Novel Method For Isolation Research Articles

Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation

To study neurons or glia-specific mitochondria one needs to isolate these organelles from primary neuronal or astrocytic cell culture. This work provides novel method for isolation of functional and morphologically intact mitochondria from neurons and astrocytes in cell cultures. In the first step, mitochondria are released from cells by disruption of cell membranes using a nitrogen cavitation technique. This technique is based on rapid decompression of a cell suspension from within a pressure vessel. Mitochondria released from cell bodies are then separated from the rest of cell homogenate by Percoll gradient centrifugation. This is a relatively rapid technique that yields to very well coupled mitochondria that exhibited functional and morphological characteristics comparable to mitochondria isolated from brain tissue using common techniques. This technique thus will allow examination of mitochondria that are exclusively cell specific in origin.

Issues and Progress in Isolation of Susceptibility Genes of Essential Hypertension

Essential hypertension (EH) is thought to be a multifactorial disease. In Japan, there are more than 20 million cases of EH, which accounts for 80 to 90% of hypertension cases in Japan. It is very difficult to isolate the susceptibility genes of EH in familial linkage analysis such as sib-pair analysis and association studies such as case-control studies of candidate genes. Whole genome scanning and haplotype analysis using genetic markers will be widely conducted in the future. The present paper contains a review of issues and progress in isolation of susceptibility genes of EH, and an introduction to an effective novel method for isolation of susceptibility genes of EH.

A novel method for isolation of endothelial cells and macrophages from murine tumors based on Ac-LDL uptake and CD16 expression

Analysis of specific properties of tumor endothelium should be useful for development of novel antiangiogenic strategies. However, the isolation of pure endothelial cells from tumor tissues is still a fundamental problem. In this study, we have attempted to develop a reliable method for the isolation of endothelial cells from murine tumors. We found that the labeling with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-acetylated-low density lipoprotein (Dil-Ac-LDL), commonly used for this purpose, can result in the contamination of isolated endothelium by macrophages due to the overlapping staining patterns of these two distinct cell types. Therefore, we chose the CD16, which is expressed on macrophages but not endothelial cells, to better distinguish them when labeled with Dil-Ac-LDL. By using this method, we obtained pure populations of endothelial cells and macrophages from murine colorectal cancer tissues, showing characteristic morphological and functional properties of the either cell type. The endothelial cells were long spindle-shaped, spread on gelatin, formed tube-like structures on Matrigel and expressed MECA-32 but not CD68. In contrast, the macrophages were round-shaped, partially spread on gelatin, formed unorganized aggregates on Matrigel and expressed CD68 but not MECA-32. The additional analysis of normal and tumor tissues revealed a positive correlation between the relative numbers of tumor endothelial cells and macrophages, calculated as % total cells, as well as the respective relative number and tumor weight. The present method is hoped to be useful for the evaluation of tumor angiogenesis and antitumor immunity.

A novel method for isolation of Campylobacter spp. from environmental samples, involving sample processing, and blood- and antibiotic-free medium.

To develop a method that involves sample processing, and blood- and antibiotic-free medium for isolation and enumeration of Campylobacter spp. from environmental samples. The sample processing (preT) was standardized to minimize the population of competing bacteria. A blood- and antibiotic-free differential, Kapadnis-Baseri medium (KB medium) was formulated and tested for isolation of Campylobacter spp. in comparison with CAT medium. PreT-KB method was evaluated in comparison with the conventional viable count method and with the conventional most probable number (C. MPN) method for enumeration of Campylobcater from environmental samples. The results indicated that sample processing significantly reduced population of competing bacteria. The KB medium selected Gram-negative bacteria and differentiated Campylobacter from lactose-fermenting competing bacteria. The population of Campylobacter detected by preT-KB method was similar to that by conventional viable count method. While, the population of Campylobacter spp. determined by preT-KB method was higher than that by C. MPN method. In addition, the preT-KB method detected antibiotic sensitive campylobacters. The preT minimizes population of competing bacteria and the KB medium selects Gram-negative bacteria and differentiates Campylobacter from them. Therefore, Campylobacter can be isolated from environmental samples without using antibiotics. The preT-KB method is simple and facilitates isolation of antibiotic sensitive and enumeration of Campylobacter in the environmental samples. Therefore, the new method will be useful for isolation and enumeration of Campylobacter from water, food and sewage samples. Besides, it would also detect antibiotic-sensitive campylobacters, which are not detected by conventional viable count and MPN methods.

The alcohol-induced conformational changes in casein micelles: a new challenge for the purification of colostrinin.

