Normalization of the adaptive avoidance reaction in rats using substances with nootropic activity

The system of double coaxial cylinders filled with water was used as a device for studying simple extrapolation behaviour of rats. The amount of rats which were able to dive under the lower edge of the inner cylinder, without reaching the bottom of the outer cylinder and the latency of this avoidance reaction were considered as a measure of extrapolation ability. This reaction was altered by the pretreatment of animals with cycloheximide, a protein synthesis inhibitor. Piracetam as a standard nootropic, sodium and lithium hydroxybutyrate as substances with a potential nootropic effect were shown to be able to antagonize the damaging effect of cycloheximide on the avoidance performance. Benzodiazepine tranquilizer, phenazepam, in contrast to nootropics, evokes additional worsening of extrapolation reaction. Normalization of avoidance disturbed by cycloheximide, can be used as an adequate and informative approach for screening of nootropics.     

Detection of apical Na(+)/H(+) exchanger activity inhibition in proximal tubules induced by acute hypertension

We previously showed that acute arterial hypertension induces an inhibition of fluid and NaCl reabsorption in proximal tubules of Sprague-Dawley rats, which is associated with a rapid reversible internalization of apical Na(+)/H(+) exchanger in brush border. To determine whether there is a corresponding inhibition of apical Na(+)/H(+) exchanger activity in proximal tubules to account for the reduced tubular reabsorption, an instrument capable of measuring intracellular pH (pH(i)) ratiometrically and repeatedly on the surface of kidney with high temporal resolution is required. We report the design and validation of such a fluorimetric system based on two ultraviolet nitrogen-pulsed lasers and a photomultiplier. pH(i) of proximal tubules in situ was measured with pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein at 5 Hz. Using the initial rate of change of pH(i) (dpH(i)/dt) during luminal Na(+) removal as an index of apical Na(+)/H(+) exchanger activity, the exchanger activity was found to be reduced by 52 +/- 11% (n = 14, P < 0.05) compared with the baseline after 20 min of induced acute hypertension. The inhibition of Na(+)/H(+) exchange activity was alleviated when the blood pressure was returned to prehypertensive level. These observations indicate that acute changes in arterial pressure can reversibly inhibit apical Na(+)/H(+) exchanger activity, which might contribute to pressure natriuresis in proximal tubule.     

Renin inhibition activity by chitooligosaccharides

Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity.     

Structural asymmetry of the F1 of Escherichia coli as indicated by reaction with dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) inhibits the ATPase activity of F1 from Escherichia coli by covalent modification of a single glutamic acid in the beta subunit. 95% inhibition was obtained after incorporation of around 1 mole of DCCD per mole F1, i.e. 1 mole of reagent per 3 beta subunits; and up to 2 moles of DCCD per mole F1 were readily incorporated into the protein. One of the 3 beta subunits per F1 can be crosslinked to the epsilon subunit by 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC). This beta subunit (beta 1) is here shown to be shielded from reaction with DCCD, presumably by its association with epsilon and also possibly the gamma subunit. Thus the three beta subunits are not equivalent in the enzyme complex.     

Antihyperglycemic, antistress and nootropic activity of roots of Rubia cordifolia Linn

Effect of alcoholic extract of roots of Rubia cordifolia was studied on elevated blood glucose level in alloxan treated animals. The extract reduced the blood sugar level raised by alloxan. Effect of alcoholic extract was also investigated on cold restraint induced stress and on scopolamine-induced memory impairment. Alcoholic extract enhanced brain gamma-amino-n-butyric acid (GABA) levels and decreased brain dopamine and plasma corticosterone levels. Acidity and ulcers caused due to cold restraint stress were inhibited by alcoholic extract. Animals treated with alcoholic extract spent more time in open arm in elevated plus maze model. It also antagonized scopolamine induced learning and memory impairment. Baclofen induced catatonia was potentiated by alcoholic extract.     

