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At least 361 records · Page 20Linked to original sources
PubMed2004

Imaging the cell surface: argon sputtering to expose inner cell structures.

Established microscopies such as Scanning Electron Microscopy (SEM) and more recent developments such as Atomic Force Microscopy (AFM) and X-ray Photo-Electron Emission spectroMicroscopy (X-PEEM) can only image the sample surface. We present an argon sputtering method able to progressively expose inner cell structures without apparent damage. By varying the sputtering time, the structure of cell cytoskeleton, vesicles, mitochondria, nuclear membrane, and nucleoli can be imaged. We compared images obtained with confocal fluorescence microscopy, transmission electron microscopy (TEM), SEM, and X-PEEM on similar samples after argon sputtering, then confirmed the similarity of reference intracellular structures, including cytoskeleton fibers, cell-cell and cell-substrate adhesion structures, and secretory vesicles. We conclude that the sputtering method is a new valuable tool for surface sensitive microscopies.

Argon↗
PubMedDate not supplied

Biosensor-based characterization of serum antibodies during development of an anti-IgE immunotherapeutic against allergy and asthma.

Antibody responses, induced in Cynomolgus monkey by recombinant IgE-derived immunotherapeutic protein against atopic allergies and asthma, were characterized using label-free, real-time protein interaction analysis. The effects of two different immunotherapeutic proteins were compared. Active concentrations of specific anti-IgE antibodies formed were determined in sera sampled at multiple time points, using conditions of total mass transport limitation that were proved to exist on the sensor surface. These concentrations varied from about 0.4 to 35 microg/ml among the monkeys and throughout the immunization period. Based on these concentrations, the rate and affinity constants for the binding of antibody populations to the antigen could be determined. The apparent equilibrium dissociation constant decreased during the immunization period, for all the monkeys, by a factor between 6 and 50, ending at values from approximately 2 x 10(-9) to approximately 2 x 10(-11) M among the animals. This affinity maturation was attributable to the changes in both rate constants, although the magnitude of the contribution of each constant depended partly on specimen, but primarily on the immunotherapeutic used. The immunotherapeutic proteins examined showed excellent immunogenic properties, providing the basis for a new and effective treatment for allergy and asthma.

Animals↗
PubMed2002

Regulation of kynurenic acid synthesis in C6 glioma cells.

Studies with brain slices have provided evidence that synthesis of kynurenic acid (KYNA) from kynurenine (KYN), which occurs in astrocytes, is modulated by changes in the ionic composition of the medium and the presence of depolarizing agents or the excitatory amino acid glutamate (Glu). The present study analyzed the effects of changes in incubation medium on KYNA synthesis in cultured C6 glioma cells. The synthesis was not affected by omission of Na(+) and raising K(+) concentration to 50 mM, conditions that in brain slices stimulate or inhibit KYNA formation, respectively. KYNA synthesis in C6 cells was inhibited by the absence of Ca(2+), which contrasts with its Ca(2+) independence in brain slices. Also, lack of Mg(2+) and addition of a chloride channel blocker, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate (SITS), did not affect the synthesis. KYNA synthesis in C6 cells was dose dependently inhibited by Glu. The inhibitory effect of Glu was not affected by GDPbetaS, an antagonist of metabotropic Glu receptors, the receptor class prevailing in C6 cells, suggesting that Glu acted intracellularly. NH(4)Cl and veratridine decreased KYNA production, mirroring the effects noted in brain slices. KYNA synthesis was strongly reduced in the presence of leucine (Leu), and the uptake of [(14)C]Leu was inhibited by the KYNA precursor KYN, which points to Leu as a potential endogenous modulator of KYNA formation in CNS cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2003

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves.

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non-pre-degenerated or 7-day-pre-degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain-derived neurotrophic factor (BDNF) or fibrin only. We examined DiI-labeled motoneurons, toluidine blue-labeled myelinated fibers in the mid-part of the chambers, and AChE-positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7-day-pre-degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.

Acetylcholinesterase↗
PubMed2004

Kynurenic acid synthesis in cerebral cortical slices of rats with progressing symptoms of thioacetamide-induced hepatic encephalopathy.