Recent advances in protein separation technology have allowed for the isolation of whey proteins and peptides of significant biological importance. In this study, we report a novel method for isolation and purification of the neuroprotective proline-rich polypeptides, also known as Colostrinin (CLN). Although CLN was first isolated from ovine colostrum and characterized as a complex of small molecular peptides, its constituents are present also in other mammal colostrums. The previous purification protocols are very tedious, time consuming, and, due to the diverse characteristics of colostrum, also very difficult to validate. Thus, the aim of this study was to develop a simple protocol with a maximum recovery rate for CLN peptides. Here we demonstrate the two-step extraction/purification method that consists of methanol extraction and ammonium sulfate precipitation as the general principles. When compared with the original material, CLN obtained by this method shows (1) similar pattern of peptides in SDS PAGE, (2) identical amino acid analysis, characterized by high content of proline (22%), a high proportion of nonpolar amino acids, a low percentage of glycine, alanine, arginine, histidine, and no tryptophan, methionine, and cysteine residues, (3) similar pattern of HPLC profiles, and (4) its ability to induce IFN gamma and TNF alpha. More importantly, the protocol for the production of high-quality CLN can be accomplished in less than a 48 h timeframe. In addition, avoidance of excessively harsh conditions preserves the structure and biological activity of the peptides.

Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity

Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.

A novel method of isolation of a bone-morphogenetic-protein-like protein from ossein.

A new bone-morphogenetic-protein (BMP)-like protein has been isolated through a new protocol from a novel source, ossein. The BMP-like protein was hydrophilic and characterized through Fourier-transform IR studies, SDS/PAGE and coupled with a neutral binder, hydroxypropylmethylcellulose (HPMC) for control release. The IR spectrum of the protein showed peaks in tandem with BMP from bone matrix, and its molecular mass was in the range 18-21 kDa. Sustained release from the surface of HPMC was achieved for a period of 3 days.

A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic Separation

Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.

Novel method for isolation of major phenolic constituents from cashew (Anacardium occidentale L.) nut shell liquid.

Commercially available cashew (Anacardium occidentale L.) nut shell liquid (CNSL) mainly contains the phenolic constituents anacardic acid, cardol, and cardanol. These phenolic constituents are themselves heterogeneous, and each of them contains saturated, monoene, diene, and trienes in the fifteen-carbon side chain. This communication describes the separation of anacardic acid, cardol, and cardanol for industrial application. Anacardic acid was selectively isolated as calcium anacardate. The acid-free CNSL was treated with liquor ammonia and extracted with hexane/ethyl acetate (98:2) to separate the mono phenolic component, cardanol. Subsequently, ammonia solution was extracted with ethyl acetate/hexane (80:20) to obtain cardol.

A novel method for isolation of Helicobacter pylori from contaminated human and environmental specimens

A novel method for isolation of Helicobacter pylori from contaminated human and environmental specimens

A novel method for isolation of Helicobacter pylori from contaminated human and environmental specimens

A novel method for isolation of Helicobacter pylori from contaminated human and environmental specimens

A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy

Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.

Mechanism of Selective Release of Membrane Proteins from Human Erythrocytes in the Presence of Liposomes

Incubation of erythrocytes with liposomes results in the release of shed vesicles rich in glycosyl-phosphatidylinositol (GPI)-anchored proteins but poor in transmembranous proteins. We investigated the mechanisms of membrane protein polarization by examining the effect of the interaction between spectrin and membrane proteins on the release of a transmembranous protein, band 3, and a GPI-anchored protein, acetylcholinesterase (AChE), from erythrocyte ghosts. Polymerization of spectrin resulted in a 30-fold decrease in the released amount of band 3 per constant amount of shed vesicles but did not affect the amount of released AChE per constant amount of shed vesicles. On the other hand, the amount of released band 3 per constant amount of shed vesicles increased by cleaving the cytoplasmic part of band 3. Our results first demonstrated that the diffusibility of membrane proteins determined by steric hindrance between membrane proteins and protein mesh primarily determines the ease of localization of membrane proteins into shed vesicles. Taken together with the recent biophysical studies, we built a “fence selection model” that retrograding spectrin mesh sweeps diffusing band 3 molecules from the tip of the membrane crenated area toward the entry of the crenated area, but not AChE molecules. Our study describes a novel method for isolation of a large number of vesicles containing special and intact membrane proteins from cells not by using detergents or organic solvents, but by utilizing the fence effect between the cytoskeleton and membrane proteins.

Electrophysiological characterization of ion channels in osteoclasts isolated from human deciduous teeth

Ion channels contribute to several important processes in osteoclasts, including proton transport and volume regulation. Although ion channels have been described in osteoclasts from several species, little is known about their properties in human osteoclasts. We devised a method for isolation of authentic human osteoclasts from deciduous teeth undergoing root resorption, and characterized currents in these cells using patch-clamp techniques. Three types of K + current were identified. Hyperpolarization elicited an inwardly rectifying K + current in most osteoclasts, which was inhibited by Ba 2+ in a voltage- and time-dependent manner. Depolarization elicited an outwardly rectifying and tetraethylammonium-sensitive current, consistent with a large-conductance Ca 2+-dependent K + channel. In addition to these basal currents, extracellular adenosine 5′-triphosphate (ATP) elicited a linear current that was identified as a Ca 2+-dependent K + current, based on its reversal potential close to that predicted for K +, its blockade by quinine, and its activation by Ca 2+ ionophore. Last, an outwardly rectifying current was observed to activate spontaneously or in response to ATP, with properties of a swelling-activated Cl − current. This current reversed direction close to the Cl − equilibrium potential and was blocked by the anion channel blocker, niflumic acid, identifying it as a Cl − current. In summary, we have developed a novel method for isolation of authentic human osteoclasts and have characterized K + and Cl − currents. Cl − current mediates charge compensation during electrogenic H + transport, so activation of Cl − current may contribute to the stimulatory effects of extracellular ATP on bone resorption.

