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PubMed1999

Determination of cows' milk in goats' milk and cheese by capillary electrophoresis of the whey protein fractions.

The use of capillary zone electrophoresis to determine the adulteration of cows' milk in goats' milk products is described. The detection and quantification of cows' milk was based on the presence of the specific whey proteins: the relative calibration curve is reported. The peaks of interest were well resolved by using sodium borate at pH 9.2 as background electrolyte in methyl-silanized capillaries. The minimum amount detectable of cows' milk was 2% in milk mixtures and 4% in cheeses. Restrictions due to genetic variability and possible heat treatments, on only one of the two types of milk employed, are taken into account. Qualitative analysis of goat-ewe-cow and goat-ewe samples are also reported.

Animals↗

PubMed2002

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library.

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.

Antibodies↗

PubMed2002

Volatile fraction and sensory characteristics of Manchego cheese. 2. Seasonal variation.

An automatic purge and trap apparatus, coupled to a GC-MS was used to study the seasonal variability of the volatile fraction of raw milk Manchego cheese. Both season and dairy significantly affected abundance of most volatile compounds. Most aldehydes, methyl ketones, n-alcohols, and secondary alcohols reached significantly higher concentrations in spring cheeses. Branched chain alcohols showed significantly higher concentrations in autumn and winter cheeses, while significantly higher amounts of diketones were found in summer cheeses. Most ethyl esters reached higher concentrations in spring and winter cheeses and lower in autumn cheeses. Lower concentrations of alpha-pinene were found in spring cheeses, and higher amounts of limonene were observed in winter cheeses. Heptane and octane were significantly more abundant in summer cheeses. No significant seasonal differences were found either for quality or intensity scores.

Alcohols↗

PubMed1990

Evaluation of occupational exposure to sulfur compounds in paper pulp kraft mills.

A study was carried out to provide a comprehensive profile of sulfur gas concentrations to which workers are exposed during the manufacture of paper pulp by the kraft process. Methods currently available--including colorimetric tubes, direct-reading monitors, and gas chromatographic methods--were compared. Gas chromatography is the only technique able to reliably measure individual sulfur gases. However, the necessity of using a gas sampling bag renders this technique inappropriate for personal monitoring. A strategy to evaluate occupational exposure was developed, based on fluctuations in workplace concentrations and the time profile of worker activities. In most samples, only three sulfur products were detected: dimethyl sulfide, methyl mercaptan, and hydrogen sulfide. Concentrations varied as a function of time and location and are correlated to process and equipment-operation variables. Using the proposed strategy, it was demonstrated that workers in the pulp and paper industry can be exposed to significant concentrations of sulfur compounds.

Chromatography, Gas↗

PubMed1998

Characterization of a major slow oscillation in the mesenteric circulation of conscious rats.

1. Little is known about spontaneous slow rhythms in regional circulations. The present study was aimed at characterizing low-frequency (LF; 78-269 mHz) oscillations in the mesenteric and hindquarter circulations of conscious rats. 2. Mean arterial pressure (MAP) and indices (pulsed Doppler technique) of mesenteric (n = 25) and hindquarter (n = 23) blood flows were recorded in conscious, freely moving rats during 1 h periods. Fast Fourier transform analysis was applied to beat-to-beat data after resampling at 10 Hz of consecutive 205 s time series. 3. A major oscillation centred at 164 +/- 4 mHz was present in the mesenteric, but not in hindquarter, circulation. Consequently, LF power accounted for approximately 43% of the overall variability of mesenteric blood flow. Cross-spectral analysis performed between MAP and mesenteric blood flow indicated that fractional changes in flow were approximately two-fold of those in MAP, in pressure, at the peak frequency. 4. Acute blockade of the autonomic, renin-angiotensin and vasopressin systems combined with noradrenaline infusion (n = 7) reduced the frequency of the mesenteric blood flow oscillation (115 +/- 6 mHz) but did not change its contribution to overall flow variability (approximately 48%). A clear oscillation was still present after acute inhibition of nitric oxide (NO) synthesis with NG-nitro-L-arginine methyl ester (n = 8), but was virtually absent in chronically guanethidine-sympathectomized rats (n = 12). 5. In conclusion, the mesenteric blood flow of conscious rats exhibits a major slow oscillation that originates in the mesenteric vasculature and is not secondary to the activity of the major pressor systems or to the cyclic release of NO. Because of the strong attenuation of the oscillation in sympathectomized rats, we suggest that adrenergic vasoconstrictor tone plays a permissive role in its genesis.

