Low-speed Differential Centrifugation Research Articles

Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC-MS/MS.

Metaproteomics is one approach to analyze the functional capacity of the gut microbiome but is limited by the ability to evenly extract proteins from diverse organisms within the gut. Herein, we have developed a pipeline to optimize sample preparation of stool obtained from germ-free (GF) mice that were gavaged a defined community of 11 bacterial strains isolated from the human gut. With 64% more proteins identified, bead-beating was confirmed to be an indispensable step for the extraction of bacterial proteins, especially for Gram-positive bacteria. Bacterial enrichment from mouse fecal samples was further optimized by evaluating three different methods: (1) a high-speed differential centrifugation (HCE) or (2) a low-speed differential centrifugation (LCE) and (3) a filter-aided method (FA). The HCE method was associated with dramatic loss of bacteria and 71% less recovery of bacterial proteins than the LCE method. Compared with LCE, the FA method also showed dramatic loss of the amount of bacteria recovered and decreased protein identifications from Gram-positive bacteria in the stool samples. Ultimately, LCE may provide an alternative and complementary method for enriching bacteria from small amounts of mouse fecal samples, which could aid in investigating bacterial function in health and disease.

Isolation, Partial Purification, and Immunogenicity of Flagella fromTritrichomonas foetus

Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens.

Isolation and characterization of flagella from Trichomonas vaginalis.

As a first step in the biochemical and biomedical analyses of flagella from Trichomonas vaginalis the flagella were isolated, purified, and analyzed. The flagella were detached by mechanical shearing and a crude flagellar preparation was obtained by low-speed differential centrifugation. The crude flagellar preparation was subjected to further purification by discontinuous sucrose density-gradient centrifugation. Electron micrographs (EM) of the purified flagella showed the typical 9 + 2 axonemal arrangement. The structural integrity and the flagellar membrane were not destroyed by the deflagellation method or the purification scheme employed. The flagellar preparations were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation contained many flagellar enriched proteins ranging from 20 to 120 kDa. Three major proteins of 65 kDa and a doublet of about 50-58 kDa were observed. The protein patterns and EM appearance of the fractions were highly reproducible.

Isolation of platelets from human blood by free-flow electrophoresis

We are using platelets as a model system in an investigation into the alterations in the calcium-stimulated ATPase which occur in primary hypertension in man. In order to make valid comparisons between platelets prepared from normal subjects and from hypertensive patients it is essential that the purity of the platelet preparation be high and reproducible. Most clinical studies on the human platelet require only 20-30 ml blood and low-speed differential centrifugation is very successful in obtaining acceptable recoveries from such samples. On the other hand many biochemical studies are carried out on platelet+ch plasma derived from one or more units of donated blood: in these cases high recoveries are relatively unimportant. For our own studies we needed to maximise the recovery of platelets from 150-200 ml of blood. Purification by differential centrifugation required multiple cycles of sedimentation, resuspension and washing which was both timeconsuming and led to progressive platelet disruption. Preliminary to our biochemical studies therefore we have investigated methods of harvesting platelets from human blood. A new method is presented in this paper which uses free-flow electrophoresis. This technique involves injection of a buffy-coat fraction into a vertically moving column of buffer across which a potential difference is imposed. Because of the lower surface charge density on the platelets compared to that on erythrocytes or white cells, the platelets can be resolved from these other blood cell elements. The functional integrity of the platelet preparation has been assessed by measurement of nucleotide levels in the cells before and after stimulation by a variety of agents which cause aggregation.

O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine.

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.

39] Preparation of inside-out thyroid follicles for studies on transcellular transport (transcytosis)

This chapter describes the preparation of inside-out thyroid follicles for studies on transcellular transport also referred to as “transcytosis.” Studies on the mechanism of transepithelial passage of thyroglobulin can be facilitated by using an in vitro system where access to the apical plasma membrane is possible. Such a system is provided by preparations of inside-out follicles from pig thyroid gland. The chapter describes the conditions that lead to the formation of inside-out follicles and allows studying the transepithelial vesicular traffic from the apical to the basolateral cell surfaces. Collagenase digestion of thyroid tissue and application of shearing forces by pipetting result in the dissociation of tissue into intact segments of follicles opened during pipetting. Fibroblasts and parafollicular and endothelial cells are separated from follicle segments by low-speed differential centrifugation. Purified follicle segments form closed follicular spheres with a reversed polarity within 24 hr of suspension culture. The polarity of all follicles in suspension is reversed so that the apical plasma membrane is oriented toward the culture medium, and the basal cell surface is directed toward the central cavity.

A marked change in insulin binding to rat thymic lymphocytes by fractionation of the cells into two subsets.

