Body metrics are associated with clinical, free-living, and self-report measures of mobility in a cohort of adults with obesity and multiple sclerosis.

BACKGROUND: Obesity is associated with multiple sclerosis (MS) onset and may contribute to more rapid disability accumulation. Whether obesity impacts mobility in MS is uncertain. Some studies find that obesity in MS is associated with poorer mobility; other studies find no relationship. Discrepant findings may be due to differences in measurement and methodology. In the present study, we employ a comprehensive battery of anthropometric and mobility measures in a sample of people with MS and obesity. METHODS: Participants with MS (N = 74) completed a battery of adiposity measurements (weight, height, waist circumference, and full body dual-energy x-ray absorptiometry [DXA] scans). They also completed validated clinical, free-living (accelerometry), and self-report measures of mobility. Spearman's Rho correlations were used to examine the associations between mobility and obesity measures with Benjamini and Hochberg correction for multiple comparisons. Multiple linear regression was used to examine if adiposity predicted mobility outcomes in people with MS when controlling for age and disease duration. RESULTS: The majority of participants (n = 70) were diagnosed with relapsing-remitting MS and reported mild MS-related disability on the Patient Determined Disease Steps (M = 0.77, SD = 1.1). Median BMI was 35.8 (SD = 5.4). Higher percentage body fat (measured via DXA) was associated with poorer self-reported physical functioning (rs = -0.52, p <0.001), less moderate-to-vigorous physical activity (rs = -0.24, p = 0.04), and worse performance on the Six Minute Walk Test (6MWT; rs = -0.44, p <0.001), the Timed 25 Foot Walk (T25FW; rs = 0.45, p <0.001), and the Timed Up and Go test (TUG; rs = 0.35, p = .003). Higher BMI and waist-to-height ratio (WtHR) were associated with worse outcomes on the 6MWT (BMI; rs = -0.35, p <0.01, WtHR; rs = -0.43, p <0.001), T25FW (BMI; rs = 0.32, p <0.01, WtHR; rs = 0.38, p <0.001), and the SF-36 (BMI; rs = -0.29, p <0.005, WtHR; rs = -0.31, p <0.05). Percentage body fat accounted for an additional 17 % of the variance in the T25FW and 6MWT performance, after controlling for age and disease duration. CONCLUSION: Higher BMI, WtHR, and percentage body fat were associated with lower levels of mobility (T25FW and 6MWT) in people with MS who have class I, class II, and class III obesity. Higher percentage body fat was associated with significantly worse performance on clinical, free-living, and self-report measures of mobility in people with MS even when accounting for participant age and disease duration. These findings suggest that people with MS and obesity may show improved mobility with weight loss.

Effectiveness comparison of multiple sticky-trap configurations for sampling Pseudacteon spp. phorid flies (Diptera: Phoridae).

A variety of traps have been developed for monitoring introduced populations of Pseudacteon spp. phorid flies (Diptera: Phoridae) across their established range in the United States. Such traps typically exploit common aspects of phorid fly biology and behavior, such as their attraction to live or dead red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), as well as the perching behavior of these parasitoids. However, populations of multiple species of phorid flies have been established in the United States to serve as biological control agents against S. invicta, and it is unclear if all trap designs are equally effective in sampling this variety of phorid species. This study investigated the effectiveness of six trap designs simultaneously during three sampling events in south-central Texas. Interactions between two species of phorid flies (Pseudacteon tricuspis Borgmeier and P. curvatus B.) and their hosts have been intensively studied at this location for over eight years. When analyzed independently, there were no significant differences in the mean number of P. curvatus or P. tricuspis phorids collected by any of the trap designs during any of the sampling events. However, when the total number of phorids collected were combined, significant trap performance differentials were observed during the October 2010 sampling event. Furthermore, there were significant differences among male flies during the September 2012 observation. Additionally, a trap component cost comparison is provided. The consistent and relatively equivalent performance of the phorid traps investigated in these trials suggests that all are appropriate for phorid surveillance, and cost and ease-of-use considerations may be the most important criteria when selecting a trap design.

