Intraperitoneal fat is associated with thickening of the thoracic aorta in individuals at high risk for cardiovascular events

Increased intraperitoneal (IP) fat is associated with increased cardiovascular (CV) risk, but mechanisms for this increase in risk are not completely established. We performed this study to assess whether IP fat is associated with ascending aortic wall thickness (AOWT), a risk factor for CV events. Four hundred and forty-one consecutive participants, aged 55-85 years, with risk factors for CV events underwent magnetic resonance measures of AOWT and abdominal fat (subcutaneous (SC) fat + IP fat). For the ascending aorta, mean wall thickness of the 4th quartile of the IP fat was higher relative to the 1st quartile (P ≤ 0.001). This difference persisted after accounting for SC fat (P ≤ 0.001), as well as age, gender, height, weight, smoking, diabetes, hypertension, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and C-reactive protein (CRP) (P < 0.03). Elevated IP fat volume is associated with an increase in ascending AOWT, a condition that promotes CV events in middle aged and elderly adults.     

Plasma nitrate/nitrite levels are unchanged after long-term aerobic exercise training in older adults

Reduced nitric oxide (NO) production and bioactivity is a major contributor to endothelial dysfunction. Animal data suggest that improvements in endothelial function in response to aerobic exercise training may depend on the duration of the training program. However, no studies have examined changes in NO (as assessed by the major NO metabolites, nitrate and nitrite, NO_x) after long-term training in humans. In addition, aging may impair the ability of the vasculature to increase NO with exercise. Thus, we determined whether 24 weeks of aerobic exercise training increases plasma NO_x levels in sedentary older adults. We also examined changes in forearm blood flow (FBF) at rest and during reactive hyperemia as a measure of vasomotor function. Plasma NO_x levels were measured in 82 men and women using a modified Griess assay. FBF was assessed in a subset of individuals (n = 15) using venous occlusion plethysmography. After 24 weeks of exercise training, there were significant improvements in maximum oxygen consumption, HDL cholesterol, triglycerides, and body fat. Changes in plasma NO_x levels ranged from ?14.83 to +16.69 μmol/L; however, the mean change overall was not significant (?0.33 ± 6.30 μmol/L, p = 0.64). Changes in plasma NO_x levels were not associated with age, gender, race, HDL cholesterol, triglycerides, body weight, body fat, or maximal oxygen consumption. There were also no significant changes in basal FBF, peak FBF, hyperemic response, total hyperemic flow, or minimum forearm vascular resistance with exercise training. In conclusion, improvements in plasma NO_x levels and FBF are not evident after long-term training in older adults.     

Cell-detaching Escherichia coli (CDEC) strains from children with diarrhea: Identification of a protein with toxigenic activity

Twenty-four strains of cell-detaching Escherichia coli (CDEC) isolated from stool specimens in different cities in Brazil were examined for virulence properties. Aerobactin production and multiple antibiotic resistance were observed in most of the isolates. In hybridization studies, the alphahly, pap, and cnf sequences, common properties of this category of E. coli, were found in a minority of isolates. Half of the CDEC isolates had enteroaggregative DNA sequences (pet, astA, aggA), six strains carried the shet1 gene, nine strains carried the daaC sequence, and one strain carried the stp gene. Thirteen strains induced fluid accumulation in the rabbit intestinal loop assay. Supernatant filtrate of one of those strains, which did not hybridize with any of the toxin probes tested, induced destructive lesions in the rabbit ileal loop and enterotoxic activity in the Ussing chamber. A 12-kDa protein purified by 60% ammonium sulfate precipitation of the supernatant filtrate demonstrated a toxigenic effect that was inhibited by the anti-12-kDa protein antiserum.     

Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.     

Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.     

Potassium inhibition of transforming protein P85gag-mos and reversal of the transformed phenotype in 6m2 cells

K+ at high concentrations (52-72 mM hypertonic KCl) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature-sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33 degrees C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39 degrees C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39 degrees C. Exposure to K+ resulted in a 90% inhibition of P85gag-mos-associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F-actin cables were affected more slowly by K+ than by a temperature shift to 39 degrees C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.     

Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient

Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci, A. P. J., and Robertson, W. 1996. Polarized growth of fungal hyphae is defined by an alkaline pH gradient. Fungal Genetics and Biology 20, 289-298. Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells. The events which are responsible for this growth are poorly understood. However, the existence of ion gradients may play an important role in establishing and driving cell polarity. Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions. Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth. These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi.     

Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore-enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.