Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

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.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

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.