Phospholipids in parasitic protozoa

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs.     

High-Content Screening Microscopy Identifies Novel Proteins With a Putative Role in Secretory Membrane Traffic

Here we describe the establishment of microscope-based functional screening assays in intact cells that allow us to systematically identify new proteins involved in secretory membrane traffic, and proteins that can influence the integrity of the Golgi complex. We were able to identify 20 new proteins that affected either secretory transport, Golgi morphology, or both, when overexpressed in cells. Control experiments with human orthologs to yeast proteins with a role in membrane traffic, or already well characterized mammalian regulators of the secretory pathway, confirmed the specificity and significance of our results. Proteins localized to the Golgi complex or endoplasmic reticulum (ER) showed preferential interference in our assays. Bioinformatic analysis of the new proteins interfering with membrane traffic and/or Golgi integrity revealed broad functional variety, but demonstrated a bias towards proteins with predicted coiled-coil domains and repeat structures. Extending our approach to a much larger set of novel proteins in the future will be an important step toward a more comprehensive understanding of the molecular basis of the secretory pathway. It will also serve as an example for similar microscope-based screens addressing different biological questions.     

The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins

The immunoglobulins which are present on the membrane of B lymphocytes of different species are mobile in the plane of the membrane itself. This mobility results in the formation of spots of immunoglobulins when the cells are treated with divalent antibodies against immunoglobulins; the spots then combine into polar caps. The process of spot formation is not inhibited by different substances that inhibit cell metabolism, but is markedly inhibited in the cold. The formation of caps is followed by the disappearance of immunoglobulins from the cell membrane but, if the cells are not left in contact with the anti-immunoglobulin antiserum, there is a rapid resynthesis of new membrane immunoglobulins. It appears that the percentage of lymphocytes that resynthesize immunoglobulins is identical to that of cells originally carrying immunoglobulins, but the amount of surface immunoglobulins of each cell is increased after antiserum treatment. Many other membrane antigens behave in a similar way after treatment with the corresponding antisera; the possible role of the mobility of membrane proteins for the immunological functions of lymphocytes is discussed.     

近端小管表面细胞膜再循环的直接证据

利用大鼠活体肾小管显微穿刺技术，将一种新研制的膜蛋白标记试剂直接注入近端小管腔，内，用电镜放射自显影方法处理组织切片，追踪被内陷的膜蛋白，最后体视学定量分析。结果表明：在近端小管表面细胞膜和内吞泡之间存在着有效的持续不断的片段的细胞膜循环。     

Two Distinctive BMP-Carriers Induce Zonal Chondrogenesis and Membranous Ossification,Respectively; Geometrical Factors of Matrices for Cell-Differentiation

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/ BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.     

Adsorption of lipase on hollow fiber membrane chips

The amount of lipase from Mucor miehei adsorption on ultrafiltration polysulfone hollow fiber membrane chips has been determined using different lipase concentrations at three different temperatures, namely 30, 35, and 40 degrees C. It was experimentally shown that adsorption of lipase increases with temperature. The results were used to evaluate the constants found in the Langmuir adsorption isotherm model coupled with the Van't Hoff's relationship. A temperature dependence correlation for the amount of adsorbed lipase activity, alip,ads, and that present in the supernatant solution, alip,free was determined. The effect of varying the concentration on a cross-linking agent, namely, glutaraldehyde, to the membrane chips was also tested. It was found that, under the same operating conditions, the amount of lipase adsorbed on polysulfone membranes was increased dramatically after pre-treating the membrane with 1% Glutaraldehyde. However, increasing the concentration of the cross-linking agent has a low effect on the amount of lipase adsorbed.     