Genomewide linkage scan for combined obesity phenotypes using principal component analysis.

Traditional whole genome linkage scans for obesity were usually performed for a number of correlated obesity related phenotypes separately without considering their correlations. The purpose of this study was to identify quantitative trait loci (QTLs) underlying variations in multiple correlated obesity phenotypes. We performed principal component analysis (PCA) for four highly correlated obesity phenotypes (body mass index [BMI], fat mass, percentage of fat mass [PFM], and lean mass) in a sample of 427 pedigrees (comprising 3,273 individuals) and generated two independent principal components (PC1 and PC2). A whole genome linkage scan (WGS) was then conducted for PC1 and PC2. For PC1, the strongest linkage signal was identified on chromosome 20p12 (LOD = 2.67). For PC2, two suggestive linkages were found on 5q35 (LOD = 2.03) and 7p22 (LOD = 2.18). This study provided evidence supporting several previously identified linkage regions for obesity (e.g., 1p36, 6p23 and 7q34). In addition, our approach by linear combination of highly correlated obesity phenotypes identified several novel QTLs which were not found in genome linkage scans for individual phenotypes.

A whole genome linkage scan for QTLs underlying peak bone mineral density.

UNLABELLED: We conducted a whole genome linkage scan for quantitative trait loci (QTLs) underlying peak bone mineral density (PBMD). Our efforts identified several potential genomic regions for PBMD and highlighted the importance of epistatic interaction and sex-specific analyses in identifying genetic regions underlying PBMD variation. INTRODUCTION: Peak bone mineral density (PBMD) is an important clinical risk predictor of osteoporosis and explains a large part of bone mineral density (BMD) variation. METHODS: To detect susceptive quantitative trait loci (QTLs) for PBMD variation including consideration of epistatic and sex-specific effects, we conducted a whole genome linkage scan (WGLS) for PBMD using 2,200 Caucasians from 207 pedigrees, aged 20-50 years. All the individuals were genotyped with 410 microsatellite markers. In addition to WGLS in the total combined sample of males and females, we conducted epistatic interaction analyses, and sex-specific subgroup linkage analyses. RESULTS: We identified several potential genomic regions that met the criteria for suggestive linkage. The most impressing region is 12p12 for hip PBMD (LOD = 2.79) in the total sample. Epistatic interaction analyses found a significant epistatic interaction between 12p12 and 22q13 (p = 0.0021) for hip PBMD. Additionally, we detected suggestive linkage evidence at 15q26 (LOD = 2.93), 2p13 (LOD = 2.64), and Xq27 (LOD = 2.64). Sex-specific analyses suggested the presence of sex-specific QTLs for PBMD variation. CONCLUSIONS: Our efforts identified several potential regions for PBMD and highlighted the importance of epistatic interaction and sex-specific analyses in identifying genetic regions underlying PBMD variation.

Enrichment during transdominant genetic experiments using a flow sorter.

BACKGROUND: Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. METHODS: Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. RESULTS: MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. CONCLUSIONS: Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS.

A protein interaction map for cell polarity development.

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Characterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

Neoglycolipids derived from phosphatidylethanolamine serve as probes in cell culture studies on glycolipid metabolism.

The neoglycolipid (NeoGL) N-acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc4Ose-PtdEtn(NAc)] and the radioactivly labeled analog [Lc4Ose-PtdEtn(N[14C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N-acetylation with unlabeled and [14C]acetic acid anhydride, respectively. Lc4Ose-PtdEtn(N[14C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc4Ose-PtdEtn(NAc) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc4Ose-PtdEtn(N[14C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc3Ose-PtdEtn(N[14C]Ac), Lac-PtdEtn(N[14C]Ac), and Glc-PtdEtn(N[14C]Ac), as well as the main anabolic product NeuGc-Lc4Ose-PtdEtn(N[14C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.

