Wheat Germ Lectin Research Articles

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

Differential glycosylation of tissue non-specific alkaline phosphatase in mesenchymal stromal cells differentiated into either an osteoblastic or adipocytic phenotype

It has long been known that tissue non-specific alkaline phosphatase (TNAP) is essential for the correct formation of bone, as altered expression or function of this enzyme results in hypophosphatasia, a disease characterised by compromised bone structure, density and strength. However, recent evidence strongly suggests that the enzyme also has a role in lipid accrual and adipogenesis, a function that seems far removed from bone formation. Given that mesenchymal stromal cells (MSCs) are progenitors of both osteoblasts and adipocytes, the question arises of how TNAP is regulated to potentially have a different function when MSCs undergo either osteogenesis or adipogenesis. As the primary protein sequence is unchanged for the enzyme during both types of differentiation, any differences in function must be attributed to post-translational modification and/or localisation. We therefore examined the location of TNAP in bone- or adipose-derived MSCs differentiated into an adipocytic phenotype and compared the glycosylation state of the enzyme in MSCs differentiated into either osteoblasts or adipocytes. TNAP was found to co-locate with perilipin around lipid droplets in MSCs from bone, subcutaneous- and visceral adipose tissue during adipocytic differentiation. Treatment of TNAP with wheat germ lectin followed by electrophoresis showed minor differences in glycosylation between the phosphatase isolated from cells from these tissues, whereas electrophoresis after neuraminidase digestion highlighted differential glycosylation between cell types and during adipogenesis and osteoblastogenesis. This infers that post-translational modification of TNAP is altered during differentiation and is dependent on the eventual phenotype of the cells.

Morphofunctional pecularrities and sialoglicans in lungs from offspring born from female rats with thyroid gland hyperfunction

Background. Over the last decade, there has been a global pending to increase the incidence of chronic obstructive pulmonary disease by 25% in men and 69% in women. Objective. To study the role of sialoglycans according to wheat germ lectin receptors (WGA) and morphofunctional features of offspring rats, which developed in conditions of hyperfunction of the thyroid gland of the maternal organism. Methods. The experimental study was performed on the offsprings of 20 females (10 control, 10 experimental) rats of the Wistar line. For 20 days before coupling the females of the experimental group received the drug L-thyroxine (Berlin-Chemie) at a dose of 150 μg / kg body weight. After euthanasia by overdose of ether anesthesia, the offspring from control and experimental females were taken lungs on the 1st, 10th, 20th and 40th day of postnatal development. Histological material was fixed in 4% neutral formalin, compacted and poured into paraffin blocks, made sections 5-7 μm thick. Review preparations were stained with hematoxylin and eosin according to standard methods. Wheat germ lectin WGA (Triticum vulgare) labeled with peroxidase-specific DGlcNAc NeuNAc was used to detect sialoglycans. Results. On the 1-40th day of postnatal development, the lungs of rats of the intact group show up typical morphology characteristic of rat lungs. On the tenth day of postnatal development in the offsprings lungs from females receiving L-thyroxine visually increases the number of alveoli, indicating an intensification of morphogenesis of lung tissue. Lectinohistochemical studies showed that on the first day of postnatal development in the lungs of rats of the intact group, receptors for sialospecific lectin WGA were identified on the surface of ciliated cells of the bronchial tree and vascular endothelium, while in the experimental group receptors for this lectin were found significantly in alveolar macrophages, visualised more often than in the lungs of the offspring from rats of the control group. Whereas on the 20th-40th day of postnatal development the number of macrophages with the presence of sialoglycans decreased. Conclusion. Studies have shown that in the lungs of offsprings from females receiving L-thyroxine, there are processes of intensification of the structural components of the lungs and the appearance of alveolar macrophages with the presence of sialospecific lectin WGA receptors, the number of which decreases during postnatal ontogenesi, that indicating a decrease of the immunity.

Preparation of Polymer Nanocarrier Material and Its Application in B-Cell Lymphoma

The β-cyclodextrin (β-CD) is coupled with polyethyleneimine (PEI 600 Da) to produce a polymer nanocarrier material (PyD-W) with good biocompatibility and high transfection rate. First, the test was performed to study the influence of different factors on the transfection efficiency of PyD-W materials in terms of cell type and transfection system. Then the effect of adding wheat germ agglutinin on the material-cell membrane binding when transfecting cells by PyD-W materials was studied. The influence of temperature and cell phagocytosis inhibitors on the entire transfection process were taken into consideration at the same time. In the end, the escape ability of PyD-W material carrying plasmid DNA in lysosomes was studied. After analyzing the performance of PyD-W material transfected cells, the appropriate transfection staining conditions was selected to be applied in the study of the inhibitory properties of mouse B-cell lymphoma cells. In the experiment, the results showed that the cell type had a great influence on the material. The N/P ratio should not be set too high. Prolong the transfecting and fostering time appropriately could increase the transfection efficiency. The addition of wheat germ lectin would significantly reduce the enrichment ability on the cell surface of plasmid DNA carried by PyD-W. Increasing the temperature could also increase the transfection efficiency of PyD-W materials, cell phagocytosis inhibitors would not have a significant impact on transfection, and the accumulation of PyD-W materials in lysosomes would be gradually released from lysosomes with time going by. According to the above results, PyD-W carrying plasmid DNA was transfected into mouse B lymphoma cells and normal B cells using similar transfection methods. The results show that B lymphoma cells (38B9) corresponded significant mRNA levels is lower than the mRNA level of normal B cells (P &lt; 0.05). It is detected by cell count and CCK-8 kit that the growth of cells in the group overexpressing PyD-W is significantly inhibited (P &lt; 0.01).

Synthesis of magnetic chitin–adsorbent for specific proteins

A simple, convenient and inexpensive method for the preparation of magnetic chitin composite, in which magnetite particles are densely covered with the polysaccharide shell has been developed. Two-step procedure for magnetic chitin preparation includes: (i) adsorption of chitosan onto magnetite particles and (ii) N-selective acetylation of chitosan to produce magnetic chitin. The composite combines the magnetic properties of magnetite and the adsorption properties of chitin. The synthesized magnetic chitin is an efficient adsorbent of β-d-GlcNAc-specific lectins and lysozyme. The adsorption capacity of magnetic chitin for wheat germ (Triticum vulgaris) and potato (Solanum tuberosum) lectins was in the range of 67–86 mg g−1 of the adsorbent. Magnetic chitin showed the high capacity for enzyme–lysozyme. Synthesized adsorbent is environmentally friendly and recyclable. Magnetic chitin may be used as a promising multi-purpose adsorbent of pollutants of organic or inorganic nature.

Trehalose mediated stabilisation of cellobiase aggregates from the filamentous fungus Penicillium chrysogenum

Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 μg of sugar/μg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins.

Determination of serum bone-specific alkaline phosphatase isoenzyme activity in captive Asian elephants (Elephas maximus) using an agarose gel electrophoresis method.

The bone-specific alkaline phosphatase (ALP) isoenzyme activity was measured in 51 serum samples from four captive Asian elephants (Elephas maximus) using a conventional method with wheat germ lectin precipitation and a commercial agarose gel electrophoresis (AGE) kit; the isoenzymes were designated as bone-specific ALP (BAP) and ALP isoenzyme 3 (ALP3), respectively. This study examined the suitability of the AGE kit for analyzing blood biochemistry in Asian elephants. The serum ALP3 and BAP activities were strongly positively correlated and met the evaluation criteria for agreement using Bland-Altman analysis. The results indicate that the AGE kit can be used to examine the blood biochemistry in Asian elephants instead of the conventional method.

Cancer cell death induced by nanomagnetolectin

Magnetic nanoparticles represent a new paradigm for molecular targeting therapy in cancer. However, the transformative targeting potential of magnetic nanoparticles has been stymied by a key obstacle-safe delivery to specified target cells in vivo. As cancer cells grow under nutrient deprivation and hypoxic conditions and decorate cell surface with excessive sialoglycans, sialic acid binding lectins might be suitable for targeting cancer cells in vivo. Here we explore the potential of magnetic nanoparticles functionalized with wheat germ lectin (WGA) conjugate, so-called nanomagnetolectin, as apoptotic targetable agents for prostate cancer. In the presence of magnetic field (magnetofection) for 15min, 2.46nM nanomagnetolectin significantly promoted apoptosis (∼12-fold, p value <0.01) of prostate cancer cells (LNCaP, PC-3, DU-145) compared to normal prostate epithelial cells (PrEC, PNT2, PZ-HPV-7), when supplemented with 10mM sialic acid under nutrient deprived condition. Nanomagnetolectin targets cell-surface glycosylation, particularly sialic acid as nanomagnetolectin induced apoptosis of cancer cells largely diminished (only 2 to 2.5-fold) compared to normal cells. The efficacy of magnetofected nanomagnetolectin was demonstrated in orthotopically xenografted (DU-145) mice, where tumor was not only completely arrested, but also reduced significantly (p value <0.001). This was further corroborated in subcutaneous xenograft model, where nanomagnetolectin in the presence of magnetic field and photothermal heating at ∼42°C induced apoptosis of tumor by ∼4-fold compared to tumor section heated at ∼42°C, but without magnetic field. Taken all together, the study demonstrates, for the first time, the utility of nanomagnetolectin as a potential cancer therapeutic.

Telocytes in exercise-induced cardiac growth.

Exercise can induce physiological cardiac growth, which is featured by enlarged cardiomyocyte cell size and formation of new cardiomyocytes. Telocytes (TCs) are a recently identified distinct interstitial cell type, existing in many tissues and organs including heart. TCs have been shown to form a tandem with cardiac stem/progenitor cells in cardiac stem cell niches, participating in cardiac regeneration and repair. Although exercise‐induced cardiac growth has been confirmed as an important way to promote cardiac regeneration and repair, the response of cardiac TCs to exercise is still unclear. In this study, 4 weeks of swimming training was used to induce robust healthy cardiac growth. Exercise can induce an increase in cardiomyocyte cell size and formation of new cardiomyocytes as determined by Wheat Germ Lectin and EdU staining respectively. TCs were identified by three immunofluorescence stainings including double labelling for CD34/vimentin, CD34/platelet‐derived growth factor (PDGF) receptor‐α and CD34/PDGF receptor‐β. We found that cardiac TCs were significantly increased in exercised heart, suggesting that TCs might help control the activity of cardiac stem/progenitor cells, cardiomyocytes or endothelial cells. Adding cardiac TCs might help promote cardiac regeneration and renewal.

Lectin-histochemical assessment of carbohydrate determinants in tumour cells of main histological types of gastric cancer

Aim. 69 cases of gastric cancer were investigated, among them 31 cases of diffuse type, 25 cases of intestinal and 13 mixed-type, using lectins of eight specificities: wheat germ, peanut, edible snail, common elder, laburnum tree, soybeans, lentils and mistletoe.Methods and results. Maximum frequency of the high lectin binding was found in the diffuse type for common elder (72.4%), edible snail and soybean lectins (67.7% each). Intestinal type of the gastric cancer was characterized by high rate of tumor cell binding to peanut lectins (52%) and wheat germ lectins (48%). In mixed type – with lectins of peanut and soybeans (53.8% each). Meanwhile lectins exhibiting high binding frequency are characterized by a more intensive binding in comparison with the normal mucosa in the same case. Cases of vascular invasion with tumour cells and without it were compared. In the intestinal type of the gastric cancer the maximum degree of staining difference was with edible snail and common elder lectins with the predominance in the group with invasion (26.4% and 20.2%, respectively). In the diffuse type of the gastric cancer, the highest frequency of intense binding was in the group with invasion with common elder (68%), edible snail (63%), laburnum tree (61.5%), and lentils lectins (57.8%). In the mixed type of the stomach cancer an intensive staining in the presence of vascular invasion was with peanut, soybean (53.8% each) and wheat germs lectins (42.2%).

Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

Lectin-decorated nanoparticles enhance binding to the inflamed tissue in experimental colitis

A major limitation in the drug treatment of inflammatory bowel disease is the inability to deliver the drug selectively towards the inflamed tissues. Nanotechnology-based drug delivery systems have led to an amelioration of the therapeutic selectivity but still the majority of the entrapped drug is eliminated without exercising a therapeutic effect. Here, lectin-decorated drug loaded nanoparticles (NP) are suggested for active targeting and selective adhesion to the inflamed tissue in experimental colitis. Peanut (PNA) and wheat germ (WGA) lectins were covalently bound to the surface of NP and were tested for their stability and degree of bioadhesion in cell culture. In-vivo, the selectivity of bioadhesion and distribution of NP throughout the intestinal tract as well as the therapeutic benefit for glucocorticoid loaded lectin-NP was studied in murine colitis models. Quantitative adhesion analyses showed that lectin-conjugated NP exhibited a much higher binding and selectivity to inflamed tissue compared to plain NP (PNA conjugates: 52.2±5.6%; WGA conjugates: 22.0±0.8%; plain NP: 18.6±9.8%). Lectin-associated NP revealed a further increase in the selectivity of bioadhesion towards inflamed tissues which partially translates into increased therapeutic efficiency. In terms of therapeutic efficiency, all glucocorticoid containing formulations revealed an enhanced therapeutic effect with lectin conjugates especially PNA-NP (myeloperoxidase: 55±37U/g; TNF-alpha: 3880±380U/g) compared to plain NP (myeloperoxidase: 145±98U/g; TNF-alpha: 6971±1157U/g). Targeted NP by using lectins, especially with PNA, as stable targeting moiety in the gastrointestinal tract appears to be a very promising tool in future treatment of inflammatory bowel disease.

Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.

The Effect of Ovine Placenta Extract, on Mammogenesis, Lactogenesis and Galactopoiesis in Sheep

The aim of the present study was to evaluate the effect of ovine placenta extract, obtained by affinity chromatography on immobilized wheat germ lectin, on mammogenesis, lactogenesis, and galactopoiesis in pregnant and lactating sheep. Placental extract (100 \mug/mL protein concentration) was subcutaneously injected to pregnant sheep in days 1, 4, 7, 10, and 13 of the experiment (totally 10 mL placenta extract/animal) alternatively on the right and left side of the neck. Placental extract induced a more intense development of udder parameters; udder circumference increase was of 16.73%, anteroposterior length increase was of 15.9%, and latero-lateral length of 11.1%. Placental extract administration resulted in 7.92% increase in milk yield. Lactating ewes were monitored for milk production for 6 days before the experiment, after placental extract administration from day 7 to day 14 of the experiment, twice a day, and they presented 14.14% higher milk yield in comparison with the control group, no significant differences in milk lactose and protein content, but an increase in milk fat (6.2%). It may be concluded that the glycoprotein or glycopeptide isolated from sheep placenta by affinity chromatography on wheat germ lectin increased mammogenesis, lactogenesis, and galactopoiesis in sheep.

