Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Demonstration of the heterozygous state for I-cell disease and pseudo-Hurler polydystrophy by assay of N-acetylglucosaminylphosphotransferase in white blood cells and fibroblasts

The biochemical abnormalities of I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III) can be explained by a deficiency of the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We demonstrate here that obligate heterozygotes for these autosomal recessive diseases have intermediate levels of this enzymatic activity in homogenates of peripheral blood white cells and in extracts from cultured fibroblasts. This finding provides further evidence that the enzyme deficiency is the primary genetic defect in these diseases. In addition, the previous observation that obligate heterozygotes for mucolipidosis III have elevations of total serum beta-hexosaminidase outside the range of normal was confirmed. In studies of three pedigrees of patients with mucolipidosis III, these techniques were used to score individuals at risk for the carrier state.     

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.     

Cloning, sequencing, and functional characterization of the murine 46-kDa mannose 6-phosphate receptor.

We have cloned and sequenced the 2175-nucleotide, full-length cDNA for the mouse 46-kDa Man 6-P receptor (46MPR) and studied its functional properties in stably transfected mouse L cells which do not express the insulin-like growth factor-II receptor/mannose 6-phosphate receptor (IGF-IIR/MPR). The 278-amino acid sequence deduced from the cDNA for the murine 46MPR shows 19 amino acid differences from that of the human 46MPR, none of which are found in the 68-amino acid cytoplasmic tail. Binding of ligand to the murine 46MPR in permeabilized cells showed a pH optimum of 6.5, was completely inhibited by Man 6-P, and was stimulated by divalent cations. Mn2+ was more effective than Ca2+ or Mg2+. Endocytosis was demonstrated at pH 6.5 and was stimulated 4-7-fold by Mn2+. In its responsiveness to divalent cations and its preference for Mn2+, the murine 46MPR resembled the bovine 46MPR more than the human 46MPR. It was even less efficient than the human receptor in its ability to mediate endocytosis in transfected murine cells. It was also no more efficient than the human 46MPR in correcting the sorting defect of IGF-IIR/MPR-deficient mouse L cells. We conclude that the previously observed relative inefficiency of the human 46MPR in sorting enzymes to lysosomes in murine cells is a property of the 46MPR itself and not a manifestation of studying its expression in a heterologous cell line.     

Effects of an Abrupt Change in Ration from All Roughage to High Concentrate upon Rumen Microbial Numbers in Sheep

When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values.     

Leaf structure and function in evergreen trees and shrubs of Japanese warm temperate rain forest I. The structure of the lamina

Information has been obtained as a part of a wider study of leaf structure, water relations and mineral status, which is to include work on a considerable variety of evergreen forests. Fifteen structural features have been investigated in well-lit leaves of 60 dicotyledonous species of Japanese Warm Temperate Rain Forest, this sample giving an 81% cover of the relevant species (Table 1). The salient features of the average leaf are summarised on p. 200. The leaves of shrubs and small trees have been compared with those of tall and medium trees; some of the differences were unexpected. For example, the leaves of the lower growing species tended to be thicker and to have thicker outer walls. The leaves of the whole Japanese sample were compared with those of 40 species in Tropical Lowland Rain Forest in New Britain. Although the average leaf area of the Japanese species was less than one tenth of that of the species from New Britain, the thickness and internal structure of the leaf and the size and density of the stomata were quite similar in the two sets of leaves (Table 2). The Japanese leaves were somewhat more xeromorphic in that they had thicker outer walls in the upper and lower epidermis.