Identification of the essential cysteine residue in Klebsiella aerogenes urease.

During reaction with [14C]iodoacetamide at pH 6.3, radioactivity was incorporated primarily into a single Klebsiella aerogenes urease peptide concomitant with activity loss. This peptide was protected from modification at pH 6.3 by inclusion of phosphate, a competitive inhibitor of urease, which also protected the enzyme from inactivation. At pH 8.5, several peptides were alkylated; however, modification of one peptide, identical to that modified at pH 6.3, paralleled activity loss. The N-terminal amino acid sequence and composition of the peptide containing the essential thiol was determined. Previous enzyme inactivation studies of K. aerogenes urease could not distinguish whether one or two essential thiols were present per active site (Todd, M. J., and Hausinger, R. P. (1991) J. Biol. Chem. 266, 10260-10267); we conclude that there is a single essential thiol present and identify this residue as Cys319 in the large subunit of the heteropolymeric enzyme.     

Hypertensive structural changes in blood vessels: do endothelial cells hold the key?

In recent decades, the complexity of the endothelium and its major role in maintaining or altering blood vessel architecture are being revealed. In contrast, the vascular smooth muscle cell previously received the most attention. I suggest support of the hypothesis that the endothelium is the key to vascular disease. An altered endothelium in diabetes mellitus likewise is likely to be pivotal in vascular complications that develop. We have demonstrated that adherent monocytes, indicators of altered endothelium, occur in deoxycorticosterone acetate induced hypertension in male Wistar rats. The coronary artery and thoracic aorta were investigated using transmission electron microscopy. Details of hypertensive changes were revealed as well as early atherogenic pathology in the absence of dietary modifications. Scanning electron microscopy of thoracic aorta showed details of the luminal endothelial surface and adherent monocyte-macrophages in hypertensive animals. There were two cell types: numerous typical monocytes with upstream tails, and larger cells that may have been free grazing macrophages or macrophages that had returned to the circulation. Debris and amorphous material were particularly evident in vessels from hypertensive animals. Monocytes squeezed between intact endothelial plasma membranes (as seen in section), and were found as subendothelial foam cells and phagocytosing macrophages. The endothelial adherence of monocytes to the aortas from diabetic animals was significantly (p less than 0.05) elevated over that found in controls (but not different from control-hypertensive or diabetic-hypertensive animals) supporting the concept of altered endothelium in diabetes.     

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

Soil invertebrate and plant responses to mowing and carbofuran application in a North American tallgrass prairie

Relationships between soil invertebrate populations and primary production of a tallgrass prairie were investigated using the insecticide-nematicide carbofuran and a range of mowing intensities to manipulate invertebrate densities and resource quantity and quality. The trophic composition of nematode populations was monitored through each of two growing seasons. Earthworm and macroarthropod densities and primary production were assessed at the end of the second season. Invertebrate densities were generally reduced in carbofuran-treated plots, although individual weights of surviving macroarth-ropod herbivores increased significantly (p<0.05). Carbofuran failed to affect estimates of above- or belowground plant biomass after two years of treatment. Changes in resource quantity and quality resulted in rapid responses by dominant invertebrate consumer populations. A 28% reduction in live root mass and a 24% increase in root detritus following two years of mowing was associated with a 54% decrease in herbivorous nematode densities, a 47% increase in microbivorous nematode densities, and a 41% increase in native earthworm biomass.     

