and 50 mg/kg of rafoxanide were 100% effective in eliminating the echinostomes on day 1 post-administration of the anthelmintics. Mebendazole and arprinocid were ineffective in eliminating worms at 1 or 2 days post drug administration.

Adenine↗
PubMed2004

Ligands of the peroxisome proliferator-activated receptor-gamma and heart failure.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. The PPAR subfamily comprises of three members, PPAR-alpha, PPAR-beta and PPAR-gamma. There is good evidence that ligands of PPAR-gamma, including certain thiazolinediones, reduce myocardial tissue injury and infarct size. The use of PPAR-gamma agonists in the treatment of heart failure is, however, controversial.

Animals↗