Effects of diet and exercise on plasma vitamin D (25(OH)D) levels in Vietnamese immigrant elderly in Sydney, Australia

Vitamin D deficiency may be associated with osteoporosis and fractures in the elderly. In Australia where there is a sizeable Vietnamese population, research has not yet clarified the roles of diet, exercise and sun exposure in determining vitamin D status. Plasma samples for 25-hydroxy-vitamin D (25(OH)D); dietary intake of vitamin D and calcium; muscle strength and sun exposure were measured and weekly dairy intake, exercise levels and smoking habits were surveyed in free-living elderly of Vietnamese and Australian/British origin. There was marginal vitamin D deficiency (<37 nmol/L 25(OH)D) in 63% of Vietnamese but only in 37% of Australian/British born. Low dairy intake and no vigorous exercise were best predictors of vitamin D deficiency in Vietnamese, taking into account age, gender, dietary intake and sun exposure. Since these migrant elderly may not get adequate sun exposure due to either clothing customs or cultural norms that encourage fair (untanned) skin, it is important to encourage increased exercise and dairy intake.     

Experimental Detritus Manipulations Unite Surface and Cave Stream Ecosystems Along a Common Energy Gradient

Subsidies of detritus from donor habitats are important energy sources for many ecosystems, but understanding their role in structuring recipient food webs requires comparative experimental studies along the full spectrum of detrital fluxes. Here we report results from an experimental addition of maize (Zea mays L.) litter to a detritus-poor cave stream ecosystem, which we then compare with analogous, past experiments using detritus-rich surface stream ecosystems that similarly have detritus-based food webs and extremely low in situ primary production. Bulk-tissue and compound-specific stable isotope analyses showed that maize litter carbon (C) was rapidly assimilated by microbes and transferred via successive trophic levels to the top of the cave stream food web (omnivorous crayfishes and predatory salamanders). All trophic levels increased in abundance and biomass, but only facultative cave taxa, that is those also found in surface streams, contributed to this numerical response. The lack of response by obligate cave species presumably occurred because evolutionary trade-offs associated with adaptations to low-C environments constrained their population-level responses during the one-year period of the litter addition. Comparison of the responses of the cave community with the analogous litter manipulation experiments in surface streams showed strong convergence in the functional relationship between invertebrate and detritus biomass (R ² = 0.72, P < 0.0001). Our results suggest that these seemingly disparate stream food webs lie along a single, common gradient of detritus supply, occupied at its extreme minimum by communities of obligate cave taxa adapted to low-energy environments.     

Dependence of In Situ Bacterial Fe(II)-Oxidation and Fe(III)-Precipitation on Sequential Reactive Transport

A study was conducted to determine in situ rates of Fe(II) oxidation and Fe(III) precipitation along a 5.0 m reach of a ferruginous groundwater discharge zone under two distinct conditions; (i) the natural state featuring abundant flocculent mats of bacteriogenic iron oxides (BIOS) produced by Fe(II)-oxidizing bacteria, and (ii) after a manual washout of the streambed to remove the microbial mat. Examination of mat samples by differential interference contrast light microscopy revealed tangled meshworks of filamentous Leptothrix sheaths and helical Gallionella stalks intermixed with fine-grained hydrous ferric oxide (HFO) precipitates. The greatest accumulation of BIOS mat was 1.0 m downstream of the groundwater spring. Redox potential (Eh) increased sharply from 200 mV to over 300 mV over the last 2.0 m of the reach. Similarly, dissolved oxygen increased from < 10% saturation to almost 100% saturation over the last 2.0 m of the reach, whereas pH increased from 6.4 to 7.3. Pseudo-first-order rate constants determined on the basis of analytical solutions to sequential partial differential advection-dispersion-reaction equations for the linear Fe(II)→Fe(III)→HFO reaction network yielded in situ Fe(II) oxidation rate constants (k_ox) of 1.70 ± 0.20 min^?1 in natural conditions and 0.48 ± 0.14 min^?1 after washout. Corresponding Fe(III)-precipitation rates (k_p) before and after washout were 3.45 ± 0.10 min^?1 and 0.90 ± 0.01 min^?1, respectively. These values for k_ox and k_p are higher than estimates obtained from closed batch microcosm and laboratory experiments, underscoring the crucial dependence of in situ Fe(II) oxidation and Fe(III) precipitation rates on advective and dispersive mass transport. The results also highlight the influence that BIOS microbial mats exert on the reaction kinetics of the multiple heterogeneous reactions contributing not only to Fe(II)/Fe(III) redox transformations in groundwater discharge zones, but also the precipitation of HFO.     

