Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.     

Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status

Background

Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL.

Methodology/Principal Findings

By introduction of 5 site-specific mutations (-DNSNN-), a β turn was incorporated between the 11^th and 12^th β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL.

Conclusions/Significance

Conversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them from dramatic yield losses from pathogenic fungi in an effective manner.     

The alpha helix dipole: screened out?

Aligned alpha helix peptide dipoles sum to a "macroscopic" dipole parallel to the helix axis that has been implicated in protein folding and function. However, in aqueous solution the dipole is counteracted by an electrostatic reaction field generated by the solvent, and the strength of the helix dipole may reduce drastically from its value in vacuum. Here, using atomic-detail helix models and Poisson-Boltzmann continuum electrostatics calculations, the net effective dipole moment, mu(eff), is calculated. Some initially surprising results are found. Whereas in vacuum mu(eff) increases with helix length, the opposite is found to be the case for transmembrane helices. In soluble proteins, mu(eff) is found to vary strongly with the orientation and position of the helix relative to the aqueous medium. A set of rules is established to estimate of the strength of mu(eff) from graphical inspection of protein structures.     

Attainment of peak bone mass and bone turnover rate in relation to estrous cycle, pregnancy and lactation in colony-bred Sprague-Dawley rats: suitability for studies on pathophysiology of bone and therapeutic measures for its management

Alteration in biochemical markers of bone turnover and bone mineral density (BMD) of whole body and isolated femur and tibia in relation to age, estrous cycle, pregnancy and lactation and suitability of use of rat as model for studies on pathophysiology of bone and therapeutic measures for its management were investigated. Immature rats (1, 1.5 and 2 month of age; weighing, respectively, 39.3 ± 1.0, 67.8 ± 2.4 and 87.2 ± 5.2 g) exhibited high rate of bone turnover, as evidenced by high serum osteocalcin and alkaline phosphatase and urine calcium/creatinine ratio. However, their BMD (whole body or of isolated long bones) was below measurable levels. Marked increase in body weight at 3 months (185.5 ± 5.2 g) was associated with low serum osteocalcin and alkaline phosphatase and urine calcium/creatinine ratio. Biochemical markers and BMD attained at puberty at 3 months were maintained until 36 month of age. No significant change in serum calcium was observed with increasing age or on any of the biomarkers during estrous cycle, and BMD of femur and tibia isolated during proestrus and diestrus stages was almost similar. Onset of pregnancy was associated with significant increase in serum total alkaline phosphatase and osteocalcin levels, but serum calcium, urine calcium/creatinine ratio or BMD of whole body or isolated long bones were not significantly different from that at proestrus stage. No marked change, except increase in body weight (P < 0.05), was also evident in these parameters between days 5 and 19 of pregnancy, irrespective of number of implantations in the uterus. A significant decrease in BMD of isolated femur (neck and mid-shaft regions) was observed on days 5 and 21 of lactation as compared to that during pregnancy or diestrus/proestrus stages of estrous cycle; the decrease being almost similar in females lactating two or six young ones. BMD of isolated tibia (global and region proximal to tibio-fibular separation point), though generally lower than that during cycle and pregnancy, was statistically non-significant. However, clear evidence of occurrence of osteoporosis during lactation, with decrease in BMD of >2.5 × S.D. in isolated femur (global, neck and mid-shaft) as well as tibia (global) was observed only when BMD data was analysed on T-/Z-score basis. Serum biochemical markers of bone turnover, too, were significantly increased in comparison to cyclic rats. Findings demonstrate marked increase in body weight and bone turnover during first 3 months of age, direct correlation between peak bone mass and onset of puberty at 3 months of age and increase in bone resorption rate during lactation. Finding of the study while might suggests possible use of rat as useful model for studies on bone turnover rate during lactation and post-weaning periods and extrapolation of the result to the human situation, but not in relation to ageing.     

Discovery and SAR of new benzazepines as potent and selective 5-HT(2C) receptor agonists for the treatment of obesity

We report on the synthesis, biological evaluation and structure-activity relationships for a series of 3-benzazepine derivatives as 5-HT(2C) receptor agonists. The compounds were evaluated in functional assays measuring [3H] phosphoinositol turnover in HEK-293 cells transiently transfected with h5-HT(2C), h5-HT(2A) or h5-HT(2B) receptors. Several compounds are shown to be potent and selective 5-HT(2C) receptor agonists, which decrease food intake in a rat feeding model.     

Synthesis and evaluation of antioxidative properties of a series of organoselenium compounds

A series of organoselenocyanate compounds 4a-d were synthesized utilizing 1,8-naphthalic anhydride as the building unit. These compounds were evaluated for their antioxidative activities against DMBA-PMA-induced oxidative stress in a two-stage mouse skin carcinogenic model. Compound 4d was found to have the maximum antioxidative property in comparison with the other compounds. Also, the pretreatment group showed better results than the concomitant treatment groups.