Percoll Density Gradient Centrifugation Research Articles

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fishDicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88±10% for gut-associated lymphoid tissue and 79±18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcRβ chain, a 203bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRVβ3. By 3′-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.

Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples

The investigation of mitochondrial abnormalities in brain commonly requires isolation of these organelles from small tissue samples. We have modified a mitochondrial isolation procedure based on Percoll density gradient centrifugation to increase the proportion of the total mitochondrial pool recovered while reducing contamination with synaptosomes and related structures containing cytoplasm. Initially, myelin was removed by centrifugation in 12% Percoll in isotonic buffer. The pellet was resuspended, treated with digitonin to break up synaptosomes and similar structures and subjected to discontinuous Percoll density gradient centrifugation. The mitochondrial fraction obtained from this procedure was highly metabolically active and well coupled, exhibiting respiratory control ratios above 5. The recovery of mitochondrial markers using a single rat forebrain as starting material was approximately 18% to 21%. When small tissue samples (approximately 50 mg wet weight) were used as starting material the recovery of the mitochondrial marker was approximately 16%. The ratio of recovery of a mitochondrial marker to the cytoplasmic marker lactate dehydrogenase exceeded 200 in preparations from a single rat forebrain. This is substantially greater than values reported for previously published procedures reflecting both an improved yield of mitochondria and a reduction in cytoplasmic contamination. Themes: Other systems of the CNS Topics: Brain metabolism and blood flow

Multiple human serum components act as bridging molecules in rosette formation by Plasmodium falciparum-infected erythrocytes

Rosetting, the binding of parasitized erythrocytes to 2 or more uninfected erythrocytes, is an in vitro correlate of disease severity in Plasmodium falciparum malaria. Although cell ligands and receptors have been identified and a role for immunoglobulin M has been suggested, the molecular mechanisms of rosette formation are unknown. The authors demonstrate unequivocally that rosette formation by P falciparum-infected erythrocytes is specifically dependent on human serum, and they propose that serum components act as bridging molecules between the cell populations. Using heparin treatment and Percoll density gradient centrifugation, they have developed an assay in which parasitized erythrocytes grown in serum-containing medium and optimally forming rosettes are stripped of serum components. These infected cells were no longer able to form rosettes when mixed with erythrocytes and incubated in serum-free medium. Rosette formation was restored by the addition of serum or certain serum fractions obtained by concanavalin A (conA) affinity, anti-IgM affinity, anion exchange, and gel filtration chromatography. The authors clearly demonstrate that multiple serum components-IgM and at least 2 others-are involved in rosette formation. Those others consist of 1 or more acidic components of high-molecular mass that binds to conA (but that is not thrombospondin, fibronectin, or von Willebrand's factor) and of at least 1 more basic, smaller component that does not bind to conA. Data on the size and number of rosettes formed support the authors' hypothesis that multiple bridges are involved in this complex cellular interaction. These findings have important implications for the understanding of pathogenic adhesive interactions of P falciparum and host susceptibility to severe malaria. (Blood. 2000;95:674-682)

The structural properties of plant peroxisomes and their metabolic significance.

Plant peroxisomes can be isolated by Percoll density gradient centrifugation at high purity and metabolic competence as well as in relatively large quantities. According to biochemical and electrophysiological analyses, plant peroxisomes have recently been shown to differ from other cell organelles in essential structural properties. Unlike mitochondria or plastids, compartmentalization of plant peroxisomal metabolism is in major parts not caused by a boundary function of the membrane but is primarily due to the specific structure of the protein matrix. The enzymes of the photorespiratory C2 cycle of leaf peroxisomes are arranged as multienzyme complexes that allow efficient metabolic channelling with high flux rates and minimum leakage of reactive oxygen species from the organelle. Transfer of metabolites, such as carboxylates, proceeds across the peroxisomal membrane via a porin-like channel, which represents a relatively unspecific but highly efficient transport system. Because all variants of peroxisomes, which all contain only a single boundary membrane, are confronted with the task of transporting a large group of metabolites while preventing the escape of reactive intermediates, it is reasonable to speculate that the unique compartmentalization feature of leaf peroxisomes also applies to peroxisomes from fungi and mammals.

Isolation of highly purified rat cerebral lysosomes using percoll gradients with a variety of calcium concentrations

We were able to isolate a lysosomal fraction from rat brain which had a higher degree of purity than in previous studies with good recovery. This has been made possible by using percoll gradients following the swelling of mitochondria in the presence of calcium which would eliminate contamination from small amounts of mitochondria. By using percoll density gradient centrifugation after the swelling of mitochondria in the presence of calcium, cerebral lysosomes were purified 312-fold with the recovery of approximately 22 %, which is the highest reported for any cerebral lysosomal preparation. The most effective procedure for the separation was achieved by using 1.25 mM calcium incubation with post nuclear fraction. As brain lysosomes may play a major role not only in degrading macromolecules but also in their transport to the deposition site, obtaining purified rat cerebral lysosomes represents an important step in the study of the generation of macromolecules which accumulate in the brain.

Isolation of chloroplasts and chloroplast-nuclei(nucleoids)from Chlamydomonas reinhardtii

Summary: We developed a method for isolating chloroplast-nuclei(nucleoids)from a unicellular green alga, Chlamydomonas reinhardtii, using cell-wall-deficient mutant(cw15)cells. Because the cells drastically change the structure of their chloroplast-nuclei during the cell division cycle, we synchronized the cell division under a 12: 12-h light: dark regimen, and collected cells in the 8th h during the dark period for use as starting material, when chloroplast-nuclei were granular in shape and scattered randomly within individual chloroplasts. The cells were converted to protoplasts and disrupted by repeated passages through a narrow-bore needle, and the intact chloroplasts were purified by Percoll density-gradient centrifugation. The chloroplast-nuclei were isolated from the purified chloroplasts following lysis with Nonidet P-40. The results of Southern and northern hybridization analyses suggested that not only structure, but also DNA synthesis and transcriptional activities of the chloroplast-nuclei might fluctuate during the cell division cycle of C. reinhardtii. Thus, the chloroplast-nuclei of C. reinhardtii may provide an ideal system for analyzing the structure-function relationships of DNA-protein complexes.

Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons.

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+-H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22 Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+-H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.

BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP‐dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD‐dependent GDH activity was observed only in the chloroplasts. Three organelle‐specific isozymes—chloroplastic NADP‐GDH1, cytosolic/mitochondrial NADP‐GDH2, and cytosolic/mitochondrial NADP‐GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). Km values of NADP‐GDH1, NADP‐GDH2, and NADP‐GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP‐GDHs and one NAD‐GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP‐GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF‐1 and BmF‐2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen‐assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.

Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy

Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, Fasta and Blast analysis revealed 100% identity to chick β 7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3′ untranslated region of β 7-tubulin. β 7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of β-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10 −6 mol/L) resulted in a higher rate of [ 3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of β-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

Human parathyroid hormone (1-34) transiently increases the excretion of lysosomal enzymes into urine and the size of renal lysosomes.

It has been reported that the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH)(1-34). We report here that hPTH(1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH(1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH(1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH(1-34). These responses to hPTH(1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH(1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro-and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH(1-34).

Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity

Objective: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. Design: Prospective, observational study. Setting: University-based infertility clinic. Patient(s): Twenty-five nonazoospermic men. Intervention(s): Semen samples (n = 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange–treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. Main Outcome Measure(s): Sperm motility and DNA integrity. Result(s): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). Conclusion(s): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles.