High-resolution linkage mapping for susceptibility genes in human polygenic disease: Insulin-dependent diabetes mellitus and chromosome 11q

Insulin-dependent diabetes mellitus (IDDM) has a complex pattern of genetic inheritance. In addition to genes mapping to the major histocompatibility complex (MHC), several lines of evidence point to the existence of other genetic susceptibility factors. Recent studies of the nonobese diabetic mouse (NOD) model of IDDM have suggested the presence, on mouse chromosome 9, of a susceptibility gene linked to the locus encoding the T-cell antigen, Thy-1. A region on human chromosome 11q is syntenic to this region on mouse chromosome 9. We have used a set of polymorphic DNA markers from chromosome 11q to investigate this region for linkage to a susceptibility gene in 81 multiplex diabetic pedigrees. The data were investigated by maximization of lod scores over genetic models and by multiple-locus affected-sib-pair analysis. We were able to exclude the presence of a susceptibility gene (location scores less than -2) throughout greater than 90% of the chromosome 11q homology region, under the assumption that the susceptibility factor would cause greater than 50% of affected sib pairs to share two alleles identical by descent. Theoretical estimates of the power to map susceptibility genes with a high-resolution map of linked markers in a candidate region were made, using HLA as a model locus. This result illustrates the feasibility that IDDM linkage studies using mapped sets of polymorphic DNA markers have, both for other areas of the genome in IDDM and for other polygenic diseases. The analytic approaches introduced here will be useful for affected-sib-pair studies of other complex phenotypes.     

Response of macroinvertebrates to blooms of iron-depositing bacteria

In field measurements and laboratory experiments we assessed the influence of high levels of iron, manganese, and concurrent blooms of iron-depositing bacteria, Leptothrix ochracea, on macroinvertebrates. Macroinvertebrate communities in five of six streams were depauerate inside blooms. Reasons for the decreased abundance vary among taxa, with our experiments demonstrating the importance, for one or more species, of (1) direct toxic effects, and,/or smothering, (2) behavioral avoidance of bacterial-coated substrates, and (3) an inability to use bacteria as food. Three mayfly species showed increased mortality when caged inside the blooms, but five trichopterans and one plecopteran did not. Five invertebrates avoided Leptothrix-coated substrate in choice trials, while three did not. Stenonema fuscum could not ingest Leptothrix, and Neophylax nacatus had reduced growth feeding on it, but Heptagenia umbratica grew equally well on diets of Leptothrix or diatoms. This study demonstrates the important role epilithic organisms play in modifying substrates, and how these changes may act to influence benthic abundance and distribution in streams.     

AGE STRUCTURE OF PHORADENDRON JUNIPERINUM (VISCACEAE), A XYLEM-TAPPING MISTLETOE: INFERENCES FROM A NON-DESTRUCTIVE MORPHOLOGICAL INDEX OF AGE

We investigated the age-structure of the xylem-tapping mistletoe Phoradendron juniperinum in relation to characteristics of its host tree, Juniperus osteosperma. We first correlated branch structure in the mistletoe with age of the mistletoe infection as determined anatomically; this correlation provided a nondestructive, field method of obtaining age structure information. We then surveyed the mistletoe plants, applying our aging index, within a population of their host trees in southwestern Utah; the majority of mistletoe plants were 2–12 years old. This peak in abundance of mistletoe infections showed no correlation to total annual precipitation within or 1 year previous to the peaks, minimum winter temperature, or to warmer than average winter temperatures. However, there was a positive correlation (r = 0.51, P < 0.06) with the amount of summer precipitation. A log-linear analysis indicated that a greater than expected number of mistletoe infections occurred at 5–7 years of age and at approximately 3 m in height among all host trees. We suggest that this pattern resulted because this canopy position had greater leaf and branch areas and was visited most frequently by seed-dispersing birds. The log-linear analysis also revealed that fewer than expected mistletoe infections occurred at ages older than 10 years, yet our data indicate that plants can reach 20 years of age. The lack of infections > 10 years of age was correlated to a period of below average precipitation, especially during the growing season, but not with cold winter temperatures, which in other studies had been suggested as a factor influencing mortality. We feel that drought may play an important role in influencing mistletoe mortality through its direct affect on host tree water status, but in addition we offer two alternative hypotheses to explain mistletoe longevity; the first is concerned with the relationship between carbon and nitrogen costs and maintaining large leaf areas in older plants, and the second addresses how increased hydraulic resistance in older and larger plants may be too costly for the plant, and stems are abscised.