The relationship of molecular weight to electrophoretic mobility of fluorescamine-labeled proteins in polyacrylamide gels

Proteins of known molecular weights were labeled with fluorescamine and then subjected to electrophoresis through polyacrylamide gels. The electrophoretic mobilities of the fluorescamine-labeled proteins were dependent upon their respective molecular weights over a range of 17,000 to 70,000 daltons. The correlation of electrophoretic mobility of fluorescamine-labeled protein to molecular weight was similar to results obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The speed with which data can be obtained with the described procedure is a definite advantage over currently employed procedures. These findings encourage the use of fluorescamine for rapid, sensitive determinations of molecular weights of proteins in polyacrylamide gels.     

The laminin β1-competing peptide YIGSR induces a hypercontractile,hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

Background

Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established.

Methods

Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro.

Results

Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR.

Conclusion

These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.     

Comprehensive Behavioral Testing in the R6/2 Mouse Model of Huntington's Disease Shows No Benefit from CoQ10 or Minocycline

Previous studies of the effects of coenzyme Q10 and minocycline on mouse models of Huntington''s disease have produced conflicting results regarding their efficacy in behavioral tests. Using our recently published best practices for husbandry and testing for mouse models of Huntington''s disease, we report that neither coenzyme Q10 nor minocycline had significant beneficial effects on measures of motor function, general health (open field, rotarod, grip strength, rearing-climbing, body weight and survival) in the R6/2 mouse model. The higher doses of minocycline, on the contrary, reduced survival. We were thus unable to confirm the previously reported benefits for these two drugs, and we discuss potential reasons for these discrepancies, such as the effects of husbandry and nutrition.     

Evolution of thermotolerance in seasonal environments: the effects of annual temperature variation and life-history timing in Wyeomyia smithii

In organisms with complex life cycles, the adaptive value of thermotolerance depends on life-history timing and seasonal temperature profiles. We illustrate this concept by examining variation in annual thermal environments and thermal acclimation among four geographic populations of the pitcher plant mosquito. Only diapausing larvae experience winter, whereas both postdiapause and nondiapause adults occur only during the growing season. Thus, adults experience transient cold stress primarily during the spring. We show that adult cold tolerance (chill coma recovery) is enhanced in spring-like conditions via thermal acclimation but is unaffected by diapause state. Moreover, adult mosquitoes from northern populations were more cold tolerant than those from southern populations largely because acclimation responses were steeper in the north. In contrast to cold tolerance, there was no significant acclimation of heat tolerance (heat knockdown), and no significant differences in heat tolerance between northern and southern populations. Field temperature data show that because of evolved differences in diapause timing, adult exposure to cold stress is remarkably consistent across geography. This suggests that geographic variation in cold tolerance may not be the result of direct selection on adults. Our results illustrate the importance of the interplay between phenological and thermal adaptation for understanding variation along climatic gradients.     

Plasma membranes of bursal, thymic and circulating lymphocytes have unique proteins

The protein subunit composition of plasma membranes of chicken bursal, thymic and circulating lymphocytes was determined by polyacrylamide gel electrophoresis. Two of the proteins common to bursa and thymus were not expressed in the circulating lymphocytes. Bursal membranes had 4 unique proteins, 2 of which were still expressed in the circulating lymphocytes. Thymic membranes had 3 unique proteins, one of which was still expressed in the circulating lymphocytes. Membranes of circulating lymphocytes had one unique protein.     

Estimation of molecular weight by polyacrylamide gel electrophoresis using heat stable fluorophors

The utilization of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) as a fluorescent label for proteins in SDS polyacrylamide gel electrophresis is described. The procedure is very sensitive and detects disks containing as little as 1 ng of protein. The fluorescence signal is linear with concentration of protein to 500 ng. Emphasis is placed on the use of MDPF-labeled proteins as standards for the estimation of molecular weights of fluorescamine-labeled proteins. Data are provided which demonstrate the need for only three standard proteins in the molecular weight curve. Finally, the relative mobilities of proteins from nonreplicate determinations exhibited an average variation of only 3.9%.     

Influence of seasonal timing on thermal ecology and thermal reaction norm evolution in Wyeomyia smithii

Evolutionary changes in the seasonal timing of life-history events can alter a population's exposure to seasonally variable environmental factors. We illustrate this principle in Wyeomyia smithii by showing that: (1) geographic divergence in diapause timing reduces differences among populations in the thermal habitat experienced by nondiapause stages; and (2) the thermal habitat of the growing season is more divergent at high compared with low temperatures with respect to daily mean temperatures. Geographic variation in thermal reaction norms for development time was greater in a warm compared with a cool rearing treatment, mirroring the geographic trend in daily mean temperature. Geographic variation in body size was unrelated to geographic temperature variation, but was also unrelated to development time or fecundity. Our results suggest that proper interpretation of geographic trends may often require detailed knowledge of life-history timing.     

Rapid population divergence in thermal reaction norms for an invading species: breaking the temperature-size rule

The temperature-size rule is a common pattern of phenotypic plasticity in which higher temperature during development results in a smaller adult body size (i.e. a thermal reaction norm with negative slope). Examples and exceptions to the rule are known in multiple groups of organisms, but rapid population differentiation in the temperature-size rule has not been explored. Here we examine the genetic and parental contributions to population differentiation in thermal reaction norms for size, development time and survival in the Cabbage White Butterfly Pieris rapae, for two geographical populations that have likely diverged within the past 150 years. We used split-sibship experiments with two temperature treatments (warm and cool) for P. rapae from Chapel Hill, NC, and from Seattle, WA. Mixed-effect model analyses demonstrate significant genetic differences between NC and WA populations for adult size and for thermal reaction norms for size. Mean adult mass was 12-24% greater in NC than in WA populations for both temperature treatments; mean size was unaffected or decreased with temperature (the temperature-size rule) for the WA population, but size increased with temperature for the NC population. Our study shows that the temperature-size rule and related thermal reaction norms can evolve rapidly within species in natural field conditions. Rapid evolutionary divergence argues against the existence of a simple, general mechanistic constraint as the underlying cause of the temperature-size rule.