Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-hemeoctapeptide conjugates.

Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c. 8-MP, which has only one reactive amine, was coupled to antibody in a two-step procedure using a bifunctional active ester, bis-succinyl succinate. In the first-step, 8-MP-succinyl succinate, a stable compound which can be stored. In a second step, the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture.     

The fine structure of the digestive system of Daphnia pulex (Crustacea: Cladocera).

The alimentary canal of Daphnia pulex consists of a tube-shaped foregut, a midgut (mesenteron) with an anterior pair of small diverticula, and a short hindgut. The foregut and hindgut are structurally similar. Each is formed by a low cuboidal epithelium 5 mum tall and lined with a chitinous intima. The midgut wall consists of a simple epithelium resting on a thick beaded basal lamina which is surrounded by a spiraling muscularis. Anteriorly the midgut cells are columnar in shape being 30 mum in height each having a basal nucleus, anteriorly concentrated mitochondria and in apical border of long thin microvilli. Posteriorly the midgut cells become progressively shorter so that in the posteriormost region of the midgut the cells are 5 mum tall and cuboidal in shape. The microvilli concomitantly become shorter and thicker. All mesenteron cells contain the usual cytoplasmic organelles. The paired digestive diverticula are simple evaginations of the midgut. The wall of each consists of a simple epithelium of cuboidal cells 25 mum in height, each with a brushed border of long thin microvilli. Enzyme secretion appears to be holocrine in mode and not confined to any one region of the mesenteron though definitely polarized anteriorly. The thin gut muscularis encircles the entire length of the midgut and caeca. Thick and thin filaments appear to be in a 6:1 ratio.     

Nephrogenic diabetes insipidus in an Australian aboriginal kindred.

Four Australian aboriginal children were found to have nephrogenic diabetes insipidus. They are the sons of 3 sisters who were shown to have a urinary concentrating defect uncorrected by vasopressin. An extensive genealogy did not reveal any caucasian genetic influence suggesting that a new genetic mutation was responsible.     

Levels and modification of methionyl-transfer RNA in preimplantation rabbit embryos.

Transfer RNA with l-methionine acceptor activity was extracted from preimplantation rabbit embryos and purified on reverse-phase-3 columns. The molar quantity of methionine acylated to RNA increases as embryo development proceeds from the 16-cell stage to the 80,000 cell blastocyst stage. However, the quantity of methionyl-tRNA per genome declines 100-fold as the embryo cell number increases. Formylation of methionyl-tRNA illustrated that approximately one-third of tRNA^Met extracted was tRNA_f^Met. Methylation of purified methionyl-tRNA by an adult rabbit liver methylase extract illustrated that two-day preimplantation embryo tRNA is highly hypomethylated relative to tRNA from later stages of development. The hypomethylated methionyl-tRNA was also less effective in ribosome binding studies than more fully methylated methionyl-tRNA present in the later stages of embryo development.     

Biochemical studies of mammalian oogenesis: possible existence of a ribosomal and poly(A)-containing RNA-protein supramolecular complex in mouse oocytes

Mouse follicles were labeled with [3H]uridine and then cultured in vitro for 3 days. When oocytes were disrupted, about 40% of the total radiolabeled RNA could be sedimented at 9,000g. Fractionation of this RNA on poly(U)-Sepharose revealed that about 30% and 60% of the total amount of radiolabeled poly(A)- and poly(A)+ RNA, respectively, were in the pellet fraction. Treatments that disrupt protein structure reduced the amount of 9,000g sedimentable RNA and affected to the same extent the distribution of Poly(A)- and poly(A)+ RNA in the pellet and supernatant fractions. CsCl centrifugation of formaldehyde-fixed pellets revealed that virtually all of the radiolabeled RNA had a density significantly lower than that of ribosomes. The sedimentable RNA appeared not to be polysomal, membrane bound or associated wih a cytoskeleton. Agarose gel electrophoresis after poly(U)-Sepharose fractionation of either the pellet or supernatant revealed the presence of 28S, 18S, 5S + 4S, and heterodisperse poly(A)+ RNA. The size of distribution of poly(A)+ RNA in the pellet and supernatant fractions was fairly similar. Pulse-chase experiments revealed that the stability of poly(A)- RNA in the pellet and supernatant fractions was the same within the experimental error and a similar situation was found for poly(A)+ RNA. RNA in pellet translated in vitro coded for discrete size classes of protein. Since the relative band intensities were similar for both total and pellet RNA translated in vitro there seemed to be no major partitioning of specific size classes of mRNA into the pellet fraction. These results are discussed in terms of a possible composition of the lattice structures that accumulate during mouse oocyte growth and have been postulated to be a storage form for ribosome (Burkholder et al., '71).     

Acute and chronic phosphate depletion as a modulator of glucose uptake in rat skeletal muscle.

The effect of acute and chronic hypophosphatemia on rat hindlimb skeletal muscle glucose uptake was examined. Acute hypophosphatemia had no effect on glucose uptake whereas chronic hypophosphatemia had a direct linear effect on glucose uptake.     

Lactate dehydrogenase in preimplantation rabbit embryos

Unfertilized eggs and early embryos up to the 2-day (16-cell) cleavage stage of development in the rabbit contain predominantly the most cathodal lactate dehydrogenase isoenzyme made up of A-type subunits. Following early cleavage there is a progressing increase in total LDH activity in the embryo as development proceeds through 4- and 6-day blastocyst stages. This is accompanied by an increase in the amount of B-type subunits and a concomitant shift in the lactate dehydrogenase isoenzyme electrophoretic pattern toward the anodal isoenzyme types.