中性粒细胞分布数据序列小波变换研究

目的　研究小波分析结合复倒谱处理血膜中中性粒细胞分布数据信号的新方法及临床意义。方法　利用“虚拟盒计数法”方法采集血膜中的细胞分布数据信号 ,然后进行小波变换结合复倒谱分析方法处理。结果　提出血膜中细胞分布状态的“密集子”新概念。计算出小波变换复倒谱主极大值 ,并以此作为不同情况下血膜中中性粒细胞分布“密集子”间距的准确参数。结论　方法简单 ,能较好的反映血膜中细胞分布状态的微观组织结构特征 ,是血膜中细胞“密集子”表达定征的一种有效方法     

Fine structure of a fifth type of epithelial cell in the thyroid gland of the C3H mouse

A new cell with relatively little cytoplasm has been identified in the second kind of follicle in the C₃H mouse thyroid gland. It has as ultrastructural characteristics the presence of clusters of fiber in the cytoplasm, vesicles near the basal plasma membrane, and half desmosomes in the basal plasma membrane. It resembles the U cell found in the ultimobranchial follicle of the Fischer rat thyroid, but has a somewhat larger amount of granular reticulum. It is found in the follicle wall between other epithelial cells and the basement membrane, but occasionally is in contact with the lumen. It also occurs as a double layer in the follicle wall. Cell debris in the lumen of the follicle contains fibrils and may be the result of desquamation of this fibril-containing cell.     

Synthesis and antithrombogenicity of heparinized polyurethanes with intervening spacer chains of various kinds

Heparin was immobilized to polyetherurethaneurea membrane by covalent or ionic bondings with intervening spacer chains having different lengths and different terminal functional groups. The amount of immobilization of heparin and the release rate of immobilized heparin were controlled by the nature and the mode of bonding of spacer chains. The heparinized polyetherurethaneurea membranes became more in vitro antithrombogenic and suppressed more strongly the adhesion and activation of platelets, as the amount of immobilization increased. It was also shown that the membrane to which the low-molecular-weight fraction of heparin was immobilized was less stimulating to platelets.     

Differential exposure of epitopes on the human red cell antigen D (Rh).

The D polypeptide of the human Rh blood group system has a number of different epitopes on its surface. It is also known that there is considerable variation in the number of D antigen sites available to different human monoclonal anti-D antibodies. For instance, certain monoclonal antibodies recognise only a small number of sites on the red cell surface (about 9,000 sites/red cell on R1R2 cells) whereas other antibodies recognise a high number (about 20,000-30,000 sites/red cell). It has been found that cholesterol enrichment of the red cell membrane increases the number of sites available to those antibodies which recognize only a few sites but has no effect on those recognising many sites. The results are consistent with the view that access to some of the D epitopes is partially hindered by neighbouring molecules in the membrane and that alteration of the lipid content of the membrane changes it in such a way as to allow increased access to these obstructed epitopes.     

Permeability of PEUU membranes: their modification towards blood compatibility

An attempt was made to develop haemodialysis membranes using polyether urethane urea synthesised in our laboratory. It was observed that the processing parameters such as precipitation medium, precipitation temperature etc. can influence the porosity of the membrane and subsequently the permeability property. It was also noted that the permeability of the dried membrane was negligible even though it was kept in distilled water overnight before use. The effect of pH on permeability through the membrane was studied by dialysis experiment using mixtures of various components such as urea, creatinine, uric acid, inulin, albumin, NaCl and KCl at various pH. Standard cellulose acetate (CA) membrane was used for comparison. Membranes were also prepared using biomer solution by precipitating in distilled water at room temperature and the monomer, 2-hydroxy ethyl methacrylate (HEMA) was grafted onto it by glow discharge technique. It was found that the permeability was increased by HEMA grafting with some loss of tensile strength and strain. A comparative study of fibrinogen adsorption during dialysis and adsorption by direct exposure of samples to a mixture containing urea, uric acid, creatinine, dextran, fibrinogen and electrolytes like sodium and potassium ions was also done with 125I labelled fibrinogen. Platelet adhesion studies indicated that the number of adhered platelets was less on the HEMA grafted samples which may enhance blood compatibility. Finally, the membranes were subjected to different sterilization processes possible under wet conditions such as glutaraldehyde treatment and autoclaving. The contact angle, permeability, mechanical property and platelet adhesion studies indicated that the sterilization method can affect the performance of the membrane.     

Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity

Surface topography appears to be an important but often neglected factor in implant performance. In this study, fibroblasts were cultured on a range of porous polycarbonate (PC) membranes with well defined surface topography (track-etched micropores, 0.2-8.0 microm in diameter) and wettability gradients. The wettability gradient on the PC membrane surfaces was produced by treating the surfaces with corona from a knife-type electrode whose power increased gradually along the sample length. The PC membrane surfaces were characterized by scanning electron microscopy (SEM) and the water contact angle measurement. Fibroblasts were cultured on the corona-treated PC membrane surfaces with different micropore sizes for 1 and 2 days. The cells attached on the membrane surfaces were examined by SEM and the cell density on the surfaces was estimated by counting the number of attached cells along the wettability gradient. It was observed that the cells were adhered and grew more on the hydrophilic positions of the membrane surfaces than the more hydrophobic ones, regardless of micropore size. It was also observed that cell adhesion and growth decreased gradually with increasing micropore size of the membrane surfaces. It seems that the cell adhesion and growth were progressively inhibited as the membrane surfaces had micropores with increasing size, probably due to surface discontinuities produced by tract-etched pores. On the membrane surfaces with smaller micropore sizes, the cells seemed to override these surface discontinuities.     

Selective plasma membrane permeabilization by freeze-thawing and immunofluorescence epitope access to determine the topology of intracellular membrane proteins

The structural and functional characterization of membrane proteins includes assessment of their topology in the bilayer. In the present work, we successfully used an approach based on comparative epitope accessibility. The classical method of detergent permeabilization of fixed cells allowed antibodies to detect epitopes distributed at either side of each cellular membrane by immunofluorescent staining. Instead, freeze-thawing followed by fixation allowed antibodies to cross only the plasma membrane whereas all intracellular membranes remained impermeable. By combining the immunofluorescence results achieved with these two methods for a variety of known membrane proteins, we showed that epitope accessibility could be accurately determined in proteins residing in the plasma membrane or in intracellular compartments, including the endoplasmic reticulum, lysosomes, peroxisomes, different Golgi regions and the nucleus. Freeze-thawing neither changed the expected distribution of each tested protein nor permeabilized intracellular membranes to antibodies. It only permeabilized the plasma membrane. Furthermore, the protocol proved to be efficient in different kinds of cells, which include MDCK and FRT polarized epithelial cells, HeLa cells and fibroblasts. If the complete topology of an integral membrane protein is known, this method would allow to assign an orientation to epitopes recognized by a panel of monoclonal antibodies. It also avoids the use of toxic reagents for permeabilization. Thus, selective permeabilization of the plasma membrane by freeze-thawing provides an inexpensive and reliable method to investigate the topology of membrane proteins as well as the distribution of soluble proteins.     

Dysfunction of AQP7 in the periadventitial fat: A novel trigger of atherosclerosis

Vascular adventitial lesion as a new etiological factor of atherosclerosis (AS) has been confirmed by the results of several different animal models, and some evidence supports adventitial inflammation as the main cause, but the exact mechanism is still elusive. The data from some studies confirm that some link exists between the adventitial inflammation (which also might happen in the periadventitial fat) and atherosclerotic lesions. Aquaporin7 (AQP7) as an aquaglyceroporin, which regulates the permeation of glycerol through the cell membrane and located in the adipose tissue, shows some relationship with obesity. The result of the studies about AQP7-knockout mice and different expression of AQP7 in the cutaneous abdominal adipose tissue among people with different body types proved this phenomenon. Meanwhile, some degree of dysfunction of AQP7 has been proved in the obese. Until now, no study has shown us the data on the correlation of the expression of AQP7 in the periadventitial fat with the severity of atherosclerotic lesions. It is proposed that dysfunction of AQP7 in the periadventitial fat may trigger the adventitial inflammation. Attempts to confirm this hypothesis may lead to new directions in the study of the pathogenesis of AS and the development of a novel agent for this disorder.     

Preparation of poly(acrylic acid) modified polyurethane membrane for biomaterial by UV radiation without degassing.