Increased ammonia is a major pathogenic factor in hepatic encephalopathy (HE), a neurologic syndrome associated with glutamatergic dysfunction. Previous studies have shown that in rat cerebral cortical slices or a glia-derived cell line, acute treatment with ammonia in vitro and in vivo inhibits the production of a broad-spectrum antagonist of excitatory amino acid receptors, kynurenic acid (KYNA). The present study analyzed KYNA synthesis in cerebral cortical slices obtained from rats with progressing HE symptoms accompanying acute liver failure induced by one, two, or three intraperitoneal administrations of thioacetamide (TAA) at 24-hr intervals. KYNA synthesis was found decreased to 83% of control 24 hr after one administration of TAA and unaffected after two TAA injections, when moderate hyperammonemia was associated by metabolic and bioelectric activation of the central nervous system, but was not accompanied by typical HE symptoms. KYNA synthesis was elevated to 155% of control after three TAA administrations, a period in which the rats showed advanced HE symptoms including stupor or coma. KYNA synthesis at the advanced HE stage was inhibited by glutamate in a degree comparable to that observed in control slices. The elevation of KYNA synthesis was associated with increased activity of a kynurenine aminotransferase (KAT) isomer, KAT-II. KYNA synthesis did not differ from control 21 days after the third TAA administration when HE symptoms receded. The results suggest that alterations of KYNA synthesis may contribute to the imbalance between neural excitation and inhibition at different stages of HE.

Animals↗
PubMed2003

Transformations of testosterone and related steroids in Absidia glauca culture.

In the following study, the course of transformations of testosterone and its derivatives with an additional C(1)-C(2) double bond and/or 17alpha-methyl group or without 19-methyl group in Absidia glauca culture was investigated. The fungi were observed to hydroxylate these compounds and to oxidise the 17beta-hydroxyl group. The products of 6beta, 7alpha, 7beta, 11alpha, 12beta or 15beta hydroxylation were obtained. 19-Nortestosterone was also hydroxylated at the 10beta position.The position and stereochemistry of the introduced hydroxyl group was dependent on the presence of the additional C(1)-C(2) double bond, while hydroxylation at the C-6beta was unaffected. The incubation of testosterone with Absidia glauca gave hydroxyderivatives of androstenedione: 7alpha-hydroxyandrostenedione and 6beta,11alpha-dihydroxyandrostenedione. When 1-dehydrotestosterone and 1-dehydro-17alpha-methyltestosterone were used, the products were hydroxylated at 6beta, 7beta or 15beta position while 17alpha-methyltestosterone and 19 nortestosterone products were hydroxylated at 6beta, 7alpha, 11alpha or 12beta position. Two metabolites: 6beta,11alpha-dihydroxyandrostenedione and 6beta,12beta-dihydroxy-17alpha-methyltestosterone were dihydroxylated. The presence of 17alpha-methyl group in 17alpha-methyltestosterone and 1-dehydro-17alpha-methyltestosterone did not influence the position of hydroxylation.

Absidia↗
PubMed2002

Pharmacokinetics and pharmacological effect of lerisetron, a new 5-HT3 antagonist, in rats.

The pharmacokinetics of lerisetron, a novel 5HT(3) antagonist, are studied together with its efficacy in inhibiting the serotonin (5-HT)-evoked transient bradycardia reflex (von Bezold-Jarisch reflex) in Sprague Dawley rats. [(14)C]Lerisetron (50, 100, and 200 microg/kg) was given to rats by intravenous (iv) injection and plasma levels of unchanged (UL) and total (unchanged + changed, TL) drug were measured by liquid chromatography with radioactivity monitoring and scintillation counting, respectively. Linearity of UL and TL pharmacokinetics over the dose range was established by noncompartmental analysis. Protein binding of lerisetron was measured in vitro by ultrafiltration. The unbound fraction was 14.4 +/- 1.4%. A nonlinear mixed effects ("population") bicompartmental pharmacokinetic analysis showed that volume of distribution and clearance (CL) were high for both forms of the drug, but CL was significantly smaller for TL [(mean +/- SEM) 0.014 +/- 0.03 L/min for UL versus 0.006 +/- 0.03 L/min for TL, p < 0.05)]. Large interindividual variabilities were observed for both forms. The response to lerisetron administration (inhibition of bradycardia) was evaluated at different doses (2, 3, 5, 6, and 10 microg/kg, iv) at times 2-180 min after dose administration and related to simulated concentrations. Inhibition was 100% 5 min after the 10-microg/kg dose and, 3 h later, it was still > 10%. Response to lerisetron was dose related in the range studied. Pharmacodynamic parameters were estimated by a sigmoid E(max) naive-pooled model. The parameters were also different between the two forms: EC(50) was 0.44 ng/mL (CV = 5.9%) for UL and 0.88 ng/mL (CV = 4.9%) for TL. We conclude that UL and TL pharmacokinetics were linear and that the differences in the kinetics and dynamics between the two forms suggest the presence of at least one metabolite.

Animals↗
PubMed2005

Capillary electrophoresis determinative and GC-MS confirmatory method for water-soluble organic acids in airborne particulate matter and vehicle emission.