As compared with the pre-separated thymic lymphocyte (TL) population, a marked reduction of insulin binding was found in the two fractions [supernatant lymphocytes (SL) and precipitate lymphocytes (PL)] separated by low-speed differential centrifugation from the TL population. The reduction in insulin binding to SL or PL appeared to be due to a decrease in the affinity rather than a decrease in the number of insulin receptors. The reduced insulin binding was enhanced to near the level of the TL population when SL and PL were mixed and then incubated for 90 min at 37 degrees C. Moreover, when the cell-free supernatant from the mixed and incubated fraction was added to SL and PL respectively, the insulin binding to each fraction was found to be enhanced.

Morphological and functional studies of human fetal liver cells in primary monolayer culture.

Livers from human fetuses between the 16th and 24th weeks of gestation were dissociated by successive dispase and collagenase digestion followed by two cycles of low-speed differential centrifugation. This improved method recovered approximately 1 X 10(7) cells (90% hepatocytes and 90% viable cells) from 4 g of liver tissue. These hepatocytes were set into primary culture and monolayer granular hepatocytes were obtained within a week. Both albumin and alpha-fetoprotein production was demonstrated in these granular hepatocytes by the immunoperoxidase method for 2 weeks and alpha-fetoprotein production in the culture medium occurred for a week by the single radial immunodiffusion method. The morphological features of the granular hepatocytes could be distinguished from those of the other type of epithelial cells with clear cytoplasm. During the cultivation period, gradual changes from granular to clear hepatocytes with high mitotic activity were found.

Localization and solubilization of a rat liver microsomal carnitine acetyltransferase

Carnitine acetyltransferase activity had been previously shown to occur in peroxisomes, mitochondria, and a membranous fraction of rat and pig hepatocytes. When components of this third subcellular fraction (plasma membranes, components of the Golgi apparatus, and microsomes) were further separated, carnitine acetyltransferase fractionated with the microsomes. Microsomes isolated by three different methods (isopycnic sucrose density zonal centrifugation, high-speed differential centrifugation, and aggregation with Ca 2+ followed by low-speed differential centrifugation) all contained carnitine acetyltransferase activity. The lability of carnitine acetyltransferase in microsomes isolated by different methods and in different isolation media is reported. When total microsomes were subfractionated into rough and smooth components, carnitine acetyltransferase activity was found to the same extent in both and was tightly associated with the microsomal membrane. The microsomal enzyme was rapidly inactivated in 0.25 m sucrose or 0.1 m phosphate, but was stable for at least 2 weeks in 0.4 m KCl. Extensive treatment with high ionic strength salt solutions, 1% Triton X-100, or a combination of the two was used to solubilize microsomal carnitine acetyltransferase activity. Carnitine octanoyltransferase activity was also found in the microsomal fractions isolated by three different methods, but no carnitine palmitoyltransferase was detected in the microsomal fractions. It is proposed that microsomal carnitine acetyl- and octanoyltransferases could be involved in the transfer of acyl groups across the microsomal membrane, thereby providing a source of acetyl and other acyl CoA's at sites of acetylation reactions and synthesis.

Effects of Asbestos Fibers on Mammalian Cells in Culture

In previous reports from this laboratory, we examined the correlation of the toxic effects of several organic pesticides, herbicides and trace metals on the plating efficiency to the ultrastructural changes found in the treated cells in culture. Since there has been a long-term interest in the effects of exposure of humans and experimental animals to asbestos, the current study was undertaken to determine if ultrastructural changes would also be found in mammalian cells grown in vitro after exposure to asbestos. In a series of preliminary experiments, suitable fractionation by low-speed differential centrifugation and concentrations of amphibole asbestos were found in which there was at least 70% to 90% viability depending on the cell type. The viability of the cells incubated in the presence of the asbestos was determined both by growth curves established from direct cell counts and from staining with trypan blue which does not penetrate viable cells.

The isolation of plasma membrane from frog cardiac muscle cells

A vesicular preparation consisting largely of the plasma membrane of frog cardiac cells was isolated and its enzymatic activities and lipid content were investigated. The enriched plasma membrane preparation was obtained by (1) mildly homogenizing washed ventricles, (2) separating away the cellular debris by low speed differential centrifugation, (3) separating the plasma membrane fraction from other membranous components by centrifugation to equilibrium in various sucrose gradients. The frog cardiac plasma membranes were found to be concentrated between specific gravities of 1.07 and 1.11. In the membrane fraction the specific activities of membrane marker enzymes: 5′-nucleotides (EC 3.1.3.5), alklaline phosphatase (EC 3.1.3.1) and Na +: K +)-activated ATPase were, respectively, 19, 15, and 14 times greater than in the homogenate. Activities of mitochondrial marker enzymes were either very low or absent. No unusual lipid types were found. Cardiolipin was less than 0.1% (by wt) in the membrane fraction. The molar ratio of phospholipid to cholesterol was approximatley 3 : 2.