Animals↗

PubMedDate not supplied

Tautomerism of isoguanosine and solvent-induced keto-enol equilibrium.

Ultraviolet and infrared absorption spectroscopy, in aqueous and non-aqueous media, have been employed to study the tautomerism of 9-substituted isoguanines, including the nucleoside isoguanosine. With the aid of a series of model compounds, it was shown that 9-substituted isoguanines, and isoguanosine, in aqueous medium are predominantly in the form N (1) H, 2-keto-6-amino. In dioxane solution the tautomeric equilibrium is shifted in the direction of the enol form. The shift towards this form is accentuated for those analogues in which the exocyclic amino group is methylated. With the aid of N6,N6, 9-trimethylisoguanine, and 9-octyl analogue, the tautomeric constant was studied as a function of concentration, temperature, and solvent polarity, and the results applied to evaluate the tautomeric equilibria of 9-methylisoguanine and isoguanosine as a function of these variables. In general the enol form is favoured by a decrease in solvent polarity, by a decrease in concentration in dioxane, or an increase in temperature in chloroform solution. Syntheses are described for several N6 amino and methylamino derivatives of 2-methoxy-9-methylpurine, and 3-methyl-5-oxo-7,8-dihydroimidazo (2,1-i) purine, which served as an analogue of the unavailable 1,9-dimethylisoguanine.

Chemical Phenomena↗

PubMedDate not supplied

Cuticular waxes and flavonol aglycones of mistletoes.

Cuticular waxes of Viscum album subspecies and of V. cruciatum have been examined for their micromorphology and chemical composition. Wax crystalloids occur preferably as irregular platelets and rodlets, while deviant structures are found in small areas. Among the triterpenoids forming the wax layer, oleanolic acid is prevailing with some 80%. The quantitative composition of the long-chain aliphatics, which comprise several classes, is rather variable. Flavonoid aglycones, occurring as very minor components of the cuticular waxes, comprise the flavonols kaempferol and quercetin and a series of their methyl derivatives, in some taxa also the flavanone naringenin. Neither the crystalloid structures nor the chemical composition of the wax allow to discriminate the 2 species, or male and female plants, or plants grown on conifers or on dicotyledoneous hosts.

Alcohols↗

PubMed2000

Association of the dopamine transporter gene with Parkinson's disease in Korean patients.

Dopamine transporters (DAT) uptake neurotoxic substances such as 1-methyl-4-phenylpyridinium (MPP+) in the dopaminergic nerve terminals and may confer susceptibility to cytotoxic effects of neurotoxic substance. The association of a variable number tandem repeat polymorphism in the DAT gene with Parkinson's disease (PD) in a Korean population was studied. The 10-copy allele was the most common, accounting for 77.2% and 81.6% of alleles in PD patients and control subjects, respectively. The rare 11-copy allele was more common in the patients (odds ratio = 2.5, 95% confidence interval = 1.1-5.7, p<0.02). It is suggested that the 11-copy allele of the DAT gene may confer susceptibility to PD for some patients in Korea.

Aged↗

PubMed2005

Differences in macrophage activation by bacterial DNA and CpG-containing oligonucleotides.

Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN >44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

Animals↗

PubMed1989

Dynamic changes of excitatory amino acid receptors in the rat hippocampus following transient cerebral ischemia.

