β-Ar-ACETYL D-GALACTOSAMINIDASE IN BULK SEPARATED NEURONS AND NEUROPIL FROM RAT CEREBRAL CORTEX

Abstract— β- N -Acetyl D-galactosaminidase was studied in isolated neuronal and neuropil fractions from cerebral cortex and subcellular fractions derived from them. Although the enzyme activity evinced some latency properties, its subcellular distribution pattern was broader than that observed with other acid hydrolases. By contrast with nine other acid hydrolases, it was more active in neuropil than neuronal fractions (neuronal/neuropil activity ratio 0.63). This ratio was preserved in lysosomal subfractions derived from the isolated cell fractions. The data is taken as further evidence for the microheterogeneity of lysosomal particles from the brain.     

An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Development of Cryptolestes ferrugineus (Stephens) and Oryzaephilus mercator (Fauvel.) on seed-borne fungi

Two stored-product insects were reared on 23 species of seed-borne fungi and 1 species of actinomycete at 33°C and 75% R.H. Cryptolestes ferrugineus (Stephens) completed development on 10 species of fungi; the shortest development period, 22 days, was on Trichothecium roseum Lk. and the longest, 34 days, on Fusarium moniliforme Sheld. Oryzaephilus mercator (Fauvel) completed development on 18 species of fungi; the shortest developmental period, 21 days, was on Mucor sphaerosporus Hagem, and the longest, 40 days, on Chaetomium funicola Cooke. None developed on Streptomyces griseus (Krainsky) Waksman & Henrici.

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Zusammenfassung Zwei Vorratsschädlinge wurden bei 33° und 75% rel. Luftfeuchtigkeit auf 23 Pilzarten und einer Actinomycetenart aufgezogen, die sich auf Samen entwickeln. Cryptolestes ferrugineus vollendete seine Entwicklung auf 10 Pilzarten in 22 bis 34 Tagen; die kürzeste Entwicklungsdauer, 22 Tage, zeigte sich auf Trichothecium roseum Lk. und die längste, 34 Tage, auf Fusarium moniliforme Sheld. Oryzaephilus mercator entwickelte sich auf 18 Pilzarten in 21 bis 40 Tagen. Die kürzeste Entwicklungsperiode ergab sich auf Mucor sphaerosporus Hagem., die längste auf Chaetomium funicola Cooke. Keine Entwicklung trat auf Streptomyces griseus (Krainsky) Waksman & Henrici ein.

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Contribution No. 154.     

Creteuchiloglanis arunachalensis,a new species of glyptosternine catfish (Teleostei: Sisoridae) from Arunachal Pradesh,northeastern India

Ichthyological Research - A new glyptosternine catfish, Creteuchiloglanis arunachalensis is described on the basis of a single specimen collected from the upper Brahmaputra River drainage,...     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Calcium effects on calmodulin lysine reactivities

The differential reactivities of individual lysines on porcine testicular calmodulin were determined by trace labeling with high specific activity [3H]acetic anhydride as a function of the molar ratio of Ca2+ to calmodulin. In progressing from the Ca2+-depleted form of the protein to a Ca2+:calmodulin molar ratio of 5:1, six of the seven lysyl residues exhibited a modest 1.5- to 3.0-fold increase in reactivity. Lys 75, in contrast, was enhanced in reactivity greater than 20-fold. When the change in reactivity of each lysine was normalized as a percentage of the maximum change, most of the residues were found to fall into two distinct classes. One class, comprising lysines 94 and 148 from the two carboxy terminal Ca2+-binding domains 3 and 4, respectively, exhibited about 90% of their reactivity change when the Ca2+:calmodulin molar ratio was 2:1, and these residues were perturbed very little upon further addition of Ca2+. The other class, encompassing lysines 13, 21, and 30 from the amino terminal domain 1 and Lys 75 from the extended helix connecting the two globular lobes of calmodulin, underwent most of their overall reactivity change (55-70%) between 2 and 5 equivalents of Ca2+ per mol of calmodulin. Lys 77 was distinct in its pattern of change, undergoing approximately equal changes with each Ca2+ increment. These results are consistent with a model where Ca2+ first binds to the two carboxy terminal sites of calmodulin with no apparent preference, concomitant with minor alterations in the microenvironments of lysines in the unoccupied amino terminal domains. The third and fourth Ca2+ ions then bind to these latter two domains, again with no evidence of preference, with little change in the lysine reactivities at the carboxy terminus of the molecule. The environments of groups in the central helix appear to undergo changes in a manner that reflects their proximity to the amino and carboxy terminal domains. In the course of this work, it was found that Lys 94 in apocalmodulin is specifically perturbed by the addition of EGTA, suggesting that the chelating agent may interact with calmodulin at or near the third Ca2+-binding domain.     

Activation of hydrazine derivatives to free radicals in the perfused rat liver: a spin-trapping study

Spin-trapping techniques and electron spin resonance spectroscopy have been used to study bioactivations of hydrazine and its derivatives by isolated perfused rat livers. Using phenylbutylnitrone (PBN) as the stable spin trap, it was found that the liver perfusion of hydrazine, acetylhydrazine and isoniazid resulted in the formation of the same carbon-centered radical which was shown to be the acetyl radical. The identity of the acetyl radical was confirmed after organic extraction of the liver perfusates, by comparing its coupling constants with those of the in vitro metal ion- or horseradish peroxidase-catalyzed oxidation products of the hydrazines in the same solvents. The liver perfusion of iproniazid formed the isopropyl radical which was previously observed to result from peroxidase-catalyzed oxidation.     

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.