Poly(acrylic acid) modified polyurethane (AA/PU) membranes were prepared by UV radiation without degassing. The chemical composition of the AA/PU membrane was studied by IR spectroscopy. In addition to those absorption peaks associated with pure PU, the absorption peak at 2400 cm-1 of poly(AA) was also found. The morphology of AA/PU membrane was studied by optical polarizing microscopy. We also measured the glass transition temperature and the decomposition temperature of the AA/PU membrane by differential scanning calorimetry and thermogravimetric analysis. A significant domain was found in the AA/PU membrane, which resulted in different glass transition temperature and decomposition temperature between AA/PU and pure PU membrane. The effect of AA content on the contact angle and water absorption of the AA/PU membrane was determined. It was found that the water content of AA/PU membrane increased with increasing AA content, whereas the contact angle decreased. By using Kaeble's equation and the contact angle data, the surface free energy of AA/PU membrane was determined. The increase of surface free energy resulted from the increase of the dispersion (gammad) term and polar (gammap) term. In order to evaluate the biocompatibility of these membranes, a cytotoxicity test and a cell adhesion and proliferation assay were conducted in cell culture. Immortal cells and primary lymphocytes were both used in this study. The results showed that these AA/PU membranes exhibited very low cytotoxicity and could support cell adhesion and growth. An animal primary test was also done in this study. It was found that the AA/PU membrane could possibly be employed in the treatment of bowel defect.     

Intracytoplasmic Lumina with and without Cilia in Both Normal and Pathologically Altered Nasal Mucosa

Two different types of intracytoplasmic lumina have been observed electron microscopically in pseudo-stratified and metaplastic human nasal rnucosa: one type contains microvilli and the other contains both microvilli and cilia, The latter type was seen only in pseudostratified columnar epithelium, while the former was also observed in different stages of metaplasia. From these observations we conclude that the type of intracytoplasmic lumina is determined by the differential potential of the cells in which they occur. Serial sectioning demonstrated that these structures some times communicate with the extracellular space, The distinctly different ultrastructure of microvilli lining the intracytoplasmic lumina and the foldlike extensions of the cell membrane favors an intracellular genesis rather than a formation by invagination of the cell membrane. It 'is suggested that intracytoplasmic lumina occur as a consequence of dysregulation in the process of epithelial maturation     

Usefulness as guided bone regeneration membrane of the alginate membrane

Alginate membrane is a new bioabsorbable, guided bone regeneration (GBR) membrane, which is placed directly on the surface of the bone defect. It is designed to drop a calcium chloride aqueous solution into the bone defect, which is filled with sodium alginate aqueous solution. Alginate membrane is an excellent agent for this procedure due to its close assimilation to the surface of the bone. In this study, we evaluated the short-term biocompatibility of alginate membrane in the bone defects of rat tibiae. GBR membrane availability was also examined. Consequently, we found that the healing process in bone defects covered with an alginate membrane was delayed in comparison with that of controls, however, the defect was restored to nearly original condition. In contrast, in the controls, bone defect repairs exhibited partitioning as a result of connective tissue involvement. Furthermore, we observed a relation between the sodium alginate concentration and the rate of absorption of the sodium alginate membrane. Absorption of a 1.5% sodium alginate membrane was slow. As a result, the compound was not absorbed completely and bone repairs resembled an hourglass. Moreover, the inflammatory response was absent surrounding the alginate membrane. The present findings suggested that the alginate membrane functions effectively as a GBR membrane. In addition, the alginate membrane derived from 3% calcium chloride and 1% sodium alginate was most suitable as a GBR membrane.     

The molecular basis of sperm-oocyte membrane interactions during mammalian fertilization

Gamete membrane interactions begin with adhesion (binding) of the sperm to the oocyte plasma membrane and culminate with fusion of the membranes of the gametes, thus creating the zygote through the union of these two very different cells. This review summarizes the molecular and cell biology of the cell-cell interactions between mammalian gametes. Recent research studies have provided new insights into the complexity of these interactions and into the importance of multimeric molecular networks and optimal membrane order in both sperm and oocytes for successful fertilization. Molecules that will be highlighted include cysteine-rich secretory protein 1 (CRISP1) and ADAMs [fertilin alpha (ADAM1), fertilin beta (ADAM2) and cyritestin (ADAM3)] on sperm, and integrins, CD9, and other integrin-associated proteins on oocytes, as well as other molecules. The characteristics of these gamete molecules are summarized, followed by discussions of the experimental data that provide evidence for their participation in gamete membrane interactions, and also of the specific roles that these molecules might play. Insights from a variety of research areas, including gamete biology, cell adhesion, and membrane fusion, are put together for a tentative model of how sperm-oocyte adhesion and fusion occur. The clinical relevance of correct gamete membrane interactions is also noted.