The changes in excitatory amino acid receptor ligand binding induced by transient cerebral ischemia were studied in the rat hippocampal subfields. Ten minutes of ischemia was induced by common carotid artery occlusion combined with hypotension, and the animals were allowed variable periods of recovery ranging from 1 day to 4 weeks. The binding of 3H-AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) to quisqualate receptors, 3H-kainic acid (KA) to kainate receptors, and 3H-glutamate to N-methyl-D-aspartate (NMDA) receptors as determined by quantitative autoradiography. One week following ischemia the CA1 region of the hippocampus displayed a severe (90%) dendrosomatic lesion with preservation of presynaptic terminals. This was associated with a 60% decrease in AMPA binding and a 25% decrease in glutamate binding to NMDA receptors. At 4 weeks postischemia, both AMPA and NMDA sites were greatly reduced. Although the dentate gyrus granule cells are resistant to an ischemic insult of this magnitude, this region showed marked changes in receptor binding. One week following ischemia, the AMPA and NMDA binding decreased by approximately 40 and 20%, respectively. Following 2 weeks of recovery, the NMDA binding was not significantly different from control level, while the AMPA binding remained depressed up to 4 weeks postischemia. The high density of KA binding sites in the inner molecular layer of the dentate gyrus was unaffected by the ischemic insult, despite an extensive degeneration of cells in the hilus of dentate gyrus which projects glutamatergic afferents to this area.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Inhibition of dihydropteridine reductase from human liver and rat striatal synaptosomes by apomorphine and its analogs.

Dihydropteridine reductase from human liver and rat striatal synaptosomes is noncompetitively inhibited by apomorphine and its analogs. The Ki or I50 values are in the range of 0.6 to 2.9 microM for R-(-)-apomorphine, R-(-)-and S-(+)-2, 10, 11-trihydroxyaporphine, R-(-)-norapomorphine, R-(-)-N-hydroxyethylnorapomorphine, R-(-)-2,10,11-trihydroxy-N-n-propylnoraporphine, R-(-)- and S-(+)-N-n-propylnorapomorphine, and R-(-)-N-chloroethylnorapomorphine; and 13 to 151 microM for R-(-)-2,11-dihydroxy- 10-methoxyaporphine, R-(-)-apocodeine, and S-(+)-bulbocapnine. Structure-activity studies reveal that 10,11-dihydroxy substitution of the D ring of apomorphine is required for the inhibitory effectiveness of these aporphines. Methylation of the 10-hydroxy group reduces, whereas the 2-hydroxyl substitution of the A ring enhances, their inhibitory potency. N-Alkylation variably affects the inhibitory potency of aporphines. In addition, S-(+)-enantiomers of aporphines and dopaminergic antagonists are equally potent as inhibitors of this enzyme, as compared to the corresponding R-(-)-enantiomers and other aporphine agonists. Haloperidol (0.1 to 10 microM) failed to reverse the enzyme inhibitory effectiveness of apomorphine when it was incubated with intact rat striatal synaptosomes prior to or after the addition of apomorphine (0.5 to 1 microM). These results suggest that the inhibitory effects of apomorphine and its analogs against this enzyme are not mediated by their stimulation of dopamine autoreceptors. Since dihydropteridine reductase is required in vivo for the hydroxylation of tyrosine, the inhibition of this enzyme by apomorphine may represent one of several mechanisms by which apomorphine inhibits catecholamine synthesis.

Animals↗

PubMed2003

Global DNA methylation in relation to hMLH1 and hMSH2 protein immunoreactivity in sporadic human endometrial carcinomas.

Overall DNA methylation status was studied in a group of 28 sporadic human endometrial carcinomas (ECs) using the [32P]-postlabeling technique. Moreover, expression of the DNA mismatch repair proteins (hMLH1 and hMSH2) was investigated in ECs using immunohistochemistry. Mean 5-methyldeoxycytosine (m5dC) content in the studied group was 3.48+/-0.37% (range, 2.89-4.12%). The mean m5dC scores were significantly different between early (3.35+/-0.33%) and advanced (3.66+/-0.36%) endometrial neoplasms (chi2-test; p=0.03). There was a markedly increased overall DNA methylation with the degree of histological differentiation and with the infiltration of the myometrium (p<0.05). Loss of hMLH1 and hMSH2 expression was reported in 7 (25%) and 5 (18%) tumors, respectively, but the immunoreactivity did not correlate with the known clinicopathological variables of cancer. In addition, no obvious correlation was found between global m5dC content and the lack of hMLH1 and hMSH2 protein expression in human uterine tumors (p=0.97 and p=0.19 for hMLH1 and hMSH2, respectively; Spearman's rank correlation test). Our results clearly show that alterations in global DNA methylation may influence tumor progression, but they are not directly associated with the inactivation of the mismatch-repair machinery in sporadic human ECs.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver.

