Copper, ceruloplasmin, and long-term cardiovascular and total mortality (the Ludwigshafen Risk and Cardiovascular Health Study)

BACKGROUND Copper and its main transport protein ceruloplasmin have been suggested to promote the development of atherosclerosis. Most of the data come from experimental and animal model studies. Copper and mortality have not been simultaneously evaluated in patients undergoing coronary angiography. METHODS AND RESULTS We examined whether serum copper and ceruloplasmin concentrations are associated with angiographic coronary artery disease (CAD) and mortality from all causes and cardiovascular causes in 3253 participants of the Ludwigshafen Risk and Cardiovascular Health Study. Age and sex-adjusted hazard ratios (HR) for death from any cause were 2.23 (95% CI, 1.85-2.68) for copper and 2.63 (95% CI, 2.17-3.20) for ceruloplasmin when we compared the highest with the lowest quartiles. Corresponding hazard ratios (HR) for death from cardiovascular causes were 2.58 (95% CI, 2.05-3.25) and 3.02 (95% CI, 2.36-3.86), respectively. Further adjustments for various risk factors and clinical variables considerably attenuated these associations, which, however, were still statistically significant and the results remained consistent across subgroups. CONCLUSIONS The elevated concentrations of both copper and ceruloplasmin are independently associated with increased risk of mortality from all causes and from cardiovascular causes.

Protein profiling of urine from dogs with renal disease using ProteinChip analysis

Measurement of total urinary proteins in individuals that tested positive by urinary dipstick is a typical method for assessing the presence of potentially serious renal disorders. In the absence of such overt proteinuria, however, measurement of specific urinary proteins may be useful in the diagnosis of nephropathies and may provide greater insight into the pathogenesis. The urine of 28 dogs (16 with renal disease and 12 healthy) was evaluated to determine whether specific low-molecular-weight proteins or the pattern of protein excretion could also be used as a marker of tubular dysfunction in dogs. Specific proteins were assessed by immunological methods, whereas protein profiles were determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (MS). In particular, changes in the excretion of retinol-binding protein (RBP) and Tamm-Horsfall protein (THP) appear to be of clinical relevance in the diagnosis of canine kidney diseases. The pattern of urinary protein and peptides revealed specific changes in abundance in dogs with renal disease at molecular masses (kD) of 11.58, 12.41, 12.60, 14.58, 20.95 (RBP), 27.85, and 65.69 (albumin). In conclusion, comparable proteins as in humans might be used as urinary markers for proximal (RBP) and distal (THP) tubular dysfunction in dogs. Surface-enhanced laser desorption/ionization time-of-flight MS is a promising tool for the study of kidney physiology and pathophysiology and might aid in the discovery of new biomarkers of renal disease.

The prevalence and predictive value of dipstick urine protein in HIV-positive persons in Europe.

INTRODUCTION Proteinuria (PTU) is an important marker for the development and progression of renal disease, cardiovascular disease and death, but there is limited information about the prevalence and factors associated with confirmed PTU in predominantly white European HIV+ persons, especially in those with an estimated glomerular filtration rate (eGFR) of 60 mL/min/1.73 m(2). PATIENTS AND METHODS Baseline was defined as the first of two consecutive dipstick urine protein (DPU) measurements during prospective follow-up >1/6/2011 (when systematic data collection began). PTU was defined as two consecutive DUP >1+ (>30 mg/dL) >3 months apart; persons with eGFR <60 at either DPU measurement were excluded. Logistic regression investigated factors associated with PTU. RESULTS A total of 1,640 persons were included, participants were mainly white (n=1,517, 92.5%), male (n=1296, 79.0%) and men having sex with men (n=809; 49.3%). Median age at baseline was 45 (IQR 37-52 years), and CD4 was 570 (IQR 406-760/mm(3)). The median baseline date was 2/12 (IQR 11/11-6/12), and median eGFR was 99 (IQR 88-109 mL/min/1.73 m(2)). Sixty-nine persons had PTU (4.2%, 95% CI 3.2-4.7%). Persons with diabetes had increased odds of PTU, as were those with a prior non-AIDS (1) or AIDS event and those with prior exposure to indinavir. Among females, those with a normal eGFR (>90) and those with prior abacavir use had lower odds of PTU (Figure 1). CONCLUSIONS One in 25 persons with eGFR>60 had confirmed proteinuria at baseline. Factors associated with PTU were similar to those associated with CKD. The lack of association with antiretrovirals, particularly tenofovir, may be due to the cross-sectional design of this study, and additional follow-up is required to address progression to PTU in those without PTU at baseline. It may also suggest other markers are needed to capture the deteriorating renal function associated with antiretrovirals may be needed at higher eGFRs. Our findings suggest PTU is an early marker for impaired renal function.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

INTRODUCTION The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Measurement of Protein in Nearshore Marine Sediments

Proteinaceous material in marine sediments which is available to proteolytic hydrolysis has been measured using a new method. This technique utilizes Coomassie Blue dye binding, which has the advantage of being sensitive only to larger polypeptides. Substantial interferences from other sedmentary organic substances are overcome by using a standard additions approach in conjunction with enzymatic digestion of the protein. Although tedious, the technique provides acceptable precision and accuracy. Measurements of protein in surficial nearshore sediments of the Gulf of Maine and St. Croix yield values ranging from 0.1 to 2.2 mg g-1, which account for a minor fraction of total nitrogen or acid-hydrolyzable amino acids. Protein decreases downcore at a faster rate than either of these 2 indicators of nitrogenous material, indicating the greater lability of the truly proteinaceous material. Biomass comprises a minor portion of the measured protein.