Biochemical and Structural Analysis of Isolated Mature Cores of Human Immunodeficiency Virus Type 1

Mature human immunodeficiency virus type 1 (HIV-1) particles contain a cone-shaped core structure consisting of the internal ribonucleoprotein complex encased in a proteinaceous shell derived from the viral capsid protein. Because of their very low stability after membrane removal, HIV-1 cores have not been purified in quantities sufficient for structural and biochemical analysis. Based on our in vitro assembly experiments, we have developed a novel method for isolation of intact mature HIV-1 cores. Concentrated virus suspensions were briefly treated with nonionic detergent and immediately centrifuged in a microcentrifuge for short periods of time. The resuspended pellet was subsequently analyzed by negative-stain and thin-section electron microscopy and by immunoelectron microscopy. Abundant cone-shaped cores as well as tubular and aberrant structures were observed. Stereo images showed that core structures preserved their three-dimensional architecture and exhibited a regular substructure. Detailed analysis of 155 cores revealed an average length of ca. 103 nm, an average diameter at the base of ca. 52 nm, and an average angle of 21.3 degrees. There was significant variability in all parameters, indicating that HIV cores are not homogeneous. Immunoblot analysis of core preparations allowed semiquantitative estimation of the relative amounts of viral and cellular proteins inside the HIV-1 core, yielding a model for the topology of various proteins inside the virion.

Novel method for isolation of adult porcine pancreatic islets with two-stage digestion procedure.

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37 degrees C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37 degrees C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 +/- 240,452 IEQ/pancreas (n = 14) and 3,294 +/- 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 +/- 13.4 microU/dish/h in response to 3.3 mM glucose and 104.1 +/- 12.9 microU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation.

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.

Transmembrane-domain trapping: a novel method for isolation of cDNAs encoding putative membrane proteins.

We have developed a method that enables us to isolate cDNAs of putative membrane proteins. The system is designed to isolate a cDNA which can provide the transmembrane domain to the extracellular part of the IL-2 receptor alpha chain. We constructed a p18Mac vector by putting part of the IL-2 receptor alpha chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRalpha. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor alpha chain will be expressed on the surface of the transfected cells. Otherwise, the chimeric protein will be either secreted or retained inside the transfected cells. We made a cDNA library using p18Mac and screened for cDNA clones which allowed the expression of the extracellular domain of the IL-2 receptor alpha chain on the cell surface. Of the 2000 clones screened, 5 clones were scored as positive. Partial sequence analysis revealed that one clone encoded the amyloid precursor protein, two others encoded mitochondrial proteins and the rest were new. These results suggest the system is effective in isolating cDNAs encoding putative membrane proteins.

A novel method for isolation of Chlamydia pneumoniae by treatment with trypsin or EDTA.

To establish a novel method for the efficient isolation of Chlamydia pneumoniae, experiments were performed to determine the effects of EDTA or trypsin treatment of C. pneumoniae on its adsorption and inclusion body formation. Treatment of C. pneumoniae with 0.1% trypsin or 1 mM EDTA significantly increased inclusion body-forming activity from 8,000- to 10,000-fold higher than that of the control. C. pneumoniae was successfully isolated in cultured cells which were inoculated with clinical specimens after treatment with 0.1% trypsin.

A novel method for isolation of magnetic bacteria without magnetic collection using magnetotaxis

A novel method was developed for isolating magnetic bacteria without magnetic collection using magnetotaxis. The method consists of incubation of sediments, enrichment of bacteria in the medium, isolation of enriched bacteria by colony formation, and optimization of conditions for growth and synthesis of magnetic particles. The water column above natural sediment, incubated at 25°C under dim light, and containing many species of bacteria, was employed as the inoculum. Collection of magnetic bacteria using magnets was not carried out. Ferric quinate was used as the main iron source in the liquid isolation medium. Due to iron sulfide precipitation, formation of black crystals was observed in the enriched culture of magnetic bacteria. Magnetic bacteria were purified by colony formation from enriched cultures which formed the black crystals. Culturing condition was optimized by addition of appropriate nutrients which behave as electron acceptors or donors. This method allows isolation of non-motile and non- or weakly-magnetotactic bacteria, which would not accumulate in the presence of an applied magnetic field. A sulphate-reducing magnetic anaerobe, RS-1, which is weakly magnetotactic, was isolated by this method. In addition, the successful isolation of RS-1 by this method suggests the presence of magnetic bacteria which exist in a non-magnetic state in sediments.