Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Plant use in three Pre-Pottery Neolithic sites of the northern and eastern Fertile Crescent: a preliminary report

The beginnings of agriculture throughout the Fertile Crescent are still not completely understood, particularly at the eastern end of the Fertile Crescent in the area of modern Iran. Archaeobotanical samples from Epipalaeolithic/PPNA Körtik Tepe in southeastern Turkey and from the Pre-Pottery Neolithic sites of Chogha Golan and East Chia Sabz in south western Iran were studied in order to define the status of cultivation at these sites. Preliminary results show the presence of abundant wild progenitor species of crops at the Iranian sites before 10600 cal. b.p., and very few wild progenitor species at Körtik Tepe dated to 11700–11250 cal. b.p. The Iranian sites also indicate size increase of wild barley grain across a sequence of 400 years through either cultivation or changing moisture conditions.     

Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dosedependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.     

Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, α-chymotrypsinogen A and α-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (ΔG ⁰ _X→D) associated with the process heat denatured (X) state ↔ GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ΔG ⁰ _N→X (native (N) state ↔ X state), ΔG ⁰ _X→D and ΔG ⁰ _N→D (N state ↔ D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ↔ D state.     

Cardioprotective role of extracellular vesicles: A highlight on exosome beneficial effects in cardiovascular diseases

Extracellular vesicles (EVs) are nano-sized vesicles, released from many cell types including cardiac cells, have recently emerged as intercellular communication tools in cell dynamics. EVs are an important mediator of signaling within cells that influencing the functional behavior of the target cells. In heart complex, cardiac cells can easily use EVs to transport bioactive molecules such as proteins, lipids, and RNAs to the regulation of neighboring cell function. Cross-talk between intracardiac cells plays pivotal roles in the heart homeostasis and in adaptive responses of the heart to stress. EVs were released by cardiomyocytes under baseline conditions, but stress condition such as hypoxia intensifies secretome capacity. EVs secreted by cardiac progenitor cells and cardiosphere-derived cells could be pinpointed as important mediators of cardioprotection and cardiogenesis. Furthermore, EVs from many different types of stem cells could potentially exert a therapeutic effect on the damaged heart. Recent evidence shows that cardiac-derived EVs are rich in microRNAs, suggesting a key role in the controlling of cellular processes. EVs harboring exosomes may be clinically useful in cell-free therapy approaches and potentially act as prognosis and diagnosis biomarkers of cardiovascular diseases.     

Adhesion of Yersinia enterocolitica to non-cultured epithelial cells from pig and rabbit ilea

A simple and reliable method was developed to isolate intact epithelial cells from pig and rabbit ilea and these were used to investigate the adhesion of Yersinia enterocolitica. Hydrophobic interaction was eliminated by treating the bacterial culture with 0.8 M tetramethyl urea (TMU). Virulent strains of Y. enterocolitica had significantly greater attachment than avirulent strains but both attached in a linear dose-dependant fashion. Epithelial cells prepared from pig ilea were attached to more readily than those prepared from rabbit ilea.     

Follicular waves during the oestrous cycle in Nili-Ravi buffaloes undergoing spontaneous and PGF2alpha-induced luteolysis

The objective of this study was to characterize the follicular waves and associated ovarian events during spontaneous and PGF(2alpha)-induced oestrous cycles in Nili-Ravi buffaloes. In Exp. 1, (n=13 oestrous cycles) follicular and luteal development was monitored by ultrasonography and jugular blood samples were collected simultaneously on alternate days. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves. The mean (+/-S.E.M.) length of the oestrous cycle was shorter (P<0.05) in buffaloes with two waves than in those with three waves (21.2+/-0.1 days versus 22.8+/-0.1 days). In Exp. 2, follicular dynamics were compared in buffaloes undergoing spontaneous (n=12 oestrous cycles) and PGF(2alpha)-induced (n=9) regression of the corpus luteum (CL). The dynamics of ovulatory follicular growth during the 3 days before oestrus were similar (P>0.05) in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis. These results show that (1) the majority of buffaloes had a two wave pattern of follicular growth and emergence of a third wave was associated with a longer luteal phase, and (2) follicular dynamics during the 3 days before oestrus were similar in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis.     

Effect of oestrus synchronization methods on oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes

The objective of this study was to determine the effect of oestrous synchronization methods on oestrous behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes. In Experiment 1, oestrous behaviour and timing of ovulation were determined from (n=34) oestruses. The mean (+/- S.E.M.) time of ovulation after the onset of standing oestrus was greater (P<0.05) in PGF(2alpha)-induced luteolysis (30.6+/-1.5h) compared to Ovsynch buffaloes (15.0+/-0.8h). In Experiment 2, pregnancy rates were compared between two methods of synchronization (detected oestrus and Ovsynch protocol) during the breeding and low breeding seasons. Pregnancy rates of buffaloes bred at detected oestrus (62.5%) or by the Ovsynch protocol (36.3%) during the breeding season did not differ significantly (P>0.05) from those which were inseminated during the low breeding season (55.5%) and (30.4%), respectively. This study demonstrates clearly that (1) timing of ovulation in Nili-Ravi buffalo is about 30h after the onset of standing oestrus and (2) buffaloes can be successfully synchronized with optimum fertility using either PGF(2alpha) alone (detected oestrus) or using (Ovsynch protocol) during low breeding season, to calve during the period when milk availability is short.     

Gene expression profiling of 3T3-L1 adipocytes exposed to phloretin

Adipocyte dysfunction plays a major role in the outcome of obesity, insulin resistance and related cardiovascular complications. Thus, considerable efforts are underway in the pharmaceutical industry to find molecules that target the now well-documented pleiotropic functions of adipocyte. We previously reported that the dietary flavonoid phloretin enhances 3T3-L1 adipocyte differentiation and adiponectin expression at least in part through PPARγ activation. The present study was designed to further characterize the molecular mechanisms underlying the phloretin-mediated effects on 3T3-L1 adipocytes using microarray technology. We show that phloretin positively regulates the expression of numerous genes involved in lipogenesis and triglyceride storage, including GLUT4, ACSL1, PEPCK1, lipin-1 and perilipin (more than twofold). The expression of several genes encoding adipokines, in addition to adiponectin and its receptor, is positively or negatively regulated in a way that suggests a possible reduction in systemic insulin resistance and obesity-associated inflammation. Improvement of insulin sensitivity is also suggested by the overexpression of genes associated with insulin signal transduction, such as CAP, PDK1 and Akt2. Many of these genes are PPARγ targets, confirming the involvement of PPARγ pathway in the phloretin effects on adipocytes. In light of these microarray data, it is reasonable to assume that phloretin may be beneficial for reducing insulin resistance, in a similar way to the thiazolidinedione class of antidiabetic drugs.     

Influence of phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter bacterium

In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 °C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes.