UDPgalactose:glucosylceramide beta 1----4-galactosyltransferase activity in human proximal tubular cells from normal and familial hypercholesterolemic homozygotes

The activity of a galactosyltransferase (GalT-2) that catalyzes the transfer of galactose from uridinediphosphogalactose to glucosylceramide in cultured normal human proximal tubular (PT) cells was characterized with respect to substrate saturation and metal ion requirements. Using a membrane-bound enzyme source, optimum activity was obtained in the presence of 1.0 mM Mn2+/Mg2+ (1:1) and a detergent mixture, Triton X-100/Cutscum (1:2, v/v), 0.1 mg/ml. The apparent Km values for glucosylceramide and UDP[14C]galactose were 3 microM and 0.5 microM, respectively. The Vmax values for glucosylceramide and UDP[U-14C]galactose were 0.12 nmol/mg protein per 2 h and 173 nmol/mg protein per 2 h, respectively. The purified 14C-labelled product comigrated with authentic lactosylceramide (LacCer) on TLC and HPLC analysis. The presence of a terminal beta-[14C]galactosyl group in the enzymatic product was proved by its cleavage (79%) by beta-galactosidase. Following the development of optimal assay conditions in normal PT cells, GalT-2 activity was next measured in urinary PT cells from homozygous familial hypercholesterolemic (FH) patients previously shown to accumulate large amounts of lactosylceramide. Urinary PT cells from familial hypercholesterolemic homozygous patients contained 35% higher GalT-2 activity as compared to control cells. We speculate that elevated GalT-2 activity may contribute to the storage of LacCer in FH-PT cells.     

Uptake and metabolism of lactosylceramide on low density lipoproteins in cultured proximal tubular cells from normal and familial hypercholesterolemic homozygotes

The metabolism of low density lipoproteins (LDL), and LDL modified by reductive methylation (M-LDL) of lysine residues, was studied in proximal tubular (PT) cells both from normal human kidney and from urine of patients with homozygous (LDL receptor-negative) familial hypercholesterolemia (FH). LDL and M-LDL was labeled either in the protein moiety with 125I or in the lactosylceramide moiety with 3H. The binding and degradation of 125I-LDL in normal cells was saturable and displaced by unlabeled LDL but not by M-LDL. The uptake of [3H]lactosylceramide (LacCer) low density lipoprotein in normal renal cells was saturable, and time and temperature-dependent. Exogenously derived [3H]LacCer on LDL was rapidly taken up and catabolized to monoglycosylceramide, or it was utilized for the endogenous synthesis of globotriaosylceramide (trihexosylceramide) and globotetraosylceramide (tetraglycosylceramide). [3H]LacCer M-LDL was taken up less avidly and metabolized less extensively than [3H]LacCer-LDL in normal cells. In homozygous FH renal cells the binding of 125I-LDL was not saturable and not displaced by unlabeled LDL. 125I-LDL degradation did not occur in FH cells. The homozygous FH PT cells took up a 2-fold greater amount of exogenously derived [3H]LacCer on LDL than normal cells. Yet, most of the [3H]LacCer taken up by FH PT cells accumulated as LacCer, and only small amounts were metabolized to monoglycosylceramide, globotriaosylceramide (trihexosylceramide), or globotetraosylceramide (tetraglycosylceramide). When normal and FH PT cells were preincubated with LDL (0-100 micrograms/ml medium), there was a 5-fold increase in cellular LacCer levels in FH cells at saturating levels of LDL, whereas there was about a 50% decrease in LacCer levels in normal cells. While the high affinity binding of LDL was not essential for the delivery of LacCer to cells, the data support the conclusion that LDL binding to the LDL receptor facilitates further LacCer processing and metabolism in normal renal cells. We speculate that [3H] LacCer is taken up by FH homozygous cells via a LDL receptor-independent mechanism and accumulates in the cells without significant metabolism. LacCer taken up by this mechanism contributes to the storage of LacCer in FH PT cells.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )     

Separation of small ciliate protozoa from bacteria by sucrose gradient centrifugation.

Small ciliate protozoa of the genus Uronema were separated from labeled bacteria by means of a discontinuous sucrose gradient. More than 98% of the radioactivity associated with the bacteria was found in the upper portion of the gradient, whereas the remaining portion of the gradient contained the ciliates.     

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.     

Separation of human endothelial cells from fibroblasts by centrifugation in Percoll gradients

Human endothelial cells from the umbilical vein and skin fibroblasts can be separated by means of centrifugation in a density gradient of Percoll. Cells show a good recovery in culture. Viability is not impaired.     