A glycosyltransferase-enriched reconstituted membrane system for the synthesis of branched O-linked glycans in vitro

Mimicking the biochemical reactions that take place in cell organelles is becoming one of the most important challenges in biological chemistry. In particular, reproducing the Golgi glycosylation system in vitro would allow the synthesis of bioactive glycan polymers and glycoconjugates for many future applications including treatments of numerous pathologies. In the present study, we reconstituted a membrane system enriched in glycosyltransferases obtained by combining the properties of the wheat germ lectin with the dialysable detergent n-octylglucoside. When applied to cells engineered to express the O-glycan branching enzyme core2 beta (1,6)-N-acetylglucosaminyltransferase (C2GnT-I), this combination led to the reconstitution of lipid vesicles exhibiting an enzyme activity 11 times higher than that found in microsomal membranes. The enzyme also showed a slightly higher affinity than its soluble counterpart toward the acceptor substrate. Moreover, the use of either the detergent re-solubilization, glycoprotein substrates or N-glycanase digestion suggests that most of the reconstituted glycosyltransferases have their catalytic domains in an extravesicular orientation. Using the disaccharide substrate Galβ1-3GalNAc-O-p-nitrophenyl as a primer, we performed sequential glycosylation reactions and compared the recovered oligosaccharides to those synthesized by cultured parental cells. After three successive glycosylation reactions using a single batch of the reconstituted vesicles and without changing the buffer, the acceptor was transformed into an O-glycan with chromatographic properties similar to glycans produced by C2GnT-I-expressing cells. Therefore, this new and efficient approach would greatly improve the synthesis of bioactive carbohydrates and glycoconjugates in vitro and could be easily adapted for the study of other reactions naturally occurring in the Golgi apparatus such as N-glycosylation or sulfation.

Three‐dimensional architecture of the renal inner medulla of the desert rodent Dipodomys merriami: potential impact on the urinary concentrating mechanism

We hypothesize that the inner medulla (IM) of Dipodomys merriami, a desert rodent that concentrates its urine to over 6000 mOsm/Kg water, provides extreme examples of architectural features most necessary for production of highly concentrated urine. Three‐dimensional architecture of vasculature and nephron segments in the IM was assessed with digital reconstructions from physical tissue sections. Descending and ascending vasa recta (DVR, AVR), descending thin limbs (DTLs), ascending thin limbs (ATLs), and collecting ducts (CDs) were identified by indirect immunofluorescence using antibodies that label segment‐specific proteins associated with solute and water transport and lectins (UT‐B, wheat germ lectin, AQP1, ClC‐K1, and AQP2, respectively). CD clusters form the central organizing motif in the IM. Transverse sections along the corticopapillary axis show nonuniform distribution of AQP‐1‐positive DTLs and uniform distribution of ATLs. Countercurrent exchange occurs only at the perimeter of CD clusters as DVR lie distant from CDs. Electron microscopy shows fenestrated AVR adhering to CDs. This architectural arrangement raises the possibility that fluid and solute reabsorbed from CDs diffuses preferentially into AVR. An additional factor increasing and defining axial compartmentation, and thereby restricting diffusive exchange, would be interstitial cell architecture. DK16294

Determination of alkaline phosphatase isoenzymes and isoforms in dog serum by a simple anion exchange chromatographic method

The well-known marker of bone formation is bone alkaline phosphatase, which is produced by osteoblasts and is required for osteoid formation and matrix mineralization. Alkaline phosphatase (ALP, EC 3.1.3.1) is a zinc-containing glycoprotein enzyme catalyzing the hydrolysis of phosphomonoesters at alkaline pHs. ALP is synthesized in a variety of tissues and has different isoenzymes and isoforms. Four variants of ALP have been identified in domestic animals: bone ALP, intestinal ALP, liver ALP, and, in dogs, corticosteroid-induced ALP. Efforts have been made to develop analytical techniques that can differentiate between the different isoenzymes of ALP in animals. These methods include heat inactivation, gel electrophoresis, precipitation with wheat-germ lectin, and, recently, radioimmunoassay and enzyme-linked immunosorbent assay. We developed a simple, fast, and less expensive chromatographic method for differential determination of ALP isoenzymes and isoforms in serum samples. Three peaks of ALP activity were identified in chromatographic fractions. These were related to bone, liver, and steroid isoenzymes of ALP.

