Biomedical subjects

A Ambrus

Publications and source records attributed to A Ambrus.

At least 19 recordsLinked to original sources

PubMed2006

Immunohistochemical evaluation of mx protein expression in canine encephalitides.

Mx proteins are a group of interferon-induced GTPases whose expression has been demonstrated in a number of human viral infections and in some idiopathic inflammatory diseases. In this study, the expression of Mx protein was evaluated in known viral, nonviral, and idiopathic encephalitides in the dog via immunohistochemistry using an antibody against human MxA. All 12 cases of confirmed viral encephalitis, including 7 cases of canine distemper, 4 cases of canine herpesvirus, and 1 case of rabies, were Mx positive. In canine distemper cases, staining was particularly strong and a variety of cell types were positive, including astrocytes, macrophages/microglia, and neurons. Immunoreactivity for Mx protein was evident in a few cases of nonviral infectious encephalitis, including neosporosis (1/1), Chagas disease (2/3), aspergillosis (1/2), and encephalitozoonosis (1/1). Consistent staining was observed in most cases of idiopathic encephalitis, including granulomatous meningoencephalomyelitis (7/7), necrotizing meningoencephalitis of pug dogs (6/7), and necrotizing encephalitis of the Yorkshire Terrier (3/3) and Maltese (1/1) breeds. Mx staining was negative in 5 normal dog brains; 3 cases of cryptococcosis; and single cases of blastomycosis, protothecosis, and bacterial meningitis.

Animals↗

PubMed2005

A cost-effective screening method for pesticide residue analysis in fruits, vegetables, and cereal grains.

This paper reports the results of studies performed to investigate the potential of applying thin layer chromatography (TLC) detection in combination with selected extraction and cleanup methods, for providing an alternative cost-effective analytical procedure for screening and confirmation of pesticide residues in plant commodities. The extraction was carried out with ethyl acetate and an on-line extraction method applying an acetone-dichloromethane mixture. The extracts were cleaned up with SX-3 gel, an adsorbent mixture of active carbon, magnesia, and diatomaceous earth, and on silica micro cartridges. The Rf values of 118 pesticides were tested in eleven elution systems with UV, and eight biotest methods and chemical detection reagents. Cabbage, green peas, orange, and tomatoes were selected as representative sample matrices for fruits and vegetables, while maize, rice, and wheat represented cereal grains. As an internal quality control measure, marker compounds were applied on each plate to verify the proper elution and detection conditions. The Rf values varied in the different elution systems. The best separation (widest Rf range) was achieved with silica gel (SG)--ethyl acetate (0.05-0.7), SG--benzene, (0.02-0.7) and reverse phase RP-18 F-254S layer with acetone: methanol: water/30:30:30 (v/v) (0.1-0.8). The relative standard deviation of Rf values (CV(Rf)) within laboratory reproducibility was generally less than 20%, except below 0.2 Rf, where the CVRf rapidly increased with decreasing Rf values. The fungi spore inhibition, chloroplast inhibition, and enzyme inhibition were found most suitable for detection of pesticides primarily for confirming their identity or screening for known substances. Their use for determination of pesticide residues in samples of unknown origin is not recommended.

Chromatography, Thin Layer↗

PubMed2005

Application of TLC for confirmation and screening of pesticide residues in fruits, vegetables, and cereal grains: Part 2. Repeatability and reproducibility of Rf and MDQ values.

This paper illustrates the effect of major factors influencing the reproducibility of thin layer chromatography (TLC) separation and detection under largely differing environmental and laboratory conditions. The optimum conditions for reproducibility and detection sensitivity was obtained on 20 x 20 cm layer in the retention factor (Rf) range of 0.2-0.7 by applying the sample in spots of 3-4 mm diameter at 2 cm from the edge of the plate. The reproducibility rapidly decreased below Rf = 0.2. Above Rf = 0.2 the within-laboratory reproducibility of 219 pesticides obtained in ethyl acetate silica gel elution system by several laboratories was typically below 10%. The among-laboratories reproducibility of the average retention factors was generally below 12%. The minimum detectable quantities (MDQ) of 219 pesticide residues were determined with nine detection methods. The MDQ values largely varied depending on the mode of detection. Bioassay methods enabled the detection down to 0.1-10 ng, while 20-100 ng could be achieved with the chemical reagents. Higher MDQ values are also reported in order to assist the identification of compounds potentially present. The between-laboratories reproducibility of MDQ values was typically 1-5 MDQmin.

Chromatography, Thin Layer↗

PubMed2004

A heritable keratinization defect of the superficial epidermis in norfolk terriers.

