Brain metabolic activity associated with long-term memory consolidation

The use of day-old chickens trained on a single-trial passive avoidance task provides a useful paradigm for investigations into cellular mechanisms underlying memory formation. Pharmacological intervention studies indicate that there are three temporally identifiable stages of memory processing leading to the consolidation of information for this task. These stages, designated as short-term (STM; up to 15 min), intermediate-term (ITM; 15-55 min), and long-term (LTM; more than 55 min) memory, have been found to be sequentially dependent (Ng and Gibbs, 1989). In addition, ITM appears to consist of two physiologically distinguishable phases, A and B. Evidence in this laboratory suggests that the crossover between these ITM phases (at 30 min after training) represents a critical time-point for the triggering of LTM.     

Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity.

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Genetic mapping of the gene for androgen-binding protein/sex hormone-binding globulin to mouse chromosome 11

Southern blot analysis of DNAs from Chinese hamster x mouse and rat x mouse somatic cell hybrids showed that the mouse gene encoding androgen-binding protein/sex hormone-binding globulin (ABP-SHBG) is on Chromosome 11. Progeny from an intersubspecies backcross were analyzed to position this locus, termed Shbg, between Il-3 and Int-4 in the middle of this chromosome. Shbg is thus closely linked to several neurological mutations, one of which, Tr, is also associated with male sterility. The recent finding that ABP-SHBG is found throughout the rat brain raises the possibility that one of these mutations may be due to a defect in Shbg.     

Control of tsetse and trypanosomiasis transmission in Uganda by applications of lambda-cyhalothrin

The pyrethroid insecticide lambda-cyhalothrin was evaluated in field trials against Glossina f.fuscipes and sleeping sickness transmission in Iyolwa sub-county, Tororo District, Uganda. The insecticide was applied selectively to the resting-sites of tsetse, by bush-spraying, using 10% wettable powder (10WP) formulation at an application rate of 11.6 g a.i./ha over an area of 28 km2, or by a 2% Electrodyn formulation (2ED) applied at 0.9 g a.i./ha over 30 km2. In a third trial area of 32 km2, 215 pyramidal traps treated with lambda-cyhalothrin 100 mg/m2 were set. The best impact was obtained with 10WP lambda-cyhalothrin which eliminated tsetse within 1-2 months, whereas G.f.fuscipes persisted at very low density in part of the area treated with 2ED lambda-cyhalothrin. In both treated areas the numbers of human sleeping sickness cases fell to no more than one per month, compared with four to twelve per month previously. The overall rate of cattle trypanosomiasis (T.brucei and T.vivax) was also reduced slightly. Insecticide-treated traps remained fully effective for at least 6 months under field conditions and catches were reduced 20-90-fold. These results in the control of tsetse and trypanosomiasis transmission lead us to recommend lambda-cyhalothrin for tsetse control operations.     

An overview of homologous pairing and DNA strand exchange proteins.

Processes fundamental to all models of genetic recombination include the homologous pairing and subsequent exchange of DNA strands. Biochemical analysis of these events has been conducted primarily on the recA protein of Escherichia coli, although proteins which can promote such reactions have been purified from many sources, both prokaryotic and eukaryotic. The activities of these homologous pairing and DNA strand exchange proteins are either ATP-dependent, as predicted based on the recA protein paradigm, or, more unexpectedly, ATP-independent. This review examines the reactions promoted by both classes of proteins and highlights their similarities and differences. The mechanistic implications of the apparent existence of 2 classes of strand exchange protein are discussed.     

Syntheses, structures and anorectic effects of human and rat amylin.

Amylin, a 37-residue polypeptide with a single disulfide bond originally isolated from the pancreas of type-II diabetic patients, has been shown to cause peripheral insulin resistance and to attenuate the inhibition of hepatic glucose output by insulin. We have also shown that amylin is present in the rat hypothalamus and that it inhibits food intake by rats. In order to further investigate the anorectic properties we synthesized both human and rat amylin by the solid phase method and purified to homogeneity in an overall yield of 10-20%. Structural analyses indicated that human amylin exhibited predominantly a beta-sheet structure at both acidic and alkaline pH, whereas no ordered structure was evident in the case of rat amylin. Intrahypothalamic injection of rat amylin resulted in a potent dose-dependent inhibitory effect on the food intake by rats adapted to eat their daily ration of food in an eight-hour period. Human amylin was less effective as an anorectic agent. Furthermore, rat amylin completely blocked the potent orexigenic effect of neuropeptide Y (NPY). These investigations show that there is a fundamental difference in the secondary structures of human and rat amylin and that rat amylin is a potent inhibitor of both basal and NPY-induced feeding by rats.     

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.