STUDIES INTO THE MOLECULAR MECHANISMS REGULATING MUSCULAR ATROPHY RESULTING FROM HINDLIMB IMMOBILIZATION IN THE RAT (PROTEIN, SYNTHESIS, RNA)

The purpose of the work performed in this dissertation was to examine some of the possible regulatory mechanisms involved in the initiation of muscular atrophy during periods of decreased muscle utilization resulting from hindlimb immobilization in the rat. A 37% decrease in the rate of total muscle protein synthesis which has been observed to occur in the first 6 h of immobilization contributes significantly to the observed loss of protein during immobilization.^ The rates of cytochrome c and actin synthesis were determined in adult rat red vastus lateralis and gastrocnemius muscles, respectively, by the constant infusion and incorporation of ('3)H-tyrosine into protein. The fractional synthesis rates of both actin and cytochrome c were significantly decreased (P < 0.05) in the 6th h of hindlimb immobilization.^ RHA was extracted from adult rat gastrocnemius muscle by modification of the phenol: chloroform: SDS extraction procedures commonly used for preparation of RNA for hybridization analysis from other mammalian tissues. RNA content of rat gastrocnemius muscle, as determined by this method of extraction and its subsequent quantification by UV absorbance and orcinol assay, was significantly greater than the RNA content previously determined for adult rat gastrocnemius by other commonly employed methods.^ RNA extracted by this method from gastrocnemius muscles of control and 6h immobilized rats was subjected to "dot blot" hybridization to ('32)P-labelled probe from plasmid p749, containing a cDNA sequence complementary to (alpha)-actin mRNA and from rat skeletal muscle. (alpha)-Actin specific mRNA content as estimated by this procedure is not significantly decreased in rat gastrocnemius following 6h or hindlimb immobilization. However, (alpha)-actin specific mRNA content is significantly decreased (P < 0.05) in adult rat gastrocnemius (alpha)-actin specific mRNA is not decreased in adult rat gastrocnemius muscle following 6h of immobilization, a time when actin synthesis is significantly decreased, it is concluded that a change in (alpha)-actin specific mRNA content is not the initiating event responsible for the early decrease in actin synthesis observed in the 6th h of immobilization. ^

The evidence behind the level of a random urine protein:creatinine in the diagnosis of preeclampsia: A systematic review

Objective. To determine the accuracy of the urine protein:creatinine ratio (pr:cr) in predicting 300 mg of protein in 24-hour urine collection in pregnant patients with suspected preeclampsia. ^ Methods. A systematic review was performed. Articles were identified through electronic databases and the relevant citations were hand searching of textbooks and review articles. Included studies evaluated patients for suspected preeclampsia with a 24-hour urine sample and a pr:cr. Only English language articles were included. The studies that had patients with chronic illness such as chronic hypertension, diabetes mellitus or renal impairment were excluded from the review. Two researchers extracted accuracy data for pr:cr relative to a gold standard of 300 mg of protein in 24-hour sample as well as population and study characteristics. The data was analyzed and summarized in tabular and graphical form. ^ Results. Sixteen studies were identified and only three studies met our inclusion criteria with 510 total patients. The studies evaluated different cut-points for positivity of pr:cr from 130 mg/g to 700 mg/g. Sensitivities and specificities for pr:cr of 130mg/g -150 mg/g were 90-93% and 33-65%, respectively; for a pr:cr of 300 mg/g were 81-95% and 52-80%, respectively; for a pr:cr of 600-700mg/g were 85-87% and 96-97%, respectively. ^ Conclusion. The value of a random pr:cr to exclude pre-eclampsia is limited because even low levels of pr:cr (130-150 mg/g) may miss up to 10% of patients with significant proteinuria. A pr:cr of more than 600 mg/g may obviate a 24-hour collection.^

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

From protein microarray to biology: The discovery of epigenetic regulatory molecules