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. We show that polyhomeotic(proximal) but not polyhomeotic(distal) is necessary for mitosis. To test if other PcG genes have roles in mitosis, we examined embryos derived from heterozygous PcG mutant females for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. We propose that these mitotic defects in PcG mutants delay cell cycle progression. We discuss possible mitotic roles for PcG proteins, and suggest that delays in cell cycle progression might lead to failure of maintenance.     

RNASEL and MIR146A SNP-SNP Interaction as a Susceptibility Factor for Non-Melanoma Skin Cancer

Immunity and inflammatory pathways are important in the genesis of non-melanoma skin cancers (NMSC). Functional genetic variation in immune modulators has the potential to affect disease etiology. We investigated associations between common variants in two key regulators, MIR146A and RNASEL, and their relation to NMSCs. Using a large population-based case-control study of basal cell (BCC) and squamous cell carcinoma (SCC), we investigated the impact of MIR146A SNP rs2910164 on cancer risk, and interaction with a SNP in one of its putative targets (RNASEL, rs486907). To examine associations between genotype and BCC and SCC, occurrence odds ratios (OR) and 95% confidence intervals (95%CI) were calculated using unconditional logistic regression, accounting for multiple confounding factors. We did not observe an overall change in the odds ratios for SCC or BCC among individuals carrying either of the RNASEL or MIR146A variants compared with those who were wild type at these loci. However, there was a sex-specific association between BCC and MIR146A in women (OR_GC = 0.73, [95%CI = 0.52–1.03]; OR_CC = 0.29, [95% CI = 0.14–0.61], p-trend<0.001), and a reduction in risk, albeit not statistically significant, associated with RNASEL and SCC in men (OR_AG = 0.88, [95%CI = 0.65–1.19]; OR_AA = 0.68, [95%CI = 0.43–1.08], p-trend = 0.10). Most striking was the strong interaction between the two genes. Among individuals carrying variant alleles of both rs2910164 and rs486907, we observed inverse relationships with SCC (OR_SCC = 0.56, [95%CI = 0.38–0.81], p-interaction = 0.012) and BCC (OR_BCC = 0.57, [95%CI = 0.40–0.80], p-interaction = 0.005). Our results suggest that genetic variation in immune and inflammatory regulators may influence susceptibility to NMSC, and novel SNP-SNP interaction for a microRNA and its target. These data suggest that RNASEL, an enzyme involved in RNA turnover, is controlled by miR-146a and may be important in NMSC etiology.     

Analysis of angiotensin-stimulated sodium transport in cultured smooth muscle cells from rat aorta

Angiotensin peptides (AI, AII, AIII) increased the rate of Na+ accumulation by smooth muscle cells (SMC) cultured from rat aorta. The stimulatory effect of AII on Na+ uptake was observed when Na+ exodus via the Na+/K+ pump was blocked either by ouabain or by the removal of extracellular K+. AII was at least ten times more potent than AIII and about 100 times more potent than AI in stimulating Na+ uptake. Saralasin had little effect on Na+ uptake by itself but almost completely blocked the increase caused by AII. The stimulation of net Na+ entry by AI, but not AII, was prevented by protease inhibitors. The stimulation of Na+ uptake was almost completely blocked by amiloride. Tetrodotoxin, which prevented veratridine from increasing Na+ uptake, had no effect on the response to AII. Angiotensin increased the rate of ouabain-sensitive 86Rb+ uptake (Na+/K+ pump activity) but had no effect on ouabain-sensitive ATPase activity in frozen-thawed SMC or in microsomal membranes isolated from cultured SMC. The stimulation of ouabain-sensitive 86Rb+ uptake by AII was blocked by saralasin. Omitting Na+ from the external medium prevented AII from increasing 86Rb+ uptake. AII had no effect on cell volume or cyclic AMP levels in the cultured SMC. These results suggest that angiotensin peptides activate an amiloride-sensitive Na+ transporter which supplies the Na+/K+ pump with more Na+, its rate-limiting substrate.     

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.     

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor

BACKGROUND: The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins. METHODS: Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein. RESULTS: Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti-GFP antiserum was negative for the N-terminal fusion protein. CONCLUSIONS: The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors.

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.