Probing Membrane Domains and Diffusion Barriers in Live Sperm Cells Using Fluorescent Particle Tracking

Polarized cells, e.g. neurones and spermatozoa, characteristically contain compositionally distinct domains in their plasma membranes that are commensurate with specialized function. Elucidating the mechanisms that generate and maintain these heterogeneities is fundamental to understanding many of the processes involved in cell differentiation. Spermatozoa are excellent models for studying membrane compartmentalization as several distinct domains are present on the surface of the head [1,2]. In this work we probe the nature of the barriers that separate these regions by analyzing the trajectories of individual fluorophore-labelled lipids and proteins as they diffuse within and between domains.The probes used were wheat germ (WGA) lectin, DOPE, DiIC16 and cholera toxin β-subunit (CTXB) and were delivered either as single molecules from a nanopipette [2] or from suspension. Results showed that single protein and lipid molecules exchanged freely between all domains on the head plasma membrane. Conversely, particles of DiIC16 and clusters of CTXB cross-linked GM1 gangliosides, ranging in size from 0.5 to 2.0 microns, showed confinement and were unable to traverse domain boundaries. We hypothesise that a mass filter is present within the membrane that is permissive to single molecules but not multimolecular complexes. Relocation and assembly of these molecules and complexes on the sperm head in response to external stimuli is likely to be important in the developmental processes that lead to successful fertilization.[1] James et al. (2004) Journal of Cell Science 117 6485-6495.[2] Bruckbauer et al. (2007) Biophys. J. 93 3120-3131.

Biomatrix on the basis of polypropylene mesh and fetal fibroblasts

A technology for obtaining a biomatrix on the basis of polypropylene mesh, used in clinical surgery, and cultured fetal fibroblasts is proposed. The method was developed on experimental animal cells. It includes culturing of rat fetal fibroblasts on fibers of a polypropylene mesh, treated with wheat germ lectin. This technology can be used for obtaining a biomatrix with fibroblasts of different origin. This biomatrix can be used in experimental and clinical surgery for dressing and stimulation of effective healing of mechanical injuries to the connective tissue.

Chitin in the Peritrophic Membrane of &lt;I&gt;Acarus siro&lt;/I&gt; (Acari: Acaridae) as a Target for Novel Acaricides

The peritrophic membrane in Acarus siro L. (Acari: Acaridae) is produced by distinct cells located in the ventriculus. In this study, the chitin inside the peritrophic membrane was detected using wheat germ-lectin conjugated with colloidal gold (10 nm). The chitin fibrils of the peritrophic membrane were a target for chitin effectors, including 1) chitinase, which hydrolyzes chitin fibers inside the peritrophic membrane; 2) calcofluor, which binds to chitin and destroys the peritrophic membrane mesh structure; and 3) diflubenzuron, which inhibits chitin synthesis. In addition, soybean trypsin protease inhibitor (STI) and cocktails of chitinase/calcofluor, diflubenzuron/calcofluor and chitinase/STI were tested. These compounds were supplemented in diets and an increase of population initiated from 50 individuals was observed after 21 d of cultivation. Final A. siro densities on experimental and control diets were compared. The chitin in the peritrophic membrane was determined to be a suitable target for novel acaricidal compounds for suppressing the population growth of A. siro. The most effective compounds were calcofluor and diflubenzuron, whereas the suppressive effects of chitinase and STI were low. The failure of chitinase could be due to its degradation by endogenous proteases. The combination of chitinase and STI suppressed A. siro population growth more effectively than when they were tested in oral admission separately. The combinations of calcofluor/chitinase or calcofluor/difluorbenzuron showed no additive effects on final A. siro density. The presence of chitin in peritrophic membrane provides a target for novel acaricidal compounds, which disrupt peritrophic membrane structure. The suitability of chitin effectors and their practical application in the management of stored product mites is discussed.