Glutamic-dehydrogenase activity inhibition by phenoxyacilic acids

Summary Experiments were carried out on the effects of some phenoxyacilic acids, used as herbicides, on the glutamic-dehydrogenase (GLDH) activity. 2,4-D,2,4,5-T, MCPA and Dichlorprop inhibited GLDH activity in a competitive way. Phenoxyacilic acids were ranked in the following order of affinity for the enzyme on the basis of the values of the inhibition constants: 2,4,5-T>2,4-D>MCPA>Dichlorprop; on the basis of chemical structure, it was established that the carboxyl dissociation degree is instrumental in linking the herbicide with the active site of the enzyme.     

Potent inhibition of macrophomate synthase by reaction intermediate analogs

Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I50 1.3-8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I50 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (-)-Enantiomers of the diacids showed 30 times stronger activity (I50 0.34, 0.41 mM) than (+)-ones. The I50/Km values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Regulation of enzyme activity by enzyme orientation: A hypothesis

In addition to substrate binding sites, many enzymes must possess supersubstrate binding sites that regulate attachment and orientation of the enzyme toward the matrix (micelle, membrane) in which the substrate molecules are embedded, the supersubstrate.     

Synthesis and inhibitory activity of acyl-peptidyl-prolinalderivatives toward post-proline cleaving enzyme as nootropic agents

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC50 values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at dose of 10-1000 micrograms/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.     

Ifosfamide induces acute renal failure via inhibition of the thioredoxin reductase activity

The present study investigated the impact of ifosfamide (IFO) on renal thioredoxin reductase (TrxR) activity. In mice treated with IFO for 6 h, TrxR activity significantly decreased in a dose-dependent manner. Subsequently, acute renal failure (ARF) occurred dose-dependently. Like IFO, the well-established TrxR-specific inhibitor auranofin suppresfssed renal TrxR activity and generated ARF too. TrxR was inactivated by IFO preferentially over other antioxidant parameters at 6 h; however, it recovered nearly to normal levels within 12 h. When auranofin was administered at 6 h after IFO treatment, the recovery at 12 h was sharply attenuated. Consequently, ARF was pronouncedly exacerbated. IFO within its maximum tolerated dose did not considerably deplete renal glutathione. However, escalating IFO dose strikingly attacked both the thioredoxin and the glutathione systems, resulting in lethality, which implies that glutathione depletion sensitizes IFO-induced nephrotoxicity and cosuppression of both systems causes more severe toxicological consequences than suppressing the thioredoxin system alone. Indeed, combining IFO with buthionine sulfoximine, an inhibitor of glutathione synthesis, induced much more severe ARF than IFO alone did. Taken together, inhibition of renal TrxR activity can be considered as a pivotal mechanism of IFO-induced ARF, and individuals with lower levels of renal glutathione are at high risk of incurring ARF after IFO treatment.     

Use of the indirect hemagglutination inhibition reaction for evaluating the specific activity of allergens

To evaluate the specific activity of house-dust allergen, the passive hemagglutination inhibition test was used. Nine commercial batches of house-dust allergen were studied by means of this test. The results of the determination of the activity of house-dust allergen in the passive hemagglutination inhibition test, the skin tests and the indirect mast-cell degranulation test were in good correlation.     

Distribution of liver plasma membrane 5' nucleotidase as indicated by its reaction with anti-plasma membrane serum

Antiserum against mouse liver plasma membranes was used to investigate the properties and distribution of the surface membrane enzyme 5′ nucleotidase.The antiserum inhibited 5′ nucleotidase but had no effect on alkaline phosphodiesterase, nucleotide pyrophosphatase, or insulin-binding activity.5′ Nucleotidase was purified from mouse liver plasma membranes and the purified enzyme was shown to be inhibited by the antiserum. The membrane-bound and the purified enzyme were both inhibited in a noncompetitive manner.The reaction of the antiserum with 5′ nucleotidase activity of mouse liver plasma membrane “light” and “heavy” subfractions, and of rat liver and pig lymphocyte surface-membrane fractions was investigated. In each case the enzyme was inhibited by the antiserum.Since a protein must be partially exposed on the membrane surface in order to react with its antibody, the results are discussed in terms of the disposition of 5′ nucleotidase within the membrane.