Chromatin, composed of repeating nucleosome units, is the genetic polymer of life. To aid in DNA compaction and organized storage, the double helix wraps around a core complex of histone proteins to form the nucleosome, and is therefore no longer freely accessible to cellular proteins for the processes of transcription, replication and DNA repair. Over the course of evolution, DNA-based applications have developed routes to access DNA bound up in chromatin, and further, have actually utilized the chromatin structure to create another level of complexity and information storage. The histone molecules that DNA surrounds have free-floating tails that extend out of the nucleosome. These tails are post-translationally modified to create docking sites for the proteins involved in transcription, replication and repair, thus providing one prominent way that specific genomic sequences are accessed and manipulated. Adding another degree of information storage, histone tail-modifications paint the genome in precise manners to influence a state of transcriptional activity or repression, to generate euchromatin, containing gene-dense regions, or heterochromatin, containing repeat sequences and low-density gene regions. The work presented here is the study of histone tail modifications, how they are written and how they are read, divided into two projects. Both begin with protein microarray experiments where we discover the protein domains that can bind modified histone tails, and how multiple tail modifications can influence this binding. Project one then looks deeper into the enzymes that lay down the tail modifications. Specifically, we studied histone-tail arginine methylation by PRMT6. We found that methylation of a specific histone residue by PRMT6, arginine 2 of H3, can antagonize the binding of protein domains to the H3 tail and therefore affect transcription of genes regulated by the H3-tail binding proteins. Project two focuses on a protein we identified to bind modified histone tails, PHF20, and was an endeavor to discover the biological role of this protein. Thus, in total, we are looking at a complete process: (1) histone tail modification by an enzyme (here, PRMT6), (2) how this and other modifications are bound by conserved protein domains, and (3) by using PHF20 as an example, the functional outcome of binding through investigating the biological role of a chromatin reader. ^

The identification and regulation of a novel interaction occurring between $\beta$-catenin and the actin -bundling protein fascin

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesion, catenins have more recently been indicated to participate in cell and developmental signaling pathways. $\beta$-catenin, for example, associates directly with receptor tyrosine kinases and transcription factors such as LEF-1/TCF, and tranduces developmental signals within the Wnt pathway. $\beta$-catenin also appear to a role in regulating cell proliferation via its interaction with the tumor supressor protein APC. I have employed the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to $\beta$-catenin's central Armadillo-repeat domain. The $\beta$-catenin-fascin interaction exists in cell lines as well as in animal brain tissues as revealed by immunoprecipitation analysis, and substantiated in vitro with purified proteins. Fascin additionally binds to plakoglobin, which contains a more divergent Armadillo-repeat domain. Fascin and E-cadherin utilize a similar binding-site within $\beta$-catenin, such that they form mutually exclusive complexes with $\beta$-catenin. Fascin and $\beta$-catenin co-localize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. Total immunoprecipitable b-catein has several isoforms, only the hyperphosphorylated isoform 1 associated with fascin. An increased $\beta$-catenin-fascin interaction was observed in HGF stimulated cells, and in Xenopus embryos injected with src kinase RNAs. The increased $\beta$-catenin association with fascin is correlated with increased levels of $\beta$-catenin phosphorylation. $\beta$-catenin, but not fascin, can be readily phosphorylated on tyrosine in vivo following src injection of embryos, or in vitro following v-src addition to purified protein components. These observations suggest a role of $\beta$-catenin phosphorylation in regulating its interaction with fascin, and src kinase may be an important regulator of the $\beta$-catenin-fascin association in vivo. The $\beta$-catenin-fascin interaction represents a novel catenin complex, that may conceivably regulate actin cytoskeletal structures, cell adhesion, and cellular motility, perhaps in a coordinate manner with its functions in cadherin and APC complexes. ^

The effects of omega -3 fatty acid -enriched fish on C -reactive protein levels in postmenopausal women

Coronary heart disease (CHD) is the leading cause of death in women and rates markedly increase among women after 65 years of age. C-reactive protein (CRP) is a new clinical indicator of atherosclerotic-related inflammation with a direct pathogenic role. Studies show lifestyle factors can modulate CRP. Omega-3 fatty acids have anti-inflammatory properties and studies suggest that eating fish high in omega-3 fatty acids may lower CHD risk in women. This study sought to assess the possible role of omega-3 fatty acids in the reduction of CHD-related inflammation by investigating the effect of fish consumption on CRP levels. Methods. Twenty-four healthy postmenopausal women were randomly assigned to a fish group (usual diet plus two servings per week of enriched fish) or control group (usual diet with no fatty fish) for eight weeks. Omega-3 fatty acid-enriched fish developed by the West Virginia University Aquaculture Division was used. Serum CRP, serum interleukin-6 (IL-6), and the fatty acid content of red blood cells (RBC) were measured before and after the study. Women also completed food records. RESULTS: Baseline levels of CRP were low (85% of the fish group had normal levels) and few changes in CRP risk category were observed. Mean IL-6 levels were reduced by 27% and 35% in the fish and control groups, respectively (p for between-group difference = 0.60). Changes in RBC fatty acid composition were not statistically significant. Compared to control women, women in the fish group had greater reductions in mean triglycerides (p = 0.08), total cholesterol (P = 0.04), and LDL cholesterol levels (p = 0.06). Baseline dietary intake of total and monounsaturated fatty acids tended to be positively associated with baseline CRP, while vitamin E intake was inversely related. Saturated fat intake tended to have a positive association with IL-6. Conclusions. Findings regarding the effect of two servings of fish on CRP and IL-6 levels are inconclusive due to low baseline levels of CRP and IL-6. However, results indicate two servings of fatty fish have favorable effects on blood lipids. The relationship of dietary components with CRP and IL-6 is complex and further research is needed to determine the varying roles of diet on the inflammatory process. ^