Treatment of vitamin D deficiency in patients with metastatic prostate cancer may improve bone pain and muscle strength.

PURPOSE: We performed a phase II study to determine whether pain associated with prostate cancer bone metastasis would respond to vitamin D replacement and parameters of muscle strength would be improved by vitamin D replacement therapy. MATERIALS AND METHODS: After a 4-week placebo period, eligible patients received orally 2,000 units vitamin D daily for 12 weeks. Pain questionnaires and measurements of muscle strength were competed at study enrollment and every 4 weeks thereafter. Serum calcium and vitamin D were measured at each clinic visit. RESULTS: A total of 16 patients with advanced hormone refractory prostate cancer were enrolled in this phase II study, of whom 7 (44%) had decreased baseline vitamin D. With vitamin D treatment, 4 patients (25%) had improvement in pain scores and 6 (37%) had improvement in muscle strength measurements. Improvement in pain scores correlated with improvement in subjective symptoms but did not result in a significant decrease in regular scheduled analgesic requirements. CONCLUSIONS: Vitamin D deficiency develops in a significant percent of patients with advanced hormone refractory prostate cancer. Supplementation with vitamin D may be a useful adjunct for improving pain, muscle strength and quality of life in this patient population.

Molecular dissection of zyxin function reveals its involvement in cell motility.

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Progress and variations in two-hybrid and three-hybrid technologies.

The original yeast two-hybrid system and its variants have proven to be effective tools for identification and analysis of protein-protein, protein-DNA and protein-RNA interactions. The two-hybrid assay is being applied to the entire complement of proteins of the yeast Saccharomyces cerevisiae to characterize the network of protein-protein interactions in the eukaryotic cell. The development of nontranscriptional cytosolic and membrane-associated two-hybrid methods has made it possible to detect and examine a number of protein-protein interactions in their normal cellular locations. Small-molecule hybrid systems have been developed which can be used to study protein-ligand interactions and to activate cellular processes by forcing protein associations.

The GCN4 leucine zipper can functionally substitute for the heat shock transcription factor's trimerization domain.

The heat shock transcription factor (HSF) is the only known sequence-specific, homotrimeric DNA-binding protein. HSF binds to a DNA recognition site called a heat shock element (HSE), which contains varying numbers of nGAAn units ("GAA boxes") arranged in inverted repeats. To investigate the role of trimerization on HSF's DNA-binding properties, we replaced the trimerization domain, which self-assembles to form a three-stranded alpha-helical coiled coil, with the GCN4 leucine zipper, which forms a two-stranded alpha-helical coiled coil. Surprisingly, this substitution did not effect the ability of HSF to function in vivo. Biochemical studies of an HSF-leucine zipper chimera in comparison to an HSF truncation show that the HSF-leucine zipper chimera, though dimeric in solution and dimeric when bound to a two-box HSE, forms a trimeric complex when bound to a three-box HSE. The ability to form trimers depends on the presence of three contiguous GAA boxes present in inverted repeats. The proximity of the leucine zippers due to the orientation of the binding sites suggests that the leucine zippers might be forming a three-stranded coiled coil and several experiments lend support to this model. The ability of the leucine zipper to change oligomeric states in context might explain why the leucine zipper can replace the trimerization domain of HSF in vivo.

Environment-sensitive labels in multiplex fluorescence analyses of protein-DNA complexes.

Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct (HSFDT385SH) of the heat shock transcription factor (HSF) was expressed that contains the DNA-binding and trimerization domains, residues 192-385 of HSF, with four additional COOH-terminal residues, GMLC, and then labeled at the COOH-terminal cysteine with fluorescein 5-maleimide to form HSFDT385-Fl. The fluorescence increase accompanying the formation of heterotrimers on titration of HSFDT385-Fl with HSFDT385SH) led to an estimate of 3 x 10(-16) M2 for the equilibrium constant for trimerization of HSFDT385SH. HSFDT385-Fl fluorescence also increased 1.7-fold on binding to specific DNA, but not to nonspecific DNA. The protein and DNA content of the several gel-shifted complexes of HSFDT385-Fl (lambdamaxem 532 nm) with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB (lambdamaxem 658 nm) were accurately determined by a two-color fluorescence emission assay with 488 nm excitation.

Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

Parathyroid hormone (PTH) secretion: stimulation of PTH secretion by a peptide derived from the adenosine diphosphate-ribosylation factor.

Using preparations of dispersed bovine parathyroid cells, we have investigated the effect of a 16-residue synthetic peptide, ARF-16, which corresponds to the N-terminus of the ADP-ribosylation factor, on the secretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrations in the range of 15-25 microM, the ARF peptide stimulated PTH secretion to a greater degree than low extracellular calcium, and at 25 microM was more effective than isoproterenol. The stimulatory effect of ARF was not dependent on the extracellular calcium concentration over the range of 0.5-3 mM. Upon testing other synthetic peptides of similar size we found no effect on PTH secretion, indicating that the ARF-16 effect is specific. In an attempt to define the structural elements of ARF that are required for activity, we tested several analogs of ARF with amino acids deleted from the N- and C-terminus. Deletion of the 2 N-terminal residues yielded a peptide with substantially reduced activity. Further deletions from the N-terminus yielded an inactive peptide. Similarly, a peptide with deletions of 3 residues from the C-terminus was inactive. Thus, the activity of ARF-16 requires both the N- and C-terminal sequence, suggesting that the 16-residue peptide is the minimal sequence required for full activity. Measurements of cAMP concentrations indicate that the stimulatory effect is not mediated via this second messenger. The ARF peptide does not alter intracellular calcium, suggesting that its effect is not mediated by calcium. Although cells incubated with ARF are vigorously stimulated to secrete PTH, this effect is reversible, as demonstrated by washing cells free of ARF, whereupon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion through a low affinity interaction at the cell surface. Other experiments, in which the capacity for ARF stimulation was abolished after a brief exposure of the cells to trypsin, support this conclusion. Characteristics of the ARF stimulatory effect, such as dose dependency and reversibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on extracellular calcium levels and is not mediated via cAMP, we believe that this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.

Processing of chromogranin A by bovine parathyroid secretory granules: production and secretion of N-terminal fragments.

Chromogranin A (CgA) is a glycoprotein located in the secretory granules of multiple neuroendocrine tissues, including the parathyroid gland. Although the function of CgA is not known, a role has been proposed for CgA as a prohormone for biologically active peptides. Using 3H-CgA as a substrate for bovine parathyroid secretory granule extracts, we demonstrate a precursor product relationship between intact CgA and multiple N-terminal fragments of CgA. N-terminal CgA fragments of mol wt 24, 26, and 33 k are generated in a time dependent manner in the presence of bovine parathyroid secretory granule enzymes. The generation of the 33 kilodalton (kDa) N-terminal CgA fragment is calcium dependent. In the presence of EDTA, intermediate CgA fragments of mol wt 36 and 45 are generated. The effect of EDTA is reversible with added calcium. Based on immunodetection on Western blots, the 26 kDa N-terminal fragment of CgA is secreted by bovine parathyroid cells in a time and calcium-dependent manner in parallel with PTH and intact CgA. The secretion of the 26 kDa N-terminal fragment of CgA increases in response to low calcium incubation conditions and is suppressed by high calcium incubation conditions. We conclude that bovine parathyroid secretory granules contain enzymatic activity capable of processing CgA to multiple N-terminal fragments. The secretion of at least one N-terminal fragment (26 kDa) is calcium responsive. The physiological significance of CgA processing in parathyroid secretory granules is as yet unknown.