Although well-characterized in man, abnormal cornification secondary to heritable superficial keratin defects is rarely reported in animals. This report describes a mild cornification defect in seven related Norfolk terrier dogs. Lesions were present at birth and pedigree analysis suggested an autosomal recessive mode of inheritance. The affected dogs had hyperpigmented skin with scaling following mild trauma. The lesions were generalized but most prominent in the glabrous skin of the axillary and inguinal regions-areas where the epidermis is not protected by hair and is subject to frequent trauma. The most striking histological change was vacuolation in the upper epidermis, which often resulted in epidermolysis and blister formation. All of the affected dogs showed similar gross and histological changes. Ultrastructural changes included abnormal keratin filament clumping, prominent clear spaces in the cytoplasm of suprabasal keratinocytes, and abnormal keratohyaline granules. Immunohistochemical labelling for keratin 10 demonstrated a lack of expression in the superficial epidermis of affected dogs. All of the morphological changes noted in the Norfolk terriers were consistent with a mild form of a heritable defect in superficial keratin synthesis.

Animals↗

PubMed2001

Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).

This study characterizes the calcium-bound CR I-II domain (residues 1-100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I-II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix-loop-helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I-II) confirms that the linker of CR I-II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)-CR(1-60) and GST-CR(61-100) fusion proteins confirm that both EF-hands of CR I-II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I-II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I-II exists as a monomer. Calb I-II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I-II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933-938]. In contrast, Calb I-II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I-II and Calb I-II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.

Amino Acid Sequence↗

PubMed2001

Calcium binding of transglutaminases: a 43Ca NMR study combined with surface polarity analysis.

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Amino Acid Sequence↗

PubMed2001

Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase.

Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.

Adenosine Triphosphate↗

PubMed2000

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons.

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.

Animals↗

PubMed2000

Within and between field variability of residue data and sampling implications.

Information on the variability of residues in individual fruits and vegetables is required to estimate the acute dietary exposure to pesticides. The distribution of residues in apples, kiwi fruits, potatoes and butter beans was studied in field experiments representing commercial farming practice. No correlation was found between the residue concentration or surface residue and the mass of apples. The relative frequency distributions of residues in crop units were continuous and skew positive. The log-normal transformation did not result in a normal distribution in any of the trials. Consequently, 299, 120 and 59 random samples should be analysed to estimate 99th, 97.5th and 95th percentile of the residues at 95% confidence level. The distribution of residues is not significantly influenced by the mean residue, pre-harvest interval, chemical and physical properties of the active ingredient, formulation of pesticide or, on top fruit, the foliar application method. However, the residue distribution is likely to be influenced by the size, shape and density of the plants, and mode of application of pesticides. The variability factor should be defined as the ratio of the highest value at a specified percentile of residues occurring in unit crops and the population mean. Generic variability factors may be determined for various groups of commodities. Variability factors of 5 and 9 are recommended for medium size fruits, and potato following granular application of pesticides, respectively.

Agriculture↗

PubMed1998

Ontogeny of calretinin expression in rat dorsal root ganglia.

The relationship between the expression of calretinin and the maturation level of peripheral sensory neurons was investigated by means of immunohistochemistry and immunoblot analysis. Our immunohistochemical results show that calretinin is expressed during two different developmental phases in rat dorsal root ganglia. The early phase lasts between embryonic days 11 and 14, when calretinin is detectable in the majority (75%) of the cells. A second phase starts at embryonic day 17 and lasts throughout the whole postnatal life, when calretinin is expressed only in a small proportion of the neurons (less than 8%). Between these two periods no calretinin is found in the ganglia. These changes in calretinin expression during embryonic development were confirmed by Western blot analysis. The early expression of calretinin in dorsal root ganglion cells suggests that calretinin may act as a calcium regulator until neurotrophins take over the precise tuning of intracellular calcium concentration.

Animals↗

PubMed1995

Capsaicin treatment induces selective sensory degeneration and increased sympathetic innervation in the rat ureter.