Effects of castration and replacement of androgen on the separation of rat ventral prostate cells by Ficoll gradient centrifugation

When regressing or growing (hypertrophic) cells from collagenase-digested ventral prostates were centrifuged on isokinetic Ficoll gradients for 6-8 min, they distributed into four fractions. Because of changes in epithelial cell morphology and density following castration to induce regression and replacement of androgens to cause cell growth, and contrary to results with normal rat ventral prostate, stromal cell fraction 2 was contaminated to a greater extent with regressing epithelial cells, as judged by their morphology and binding of radioactive androgens. However, centrifugation for 3 min increased the purity of epithelial cell fraction 4, although the yield of desired cells was reduced. Most cells from endocrine-manipulated rats were viable, as judged by exclusion of trypan blue and the initial incorporation of 3H-uridine. Cells centrifuged on a similar gradient of Percoll separated by a 'sieving' effect, which inverted the order of cellular fractions and removed red blood cells from fraction 2. Metrizamide offered no advantages, compared with Ficoll or Percoll. Neither physiologic nor pharmacologic amounts of testosterone returned the morphology of isolated epithelial cells to normal. To obtain consistent results with prostates from normal or hormone-manipulated rats, one should take care to select an active preparation of collagenase, avoid the use of very old animals, cool the tissue after it is dissociated, and do not apply undigested clumps of cells or overload the gradient. If attention is paid to these details, populations enriched in viable regressing or growing prostate epithelial or stromal cells can be obtained from hormonally manipulated rats.     

Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

A new method designed for the specific isolation and characterization of ligand-receptor complexes using a heterobifunctional crosslinking agent and immunoprecipitation is described. The complexes are first covalently crosslinked by photoactivation of the crosslinking agent. After lysis of the cells, the crosslinked complexes are immunoprecipitated using an antiserum directed against the crosslinking agent. With this method, ligand-receptor complexes formed in only minute amounts become available for further investigation. By using this anticrosslinker antiserum, different receptor systems can be investigated without raising new receptor- or ligand-specific antibodies for each system. As a test system, a radioiodinated lectin was used as ligand molecule and erythrocyte membranes acted as receptor carriers.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

Enrichment of human heterokaryons by Ficoll gradient for complementation analysis of iduronate sulfatase deficiency.

Ficoll gradients have been used to enrich for heterokaryons in cultures of human skin fibroblasts following polyethylene glycol (PEG) induced fusion. These gradients provide a simple and consistent method for obtaining populations of multinucleated cells, at least twofold greater than those resulting from fusion alone. Formation of glucose-6-phosphate dehydrogenase (G6PD) heteropolymers has been used as a functional assay for the presence of heterokaryons. Analysis of cell populations enriched for multinucleated cells has revealed complementation leading to iduronate sulfatase activity in heterokaryons derived from iduronate sulfatase-deficient fibroblasts expressing the Hunter and multiple sulfatase-deficiency mutations.     

Separation of immunoreactive lymphocytes from human pluripotent stem cells (CFU-GEMM) by means of counterflow centrifugation

Counterflow centrifugation with continuous monitoring of the output for cell number and cell scatter was used to separate low density (d less than 1.070 g/ml) human bone marrow cells in two fractions: one containing the majority of small size lymphocytes and the other the majority of the larger sized committed progenitor cells. The recovery of the pluripotent stem cells (CFU-GEMM) in the large cell fraction was complete. The mitogenic reactivity of this putative stem cell fraction had decreased to 6% and 11%, of the original value as measured with phytohemagglutinin stimulation and one way mixed lymphocytic culture respectively. Counterflow centrifugation offers a physical separation technique, by which the majority of the immunoreactive cells can be separated from the pluripotent hematopoietic stem cells.     

离心法分离毕赤酵母活细胞和死细胞

本文以毕赤酵母为研究对象,探索出一种分离活酵母细胞的新方法。研究发现,通过改变淋巴细胞分离液和50%聚蔗糖溶液的比例,获得不同密度的酵母细胞分离液,进而通过离心分层的方法可使毕赤酵母活细胞主要存留于离心液的上层。当酵母细胞分离液的密度为1.1467 g/mL (27.5%淋巴细胞分离液+72.5%聚蔗糖溶液),分离液上下层中酵母活细胞的分配比例差别达到最大,分别为94.67%(分离液上层)和5.33%(分离液下层)。毕赤酵母细胞浓度为4.35×108~1.13×109/mL时,活细胞在分离液上下两层的分配比例约为95%和5%。低毕赤酵母细胞存活率有利于离心分离。本方法可用于有效分离培养液中的毕赤酵母活细胞。     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.