Urban fine airborne particulate matter (PM2.5) and vehicle emission samples were studied for water-soluble low-molecular-weight carboxylic acids using CE with indirect UV detection. Further identification of these acids was achieved using GC-MS as their butyl esters (after derivatization with BF3/butanol). Several dicarboxylic acids in the range C2-C10 including straight-chain, branched-chain, cis- and trans-unsaturated, and aromatic acids were confirmed by GC-MS. In addition, aromatic acids such as benzoate, phthalate, terephthalate, isophthalate, and 4-methylphtalate were present in such samples, but some of these were not well resolved by the used CE method. Oxocarboxylic acids (Cn(w) with n > 4) were also identified by GC-MS but not determined by CE due to lack of standards. The rapidity and simplicity of the CE method were clearly demonstrated, and the method was observed to be advantageous for routine monitoring of water-soluble organic acids in airborne PM2.5 and vehicle emission at low microg/L levels.

Air Pollutants↗
PubMed2005

Determination of melatonin and its precursors and metabolites using capillary electrophoresis with UV and fluorometric detection.

A micellar electrokinetic chromatography method has been developed for simultaneous determination of melatonin and its precursors and metabolites. A 20 mM borate buffer pH 9.5 with 50 mM SDS served as the electrolyte. Tryptophan, 5-methoxyindoleacetic acid, 6-hydroxymelatonin, melatonin, serotonin, and 5-methoxytryptamine were baseline separated in less than 13 min. The limits of detection for UV detection and fluorometric detection based on native fluorescence of analytes were at the sub-ppm level. The proposed method with UV detection was applied to melatonin content control in pharmaceutical tablets with a precision expressed as RSD (n = 7) = 1.6%. For biological samples extraction with chloroform and ethyl acetate was examined. With ethyl acetate and chloroform recoveries of 87.2% and 82.1% melatonin, respectively, were obtained from plasma samples. The recovery of melatonin from spiked urine samples was 80.0% for ethyl acetate and 82.5% for chloroform. Fluorometric detection provides about two-fold improvement over UV in the detection of melatonin and minor improvements for three other analytes, but is much poorer than UV for tryptophan and 6-hydroxymelatonin in applied conditions.

Animals↗
PubMed2006

Rapid identification of Escherichia coli and Helicobacter pylori in biological samples by capillary zone electrophoresis.

The differences in surface charge of different bacteria can be exploited for their separation by capillary electrophoresis. However, this method of separation of microorganisms is beset with various drawbacks such as adhesion of bacteria to the fused silica surface or cluster formation. To overcome these phenomena we investigated the addition of poly(ethylene oxide) as a focusing agent to the running buffer and used calcium and myoinositol hexakisphosphate as specific ions that interact with the bacterial surface, changing its electrical properties and electrophoretic mobilities. In the present work, we applied CZE to identification of E. coli in infected urine (direct injection) from patients with urinary tract infections and to identification of Helicobacter pylori, which is a gram-negative bacillus responsible for one of the most common infections found in humans worldwide. Helicobacter pylori colonize the stomach and are responsible for severe diseases of the gastric tract, ranging from chronic gastric ulcer to gastric cancer.

Electrophoresis, Capillary↗
PubMed2004

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.

Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.

Anticarcinogenic Agents↗
PubMed2003

In vivo electroporation of the testis versus transgenic mice model in functional studies of spermatocyte-specific hst70 gene promoter: A comparative study.

To determine whether DNA transfer to mouse testes by in vivo electroporation could be useful method for studying regulatory elements of genes specifically active in spermatocytes first we compared the expression pattern of a construct containing the EGFP reporter gene ligated to a fragment of the heat shock testis-specific hst70 gene promoter, both in testis of transgenic mice and in testis electroporated in vivo. While in transgenic mice the EGFP was expressed in all seminiferous tubules in a cell- and stage-specific manner, in the testes electroporated in vivo only small fraction of cells expressed this marker protein. In order to make a quantitative comparison between the specificity of these two experimental systems we used several vectors containing the CAT gene ligated to fragments of the hst70 gene 5' upstream of DNA sequences which either promoted or did not activate expression of the reporter gene in the testes of transgenic mice. Also, as a reference opposite to spermatogenic cells we examined the expression pattern of the same set of vectors in the rat hepatoma FTO 2B cells. Although electroporated testes retain some spermatocyte-specific features such as the ability to repress promoters which do not contain regulatory elements responsible for testis-specific transcription, several important drawbacks of the method are evident. They include basal activity of constructs which are not transcribed in testes of transgenic mice and low overall transfection efficiency. This may hamper studies in which subtle changes in the expression pattern are under investigation. However, the in vivo electroporation of the testis can be useful for preliminary screening of constructs aimed to study in transgenic mice.

Animals↗
PubMed2005

Fully grown mouse oocyte contains transcription inhibiting activity which acts through histone deacetylation.