Quantitative immunohistochemistry was used to study the innervation of the ureter in adult rats pretreated with capsaicin as neonates (50 mg/kg) or as adults (100-150 mg/kg, 10-22 days prior to being killed) using antibodies against protein gene-product 9.5, neuron-specific enolase, substance P, calcitonin gene-related peptide, neuropeptide Y, dopamine-beta-hydroxylase and vasoactive intestinal polypeptide. The number of calcitonin gene-related peptide- and substance P-containing fibres was reduced in the subepithelial plexus (adult capsaicin treatment < 1%, neonatal treatment < 5% of control), the submucosa (adult treatment < 11%; neonatal treatment < 51%) and in the smooth muscle layer and adventitia (adult treatment < 11%; neonatal treatment < 58%). Fibres immunoreactive for protein gene-product 9.5, a general neuronal marker, were reduced to 11% (adult treatment) or 0.5% (neonatal treatment) in the subepithelial plexus, but unchanged in the other layers, indicating a selective regional degeneration. In the smooth muscle layer the number of neuropeptide Y- and vasoactive intestinal polypeptide-containing nerve fibres was not altered by capsaicin. The number of neuropeptide Y fibres in the subepithelial plexus, however, was significantly increased after adult treatment (174% of control). After neonatal capsaicin treatment the intensity of the neuropeptide Y immunoreactivity was increased, more neuropeptide Y-positive nerve bundles were found and immunoreactive cell bodies were observed regularly in the adventitia of the ureter. The data indicate that capsaicin produces a selective degeneration of most afferent fibres in the subepithelial plexus of the rat ureter. This loss of capsaicin-sensitive afferent nerves evokes neuroplastic changes resulting in a hyperinnervation by neuropeptide Y-immunoreactive, presumably sympathetic fibres. The results suggest a mutual regulation of the pattern and density of innervation of peripheral target tissues by sensory and sympathetic neurons.

Animals↗

PubMed1993

Classification of drugs based on visually evoked responses in rats.

Quantified parameters of photically evoked afterdischarge and EEG background activity of visual cortex of freely moving rats were used as variables to obtain a t profile for each drug dosage. Behavioural scores were also used. The determination of the dose/time-effect profile of a compound was based on normalization of data and on calculation of the first two factor values in comparison with pooled data of representatives of neuroleptic, anxiolytic, convulsant, petit mal anticonvulsant, antidepressant and psychostimulant drug classes. The compounds were investigated in different dosages and/or routes of administration. The model and procedure are validated for antipsychotic, anxiolytic and vigilance enhancer effect, and comprise the predictability of proconvulsive and petit mal anticonvulsive effects.

Animals↗

PubMed1993

Appearance of diaminobenzidine-staining glial cells in the rat CNS: an ontogenetic histochemical study.

The postnatal appearance of diaminobenzidine (DAB)-staining glial cells was studied in certain regions of the rat central nervous system. DAB-positive cells appeared in the hypothalamus, the nucleus of the solitary tract and the most superficial laminae of the spinal dorsal horn on the 7th, 8th and 14th postnatal day, respectively. The number and staining intensity of these DAB-positive cells progressively increased and reached the adult levels at the end of the third postnatal week. Glial staining is likely to result from a non-enzymatic oxidation of DAB, since it was resistant to heat pretreatment, but was abolished if hydrogen peroxide was omitted from the incubation medium. The present results reveal that a particular population of glial cells exhibit a characteristic developmental change which can be detected by means of peroxidase histochemistry. The close topographical relationship is suggestive of a functional association between capsaicin-sensitive primary afferents and DAB-positive glial cells.

Animals↗

PubMed1988

Effects of thyrotropin-releasing hormone and its analogue L-6-ketopiperidine-2-carbonyl-leucyl-L-prolin-amide on behaviour and electroencephalogram.

A comparative dose-response investigation of thyrotropin-releasing hormone (TRH) and L-6-ketopiperidine-2-carbonyl-leucyl-L-proline-amide (RGH-2202) was carried out on rats and cats. RGH-2202 reduced the occurrence of photically evoked after-discharge in rats more significantly than TRH. Neither of the two compounds influenced the parameters of visually evoked potentials. Both compounds desynchronized the background electrocortical activity in rats. The amplitude of the acoustic startle reflex in rats was decreased dose-dependently only by RGH-2202, and this effect was nearly 3 times more potent than that of TRH. Both compounds increased the time spent awake, the latency to light sleep and the proportion of light sleep in a dose-dependent manner. The higher doses of TRH equalled the effects of the lower doses of RGH-2202. In cats the latency increasing effect of TRH on paradoxical sleep was about 30% less than that of RGH-2202; furthermore, the relative increase in the proportion of light sleep coupled with a corresponding decrease of deep sleep and paradoxical sleep was significant only in the case of RGH-2202. In a complex conditioned reflex situation in cats, the dose-dependent motivation decreasing effect on food intake and, as a consequence, on spontaneous motor activity was more pronounced in the case of TRH. The effective doses of RGH-2202 and TRH in rats were nearly equal, while in cats RGH-2202 showed about 1/10 of the potency of TRH. This finding suggests a considerably lower species variability of the effects of RGH-2202.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988