D-2 dopamine receptors in the frontal cortex of rat and human

D-2 dopamine receptors and serotonin receptors in the frontal cortex of rat and human were labelled with 3H-spiroperidol. The D-2 receptors were then distinguished in 4 ways. Dissociation of spiroperidol was biphasic, indicating two populations of sites. Cinanserin in competition with 3H-spiroperidol exhibited high (75%) and low (25%) affinity sites. Dopamine and LY 141865 in competition with 1.25 nM 3H-spiroperidol exhibited high (20-25%) and low (80-75%) affinity sites in the absence of cinanserin, while in the presence of 300 nM cinanserin only the high affinity sites remained. Lesioning of the dopaminergic meso-cortical pathway increased the number of cinanserin-resistant sites by 26%. Thus 3H-spiroperidol binding in the presence of cinanserin can be used to selectively label D-2 receptors in the frontal cortex.     

Characterization of beta-lactamase from Mycobacterium butyricum ATCC 19979

beta-lactamase has been purified to a homogeneous state from Mycobacterium butyricum ATCC 19979. The molecular weight (Mr = 29,000) and the isoelectric point (4,0) of the enzyme have been determined. The enzyme showed both penicillinase and cephalosporinase activity, but had relatively more of the former. With respect to substrate-profile the enzyme resembled the plasmid specified TEM-type beta-lactamases commonly encountered in Gram-negative bacteria. The enzyme was insensitive to p-chloromercuribenzoate, sodium chloride, or iodine inhibition.     

Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.     

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Influence of intertree distance on foraging behaviour of Rhagoletis pomonella in the field

Abstract. 1. We tested a prediction from contemporary foraging theory that animals should decrease their allocation of energy to the searching of individual patches when interpatch travel costs decrease.
2. We used individual Rhagoletis pomonella Walsh (Diptera: Tephritidae) females foraging for oviposition sites (= Crataegus fruit) in a host tree which was surrounded by four other trees at varying distances.
3. We found that flies generally invested less search, measured as time spent searching a tree or number of leaves visited on a tree, when neighbouring trees were nearby than when farther away.
4. Under our test conditions, flies appeared to have difficulty locating neighbouring trees at a distance of more than 1.6 m.
5. Our study calls into question the interpretation of search effort by insects within resource patches in the absence of information on interpatch distances.     

Degradation of sugar cane bagasse by several white-rot fungi

Forty-two white-rot fungi isolated in South America were incubated with long fibre sugar cane bagasse (LFB). The residual composition of LFB was determined after white-rot decay at 30 and 60 days. The ratio of residual lignin to residual lignin to residual cellulose (RL/RC) of untreated material (LFB) was 0.48. After white-rot-decay, the residual material with lower RL/RC ratios indicated that mainly lignin was degraded. In only 30 days, Phlebia sp. MVHC 5535, Athelia sp. MVHC 5509 and Spongipellis pachyodon MVHC 5019 caused a decrease in the RL/RC ratio to 0.36, 0.37 and 0.38, respectively, while it took 60 days for Ganoderma applanatum MVHC 5347, Hyphodontia sp. MVHC 5544, Panus tigrinus MVHC 5400, Stereum sp. MVHC 5113, Phellinus punctatus MVHC 5346 and MVHC 6388 to reach a ratio lower than 0.40. No correlation was found between the amount of some ligninolytic enzymes secreted and the residual composition of bagasse after white-rot fungi fermentation. Most of the fungal strains caused an increase in the relative amount of residual cellulose, indicating that hemicellulose was the preferred energy source.