BACKGROUND: Inactivation of the tumour suppressor gene p16 (CDKN2/MTS-1/INK4A) and K-ras mutations are among the most frequent genetic alterations in human malignancies. AIMS: To investigate the tumour suppressor gene p16 and its possible association with K-ras mutations in intrahepatic cholangiocarcinomas of the liver. METHODS: The status of p16 was evaluated in 41 cholangiocarcinomas by methylation specific polymerase chain reaction, microsatellite analysis, DNA sequencing, and immunohistochemical staining. K-ras mutations were determined by direct DNA sequencing analyses after microdissection. The results obtained were correlated with histopathological variables and patient survival. RESULTS: Hypermethylation of the 5' CpG island of the p16 gene was found in 34 of 41 (83%) carcinomas. Homozygous deletion at the p16 region was present in two (5%), and loss of heterozygosity (LOH) in eight cases (20%). We failed to detect p16 gene missense mutations. K-ras mutations were found in 22 of 41 (54%) cholangiocarcinomas and in two cases of tumour surrounding non-neoplastic liver tissue. All 22 cancers with K-ras mutations also exhibited methylated p16. We failed to observe a correlation between K-ras or p16 status and histopathological factors or prognosis of patients. CONCLUSION: These data suggest that inactivation of the p16 gene is a frequent event in cholangiocarcinoma. The most common somatic alteration is promotor methylation of the p16 gene which is closely associated with K-ras mutations. We failed to establish p16 or K-ras status as independent prognostic factors in these tumours.

Bile Duct Neoplasms↗

PubMed2006

HPLC analysis of ADMA and other methylated L-arginine analogs in biological fluids.

Post-translational methylation of arginine residues in proteins leads to generation of N(G)-monomethylarginine (MMA) and both symmetric and asymmetric dimethylarginine (SDMA and ADMA), that are released into the cytosol upon proteolysis. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Plasma concentrations of ADMA and SDMA are very low, typically between 0.3 and 0.8 microM, making their quantification by HPLC an analytical challenge. Sample preparation usually involves a cleanup step by solid-phase extraction on cation-exchange columns followed by derivatization of amino acids into fluorescent adducts. Because ADMA and SDMA concentrations in healthy subjects show a very narrow distribution, with a between-subject variability of 13% for ADMA and 19% for SDMA, very low imprecision is an essential assay feature. Procedures for sample cleanup, derivatization, and chromatographic separation of arginine and its methylated analogs are the main topics of this review. In addition, important aspects of method validation, pre-analytical factors, and reference values are discussed.

Animals↗

PubMed1998

Studies of the antagonist actions of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid (ATPO) on non-NMDA receptors in cultured rat neurones.

Whole-cell patch-clamp recordings from single cultured cortical neurones have been used to study the action of (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. ATPO competitively reduced peak responses evoked by semi-rapid applications of AMPA (Ki = 16 microM) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 microM), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors. Semi-rapid application of kainic acid (KA) and the KA receptor-selective agonist, (2S,4R)-4-methylglutamic acid (MeGlu) evoked non-desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 microM, respectively). Responses to MeGlu were unaffected by CTZ (100 microM), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 microg ml(-1)). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A. Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 microM). The remaining component was further reduced by ATPO with a Ki of 36 microM, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA. It is concluded that ATPO is a moderate-potency competitive inhibitor of naturally expressed non-NMDA receptors.

Adrenal Cortex Hormones↗

PubMed1995

Cerebrospinal fluid levels of kynurenine pathway metabolites in patients with eating disorders: relation to clinical and biochemical variable.