Mouse ovarian oocytes remain transcriptionally active during the entire period of oocyte growth. In the fully grown antral oocytes, RNA synthesis is terminated before initiation of maturation. The mechanism involved in this process remains unknown. It was proposed that some signals provided by companion granulosa cells, and accumulating in the cytoplasm of the oocyte, play a role in the termination of RNA synthesis. It seems possible that under influence of these signals, the oocyte cytoplasm becomes transcriptionally nonpermissive. In the present study, transcriptionally active, single blastomeres of the 8-cell mouse embryo were fused with nontranscribing fully grown oocytes, and RNA synthesis was analyzed in the resulting hybrids. We show that after 27 hr of culture of hybrid cells, transcription in the blastomere nuclei was finally silenced. This process occurs without nuclear envelope breakdown and is accompanied by progressive condensation of the chromatin in the blastomere nuclei. We show also the involvement of core histone deacetylation in the modification of chromatin configuration and termination of RNA synthesis in the blastomere nuclei.

Animals↗
PubMed2005

T1, T2 relaxation and magnetization transfer in tissue at 3T.

T1, T2, and magnetization transfer (MT) measurements were performed in vitro at 3 T and 37 degrees C on a variety of tissues: mouse liver, muscle, and heart; rat spinal cord and kidney; bovine optic nerve, cartilage, and white and gray matter; and human blood. The MR parameters were compared to those at 1.5 T. As expected, the T2 relaxation time constants and quantitative MT parameters (MT exchange rate, R, macromolecular pool fraction, M0B, and macromolecular T2 relaxation time, T2B) at 3 T were similar to those at 1.5 T. The T1 relaxation time values, however, for all measured tissues increased significantly with field strength. Consequently, the phenomenological MT parameter, magnetization transfer ratio, MTR, was lower by approximately 2 to 10%. Collectively, these results provide a useful reference for optimization of pulse sequence parameters for MRI at 3 T.

Animals↗
PubMed2005

MR properties of excised neural tissue following experimentally induced demyelination.

Changes in the magnetic resonance (MR) parameters of demyelinated neural tissue were measured in vitro using an experimental animal model. A tellurium (Te) diet was applied to weanling rats to induce the demyelination process in the sciatic nerve. The quantitative MR parameters, such as T(1), T(2) relaxation time constants and magnetization transfer (MT) were measured each day after applying the Te diet (up to 7 days) and were found to be substantially different from those of normal nerves. An increase in the average T(1) and T(2) was observed along with a decrease in the MT ratio (MTR) and the quantitative MT parameter M(0B), which describes the semisolid pool of protons. Most of the MR parameters correlated very well with the myelin fraction of neural tissue evaluated by quantitative histopathology. The T(2) relaxation spectrum provided the most efficient quantitative assessment of changes in neural tissue microstructure and its analysis resulted in a powerful tool to distinguish the processes of demyelination and inflammation. In comparison, the MT measurements were less successful.

Animals↗
PubMed2003

Leydig cell micronodules are a common finding in testicular biopsies from men with impaired spermatogenesis and are associated with decreased testosterone/LH ratio.

To assess the biological significance of Leydig cell 'hyperplasia' in man, Leydig cell distribution, volume, and function were studied in patients with infertility or testicular cancer and in suddenly deceased controls. A total of 156 biopsies from 95 patients and 18 necropsies from 13 controls were examined using a semi-quantitative stereological method. In patients, serum concentrations of testosterone, sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and inhibin-B were correlated with the findings on histological examination. Leydig cell clusters of more than 15 cells in a cross-section, for which we proposed the name 'micronodules', were frequently seen in testicles exhibiting Sertoli-cell-only syndrome (SCO), a mixed pattern of impaired spermatogenesis, or complete spermatogenesis in combination with elevated FSH. Median numbers of micronodules per 1.77 mm(2) (four fields of vision) in these three histological patterns were 6, 4, and 3.5, respectively. In contrast, micronodules were only occasionally observed in testicular biopsies from patients with complete spermatogenesis and normal gonadotrophin levels (median 1), and were rare in testes from controls (median = 0, p = 0.02). The proportion of testicular tissue occupied by Leydig cells increased with decreasing spermatogenic capacity. In contrast, the total volume of Leydig cells per testis was roughly comparable irrespective of the histological pattern, with the exception of testes with bilateral micronodules, which had significantly increased Leydig cell volume compared to those without micronodules. The number of micronodules correlated positively to LH (r = 0.577, p < 0.01) and FSH (r = 0.595, p < 0.01) and the presence of micronodules was most pronounced in the hyperstimulated testes, as reflected by an increased LH/testosterone ratio. In conclusion, Leydig cell micronodules were more frequent in biopsies with impaired spermatogenesis and associated with decreased ratios of testicular hormones to gonadotrophins. The presence of micronodules thus seems to be a histological marker of testicular failure in man.

Adolescent↗