Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins

Budding yeast septins assemble into hetero‐octamers and filaments required for cytokinesis. Solvent‐exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site‐directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid‐expressed, each Cys‐less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys‐less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys‐less septins were co‐expressed as the sole source of these proteins. Cells grew less well when three Cys‐less septins were co‐expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys‐less Cdc10, Cdc11, and Cdc12, and expressing Cys‐less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys‐less septins—or where one of the Cys‐less septins contained a single Cys introduced at a new site—displayed assembly properties in vitro indistinguishable from wild‐type. Proteins 2013; 81:1964–1979. © 2013 Wiley Periodicals, Inc.     

A Stochastic Regression Model for General Trend Analysis of Longitudinal Continuous Data

A predictive continuous time model is developed for continuous panel data to assess the effect of time‐varying covariates on the general direction of the movement of a continuous response that fluctuates over time. This is accomplished by reparameterizing the infinitesimal mean of an Ornstein–Uhlenbeck processes in terms of its equilibrium mean and a drift parameter, which assesses the rate that the process reverts to its equilibrium mean. The equilibrium mean is modeled as a linear predictor of covariates. This model can be viewed as a continuous time first‐order autoregressive regression model with time‐varying lag effects of covariates and the response, which is more appropriate for unequally spaced panel data than its discrete time analog. Both maximum likelihood and quasi‐likelihood approaches are considered for estimating the model parameters and their performances are compared through simulation studies. The simpler quasi‐likelihood approach is suggested because it yields an estimator that is of high efficiency relative to the maximum likelihood estimator and it yields a variance estimator that is robust to the diffusion assumption of the model. To illustrate the proposed model, an application to diastolic blood pressure data from a follow‐up study on cardiovascular diseases is presented. Missing observations are handled naturally with this model.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

Synthesis and structure-activity relationships of novel dipeptides and reduced dipeptides as ligands for melanocortin subtype-4 receptor

A series of benzylic piperazines (e.g., 4 and 5) attached to an 'address element', the dipeptide H-D-Tic-D-p-Cl-Phe-OH, 3 has been identified as ligands for the melanocortin subtype-4 receptor (MC4R). We describe herein the structure-activity relationship (SAR) studies on the N-terminal residue of the 'address element'. Several novel dipeptides and reduced dipeptides with high MC4R binding affinities and selectivity emerged from this SAR study.     

A molecular basis for the physiological variation in shade avoidance responses: A tale of two ecotypes

Using two ecotypes of Stellaria longipes with contrasting responses to shade, we found that plants can differ in their responses to similar light cues, reflecting adaptations to their natural habitat. It was also observed that the plants could distinguish between distinct shade signals. Furthermore, the activity of wall modifying proteins, expansins and xyloglucan endotransglucosylase/hydrolase(s) (XTHs) was regulated during these responses. However, only expansin activity and gene expression profiles correlated with observed growth trends. The differential expression of expansins was light signal specific and ecotype specific and could account for both the trends in growth and their magnitude. We have thus established a potential molecular basis for the observed plasticity in responses to shade.Key words: shade avoidance, cell wall modification, expansins, XTHs, Stellaria longipes, phenotypic plasticity, light quality     

A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells

Uniform patterning of cells is highly desirable for most cellular studies involving cell-cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6x6 PIRE array show that cells patterned within minutes with good uniformity (48+/-6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1+/-0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.