In brain, most L-tryptophan is metabolized to indoleamines, whereas in systemic tissues L-tryptophan is catabolized to kynurenine pathway metabolites. Among these latter compounds are: quinolinic acid, an N-methyl-D-aspartate receptor agonist; kynurenic acid, an antagonist of excitatory amino acid receptors that also reduces quinolinic acid-mediated neurotoxicity; and L-kynurenine, a possible convulsant. Because the metabolism of L-tryptophan through the kynurenine pathway is dependent upon adequate nutrition, we sought to determine whether the impaired nutrition characteristic of eating-disordered patients might be associated with specific disturbances in this metabolic pathway. Cerebrospinal fluid levels of L-tryptophan, quinolinic acid, kynurenic acid, L-kynurenine, and 5-hydroxyindoleacetic acid were measured in medication-free female patients meeting DSM-III-R criteria for either anorexia nervosa (n = 10) or normal-weight bulimia nervosa (n = 22), studied at varying stages of nutritional recovery. Eight healthy, normal-weight females served as a comparison group. Cerebrospinal fluid levels of kynurenic acid were significantly reduced in underweight anorectics, compared to normal females, but returned to normal values with restoration of normal body weight. Although cerebrospinal fluid quinolinic acid levels were not different from controls, the ratio of quinolinic acid to kynurenic acid was significantly increased during the underweight phase of anorexia nervosa. Furthermore, in the eating-disordered patients, kynurenic acid levels in cerebrospinal fluid correlated positively with percent-of-population average body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2003

Investigation of alternatives to methyl bromide for management of Meloidogyne javanica on greenhouse grown tomato.

The root galling index and the densities of eggs in roots and juveniles in soil of the root-knot nematode Meloidogyne javanica (Treub) Chitwood on tomato, and the effect of these on crop yield were assessed in greenhouse experiments applying various treatments at two different sites in Crete, Greece. Tomato crops were grown for four cycles by rotating nematode-resistant (first and third spring crops) with susceptible (second and fourth autumn crops) cultivars and receiving the following treatments: (a) untreated control; (b) methyl bromide application before the first and third crops; (c) application of the fungus Pochonia chlamydosporia (Goddard) Zare, Gams & Evans before planting the first, third and fourth crops with a supplementary application three weeks after the beginning of the fourth crop; (d) application of oxamyl in both sites and fenamiphos in site 1 only at the second and fourth crops; (e) combination of treatments (c) and (d). The fungus density in soil was monitored three weeks after application and at the end of each crop, when roots were lifted. Pochonia chlamydosporia had a variable establishment and did not control the nematode. Its pathogenicity on eggs was not demonstrated, as in all cases galls were big, with all egg masses inside and protected from infection. The methyl bromide treatment significantly reduced root galling and egg production compared to other treatments in all crop cycles and the yield of the fourth crop was significantly greater. Nematicides reduced nematode densities compared with untreated controls and the fungus treatment, but they were less effective than methyl bromide and resulted in increased yield in one site only.

Animals↗

PubMed2003

[Superinstability (TCT/TCC)n of microsatellite DNA in parthenogenetic lizards Darevskia unisexualis (family Lacertidae)].

Using multilocus DNA fingerprinting, we have examined variability of (TCT)n microsatellite and M13 minisatellite DNA repeats in populations, families, and tissues of Caucasian parthenogenetic rock lizards Darevskia unisexualis (Lacertidae). It has been shown for the first time that population and family DNA samples of D. unisexualis (75 samples in total) have individually specific DNA fingerprinting patterns of (TCT)n fragments. Analysis of inheritance of (TCT)n microsatellites in 46 first-generation progeny in 17 parthenogenetic D. unisexualis families revealed their extremely high instability. Mutant TCT fingerprint phenotypes were found in virtually each animal of the progeny. Moreover, varying fragments in the progeny and their original variants in the mothers were shown to simultaneously contain (TCT)n and (TCC)n polypyrimidine clusters. At the same time, no variability of (TCT)n fragments has been detected in the tissues and organs of mature parthenogenetic lizards and in the analogous tissues of the two-week-old progeny of this year. This suggests the absence of somatic mosaicism and methylation of the corresponding loci in the samples. Along with the hyperinstability of (TCT/TCC)n polypyrimidine clusters, we have shown that the population and family DNA fingerprinting patterns of M13 minisatellites were invariable and monomorphic in the same DNA samples of D. unisexualis. Our results indicate that mutations at loci containing polypyrimidine microsatellites significantly contribute to the total genomic variability of parthenogenetic lizards D. unisexualis.

Animals↗