Application of discriminant analysis and quantitative cytologic examination to gastric lesions

OBJECTIVE: To investigate of the potential value of morphometry and discriminant analysis for the classification of benign and malignant gastric cells and lesions. STUDY DESIGN: The data set consisted of 13,300 cells from 120 cases composed of 30 cases of cancer, 26 cases of gastritis and 64 cases of ulcer according to the final histologic diagnosis. The cytologic diagnosis was divided into 5 categories (gastritis, ulcer, inflammatory dysplasia, cancer and true dysplasia). Classification was attempted at 2 levels: the cell level to classify individual cells and the case level to classify individual cases. For the cellular classification the measured cells from 50% of available cases were selected as a training set to construct a model. The cells from the remaining cases were used as a test set to validate the model. Similarly for case classification, the same 50% of cases that were used for cell classification were used as a training set and the remaining cases as a test set. Images of routinely processed gastric smears stained by the Papanicolaou technique were analyzed by a customized image analysis system. RESULTS: Application of discriminant analysis on the test set gave correct classification of 98.4% of benign cells and 67.1% of malignant cells. On case classification, 100% accuracy was achieved for benign and malignant cases, both for the training and test sets. CONCLUSION: The application of discriminant analysis described in this paper could produce significant classification results at the cellular and individual case level.     

Regulation of dihydropyridine receptor gene expression in mouse skeletal muscles by stretch and disuse

This study examined dihydropyridine receptor (DHPR) gene expression in mouse skeletal muscles during physiological adaptations to disuse. Disuse was produced by three in vivo models—denervation, tenotomy, and immobilization—and DHPR _1s mRNA was measured by quantitative Northern blot. After 14-day simultaneous denervation of the soleus (Sol), tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius (Gastr) muscles by sciatic nerve section, DHPR mRNA increased preferentially in the Sol and TA (+1.6-fold), whereas it increased in the EDL (+1.6-fold) and TA (+1.8-fold) after selective denervation of these muscles by peroneal nerve section. It declined in all muscles (–1.3- to –2.6-fold) after 14-day tenotomy, which preserves nerve input but removes mechanical tension. Atrophy was comparable in denervated and tenotomized muscles. These results suggest that factor(s) in addition to inactivity per se, muscle phenotype, or associated atrophy can regulate DHPR gene expression. To test the contribution of passive tension to this regulation, we subjected the same muscles to disuse by limb immobilization in a maximally dorsiflexed position. DHPR _1s mRNA increased in the stretched muscles (Sol, +2.3-fold; Gastr, +1.5-fold) and decreased in the shortened muscles (TA, –1.4-fold; EDL, –1.3-fold). The effect of stretch was confirmed in vitro. DHPR protein did not change significantly after 4-day immobilization, suggesting that additional levels of regulation may exist. These results demonstrate that DHPR _1s gene expression is regulated as an integral part of the adaptive response of skeletal muscles to disuse in both slow- and fast-twitch muscles and identify passive tension as an important signal for its regulation in vivo. dihydropyridine receptor mRNA; decreased use; passive tension; denervation; tenotomy; hindlimb immobilization     

Dimethyl Sulfoxide and Ebselen Prevent Convulsions Induced by 5-Aminolevulinic Acid

We investigated whether intrastriatal (i.s.) administration of 5-aminolevulinic acid (ALA) induces oxidative damage and whether behavioral alterations induced by i.s. administration of ALA could be affected by antioxidants. Unilateral injection of ALA (6 micromol/striatum) increased (approximately 30%) thiobarbituric acid-reactive substances (TBARS), but did not affect striatal content of total thiol groups. ALA-induced body asymmetry was not prevented by pretreatment with ascorbic acid (100 mg/kg, s.c.), dimethyl sulfoxide (DMSO, 0.5 microl/striatum, i.s.) or ebselen (10 nmol/striatum, i.s.). ALA-induced convulsions were not prevented by ascorbic acid, but were partially prevented by DMSO and completely prevented by ebselen. Ebselen completely prevented the increase of striatal TBARS induced by ALA. Results obtained suggest the involvement of reactive species in ALA-induced convulsions and may be of value in understanding the physiopathology of neurological dysfunctions associated to ALA overload.     

Immunostimulating activity of polycomponent vaccine "Immunovac-VP-4" and grippol after their combined administration

The experimental study of the immunostimulating activity of therapeutic bacterial polycomponent vaccine VP-4 and prophylactic vaccine grippol, introduced both separately and in combination, on mice infected with Salmonella typhimurium, used as a model. Both preparations were found to produce an immunomodulating effect. The combined subcutaneous injection of VP-4 and grippol did not decrease their immunostimulating activity, but their separate administration at an interval of 14 days resulted in essential decrease in the protective activity of each of these two preparations. As shown on the model of Klebsiella infection in mice, challenged 4 weeks after immunization, VP-4 ensured the survival of 78.6% of mice, while after the injection of grippol their survival rate was not different from that of the group of intact animals. The evaluation of the immunostimulating activity of these preparations under the conditions of the prophylaxis of influenza and acute respiratory infections in organized groups of children revealed that the use of VP-4 alone or grippol in combination with VP-4 considerably decreased the number of secondary bacterial complications in children.     

Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.     

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete <Emphasis Type="Italic">Pseudonocardia thermophila</Emphasis>

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg^–1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K_m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Main structural and stereochemical aspects of the antiherpetic activity of nonahydroxyterphenoyl-containing C-glycosidic ellagitannins

Antiherpetic evaluation of five nonahydroxyterphenoyl-containing C-glycosidic ellagitannins, castalagin (1), vescalagin (2), grandinin (3), roburin B (5), and roburin D (7), was performed in cultured cells against four HSV-1 and HSV-2 strains, two of which were resistant to Acyclovir. All five ellagitannins displayed significant anti-HSV activities against the Acyclovir-resistant mutants, but the monomeric structures 1-3 were more active than the dimers 5 and 7. Vescalagin (2) stands out among the five congeners tested as the most potent and selective inhibitor, with an IC50 value in the subfemtomolar range and a selectivity index 5x10(5) times higher than that of Acyclovir. Molecular modeling was used to provide a rationale for the surprisingly lower activity profile of its epimer castalagin (1). These ellagitannins have promising potential as novel inhibitors in the search for non-nucleoside drugs active against Acyclovir-resistant herpes viruses.     

Molecular characterization of the <Emphasis Type="Italic">singed wings</Emphasis> locus of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes.     

Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid

Several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. The new heterocycles were evaluated for their activity in vitro against several RNA and DNA viruses.