Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body¹. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes^2,3.The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.     

Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d 33.4 g/dl) has only about 5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d 24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d_50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d_50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r² = 0.995) and sickle erythrocytes (r² = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity. phosphorylation; protein phosphatase; urea; cell size; density     

Ear reconstruction after auricular chondritis secondary to ear piercing

The recent fad of high ear piercing in the pinna has led to an increased incidence of auricular chondritis, which leads to dissolution of the cartilage and residual ear deformity. The typical postpiercing chondritis deformity presents as a structural collapse of the superior helical rim, scaphal cartilage, and the adjacent antihelix. The skin envelope is usually preserved, but it may be scarred from the infectious process and from previous drainage incisions. In the present article, the authors present a systematic approach to reconstruction of these acquired ear deformities. Careful assessment of the residual tissue is requisite to planning and appropriate reconstruction. The greater the cartilage loss, the more structural support is required to expand the skin envelope to its normal size and shape. The choice of cartilage donor site is made on the basis of the size of the defect and may include ipsilateral or contralateral conchal cartilage, bilateral conchal cartilage, or costal cartilage. Redraping of the carefully dissected skin and fixation of the flaps to the newly reconstructed cartilaginous framework usually provide sufficient soft-tissue coverage. A temporal-parietal fascial flap is preserved for the rare cases of extensive full-thickness skin loss or badly damaged and scarred auricular skin.     

The Morphogenetic Potential of Transgenic Tobacco Plants Expressing Genes of Bacterial Glucanases with Distinct Substrate Specificity

The expression of the bacterial gene for thermostable -1,3-glucanase in transgenic tobacco plants was shown to induce substantial changes in plant morphogenetic potential, whereas the expression of -1,3; 1,4-glucanase did not affect essentially plant morphogenesis. Our results permit the suggestion that the expression of bacterial -1,3-glucanase in plants elevated the level of endogenous auxin.     

Esterase activity in summer population of sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae)

A key constraint on increasing wheat production in Iran and some neighbouring countries is Sunn pest which cause severe damage to vegetative growth stage of plant in the early season. It also feeds on wheat grain in late growth stage of plants thus damaged wheat grains loose their bakery properties. Because of injecting protease enzymes into the grain during feeding, enzymes degrade gluten proteins and cause rapid relaxation of dough which results in the production of bread with poor volume and texture. Organophosphorus insecticides are the main pesticides used to control the insect pest. However, suitable reduction in pest population has not been achieved partly due to resistance to pesticides. Esterase plays crucial roles in insect physiology and detoxifies a broad range of xenobiotics including insecticides. Enhanced esterase activity is a major mechanism if insecticide resistance and has been detected in a number of insects. To evaluate esterase activity adult bugs were collected from wheat field in Karaj area of Iran and transferred to the laboratory. For biochemical assay, two adult bugs (either males or females) were homogenized in 500 microl Na-phosphate buffer pH 7.2. The homogenates were centrifuged at 14000 g for 10 minutes at 4 degrees C. The supernatants as the enzyme source were pooled and stored at -20 degrees C for later use. For enzyme assay, 300 microl of supernatant was mixed with equal volume of substrates (30 mM alpha-naphthyl acetate or 30 mM beta-naphthyl acetate) and incubated at 30 degrees C for 30 minutes. Then, 50 microl of fast blue solution (150 mg fast blue B in 15 ml distilled water plus 35 ml 5% SDS) was added and esterase activity was determined in a spectrophotometer at 595 nm. Data showed that there are no differences in esterase activity between male and female. However, There was significant differences between hydrolysis of substrates, alpha-naphthyl acetate and beta-naphthyl acetate. Insect esterase hydrolyzes alpha-naphthyl acetate much more than beta-naphthyl acetate.     

Synthesis of the racemate of (Z)-exo-alpha-bergamotenal,a pheromone component of the white-spotted spined bug,Eysarcoris parvus Uhler

The racemate of (Z)-exo-alpha-bergamotenal, a sex pheromone component of the white-spotted spined bug, was synthesized from racemic exo-alpha-bergamotene by a five-step sequence involving regioselective epoxidation and (Z)-selective Wittig olefination reactions. The 1H- and 13C-NMR spectra of the synthetic sample were identical with those of the natural material.     

Preparation of N-substituted aryl and alkyl carbamates and their inhibitory effect on oat seed germination

A series of N-substituted aryl and alkyl carbamates (RNHCOOR'; R: aryl, alkyl; R': aryl, alkyl) was prepared and screened for inhibitory activity toward the germination of oat seeds. The activity of each compound was compared with that of chlorpropham (isopropyl 3-chlorocarbanilate). Some of the synthetic carbamates possessing the N-(phenylthio)methyl group, PhSCH2NHCOOR', showed inhibitory activity close or comparable to that of chlorpropham.