Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies

Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 microm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from two-dimensional (2D) gels with M(R)'s of 20-100 kDa and pI's of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) alpha chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.     

Sialin, an anion transporter defective in sialic acid storage diseases, shows highly variable expression in adult mouse brain, and is developmentally regulated

Sialin is a lysosomal membrane protein encoded by the SLC17A5 gene, which is mutated in patients with sialic acid storage diseases (SASD). To further understand the role of sialin in normal CNS development and in the progressive neuronal atrophy and dysmyelination seen in SASD, we investigated its normal cellular distribution in adult and developing mice. Overall, sialin showed granular immunoreactivity, consistent with a vesicular protein. Adult mice showed widespread sialin expression, including in the brain, heart, lung, and liver. High-level immunoreactivity was seen in the neuropil of the hippocampus, striatum, and cerebral cortex, as well as in the perikarya of cerebellar Purkinje cells, globus pallidus, and certain thalamic and brainstem nuclei. In mouse embryos, the highest levels of expression were observed in the nervous system. We discuss the possible role of sialin in normal development and in SASD pathogenesis, as a framework for further investigation of its function in these contexts.     

Effects of ambient particulate matter on peak expiratory flow rates and respiratory symptoms of asthmatics during Asian dust periods in Korea

OBJECTIVE: Dust generating events frequently produce ambient dust particles that are less than 10 microm in diameter, and these have been linked to adverse effects in the general population. However, the evidence linking these particles to adverse effects on the airways of asthmatic individuals is limited. The objective of this study was to investigate the possible adverse effects of Asian dust events on the respiratory functions and symptoms of subjects with bronchial asthma. METHODOLOGY: From March to June 2002, individuals were enrolled who had been diagnosed as having asthma by bronchial challenge or by their bronchodilator response. The patients were divided into three groups according to asthma severity: mild, moderate and severe. Patients with other major disease states were excluded. Patients completed twice-daily diaries monitoring PEF, respiratory symptoms, and daily activities. The daily and hourly mean levels of particulate matter <10 microm in diameter (PM(10)), as well as nitrogen dioxide (NO(2)), sulphur dioxide (SO(2)), ozone (O(3)) and carbon monoxide (CO) were measured at 10 monitoring sites in Incheon, Korea. RESULTS: Dust events occurred 14 times during the study period. On dusty days there were increased levels of PM(10), decreased levels of NO(2) and SO(2), and no change in the CO levels, compared to the levels on control days. An increase in PM(10) concentration was associated with increases in PEF variability of >20% (P<0.05), more night-time symptoms (P<0.05), and a decrease in the mean PEF (P<0.05), calculated by longitudinal data analysis. In contrast, there was no association between the PM(10) levels and bronchodilator inhaler use or daytime respiratory symptoms. Using a general additive Poisson regression model, a borderline association was noted between PM(10) and respiratory symptoms, with a relative risk of 1.05 (95% confidence interval (CI), 0.99--1.17). There was no association between the PM(10) concentrations and PEF measurements, with PEF variability of >20% between the morning and evening values and a relative risk of 1.05 (95% CI, 0.89--1.24) in all subjects. CONCLUSIONS: This study provides evidence that Asian dust events are impacting on the respiratory symptoms of subjects with bronchial asthma, and ambient air pollution, particularly elevated PM(10), might be one of the aggravating factors.     

Inverse relationship between plasminogen activator inhibitor-I activity and adiponectin in overweight and obese women. Interrelationship with visceral adipose tissue, insulin resistance, HDL-chol and inflammation

Adipose tissue is an active endocrine organ secreting different adipokines such as plasminogen activator inhibitor-1 (PAI-1) and adiponectin, among many others. In this study, we investigated the association between PAI-1 activity and serum adiponectin levels in a group of 444 overweight and obese women and assessed the interrelationship with visceral adipose tissue (VAT; CT-scan L4-L5), insulin resistance (HOMA-IR), HDL cholesterol (HDL-chol) and inflammation (hs-CRP). PAI-1 was inversely related to adiponectin (r = -0.25, p < 0.001; adjusted for age and BMI). After adjustment for age, VAT, HOMA-IR and hs-CRP, the relationship remained significant (r = -0.15; p = 0.001), but disappeared after additional adjustment for HDL-chol (r = -0.09; p = 0.067). Subjects were divided in two groups according to the median levels of adiponectin or PAI-1 levels. PAI-1 activity (19.1 +/- 11.4 vs. 15.8 +/- 8.6 AU/ml; p = 0.003) and adiponectin levels (9.8 +/- 4.6 vs. 8.4 +/- 4.0 microg/ml; p < 0.001) were significantly higher in the low adiponectin/PAI-1 groups. The difference in PAI-1 remained significant after adjustment for age and BMI (p = 0.001), became borderline significant after adjustment for age and VAT (p = 0.052), and disappeared after adjustment for age and HOMA-IR (p = 0.116) or age and HDL-chol (p = 0.443). The difference in adiponectin levels remained significant after adjustment for age, VAT, HOMA-IR and hs-CRP (p = 0.006), but disappeared after additional adjustment for HDL-chol (p = 0.089). Further analyses suggest a contribution of HOMA-IR and/or HDL-chol in the relationship between PAI-1 and adiponectin. HDL-chol was found to be the only factor independently determining both factors. In conclusion, in overweight and obese women, PAI-1 activity was inversely related to serum adiponectin, independent of visceral adipose tissue.     

Evidence of major genes for plasma HDL, LDL cholesterol and triglyceride levels at baseline and in response to 20 weeks of endurance training: the HERITAGE Family Study

This study assessed major gene effects for baseline HDL-C, LDL-C, TG, and their training responses (post-training minus baseline) in 527 individuals from 99 White families and 326 individuals from 113 Black families in the HERITAGE Family Study. The baseline phenotypes were adjusted for the effects of age and BMI, and the training response phenotypes were adjusted for the effects of age, BMI, and their respective baseline values, within each of the sex-by-generation-by-race groups, prior to genetic analyses. In Whites, we found that LDL-C at baseline and HDL-C training response were under influence of major recessive genes (accounting for 2--30 % of the variance) and multifactorial (polygenic and familial environmental) effects. Interactions of these major genes with sex, age, and BMI were tested, and found to be nonsignificant. In Blacks, we found that baseline HDL-C was influenced by a major dominant gene without a multifactorial component. This major gene effect accounted for 45 % of the variance, and exhibited no significant genotype-specific interactions with age, sex, and BMI. Evidence of major genes for the remaining phenotypes at baseline and in response to endurance training were not found in both races, though some were influenced by major effects that did not follow Mendelian expectations or were with ambiguous transmission from parents to offspring. In summary, major gene effects that influence baseline plasma HDL-C and LDL-C levels as well as changes in HDL-C levels in response to regular exercise were detected in the current study.