Infectivity of feline enteroepithelial stages of Toxoplasma gondii isolated by Percoll-density gradient centrifugation

The infectivity of the feline enteroepithelial stages of Toxoplasma gondii isolated by Percoll-density gradient centrifugation was examined by the trypan blue dye exclusion method by assaying their penetration into feline fibroblast cells in vitro and by inoculation of the intestinal mucosa of cats. A large population of the parasites showed trypan blue dye exclusion activity. When feline fibroblast cells were inoculated with feline enteroepithelial stage parasites, no intracellular parasites were found 18 h post-inoculation. Kittens inoculated intraduodenally with 2 × 10 6 feline enteroepithelial stage parasites shed oocysts between 2 and 8 days post-inoculation. These results indicate that the isolated feline enteroepithelial stage parasites display infectivity towards enterocytes of cats and are capable of gametogenesis.

Regulation of Estrogen Sulfotransferase Expression in Leydig Cells by Cyclic Adenosine 3',5'-Monophosphate and Androgen

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 μm 8-bromo-dibutyryl cAMP [(Bu)2cAMP] increased EST expression 3- to 5-fold. The effect of (Bu)2cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5α-dihydrotestosterone (5-DHT). The activity of 5-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)2cAMP-induced EST expression. Further evidence came from the study with interleukin-1β, another agent known to suppress Leydig cell steroidogenesis by down-regulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1β inhibited (Bu)2cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3β-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.

Regulation of estrogen sulfotransferase expression in Leydig cells by cyclic adenosine 3',5'-monophosphate and androgen.

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 microM 8-bromo-dibutyryl cAMP [(Bu)2cAMP] increased EST expression 3- to 5-fold. The effect of (Bu)2cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5alpha-dihydrotestosterone (5-DHT). The activity of 5-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)2cAMP-induced EST expression. Further evidence came from the study with interleukin-1beta, another agent known to suppress Leydig cell steroidogenesis by down-regulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1beta inhibited (Bu)2cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3beta-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.

Isocitrate lyase of Ashbya gossypii – transcriptional regulation and peroxisomal localization

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62 584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C 2 compounds ethanol and acetate, and fully induced by soybean oil.

Rapid particle size distribution analysis of Bacillus spore suspensions

Knowledge of the homogeneity of a spore crop is an essential criteria for the subsequent investigation of their properties. Two methods of analysis have been compared; particle size distributions of Bacillus spores suspensions were analysed by laser light scattering with a Malvern Mastersizer; and scanning transmission electron microscopy (STEM) was used to record images of 50–100 individual spores. With an image particle sizing analysis software the dimensions (length, breadth, perimeter) of single spores were determined. Good agreement between the average individual spore dimensions determined by electron microscopy and those obtained by light scattering was evident. The Mastersizer was convenient and rapid to use but the image analysis provided the opportunity to distinguish between spores, vegetative cells and other particles. Spore suspensions were further separated from vegetative cells by centrifugation in continuous and discontinuous Percoll (modified colloidal silica) density gradients (1.09–1.13 g/ml). Electron microscopy revealed an adhesion of colloidal silica particles to the spore surface which could only be partially removed by repetitive washing. High silica contents were therefore detected by X-ray microanalysis. Percoll did not effect differential scanning calorimetric analysis of spores.

Molecular Cloning and Characterization of a Plasma Membrane-associated Sialidase Specific for Gangliosides

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19-38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha2-->3 and alpha2-->8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.

An improved method for the purification of stellate cells from rat liver with dichloromethylene diphosphate (CL2MDP).

Hepatic perisinusoidal cell population consists of hepatic stellate cells, Kupffer cells, endothelial cells, and Pit cells. These cells are isolated by enzymic digestion and purified by density gradient centrifugation. With isolation of stellate cells, conventional method is unable to eliminate the contamination of Kupffer cells because the densities of these two cells are similar. We report here an improved method for isolation of highly purified hepatic stellate cells, using dichloromethylene diphosphate (CL2MDP), which has selective cytotoxicity of Kupffer cells. Three days after the single intravenous administration of liposome-encapsulated CL2MDP, the Kupffer cells disappeared almost completely from the liver. Following Percoll density gradient centrifugation, the purity of the hepatic stellate cells exceeded 98% without any contamination of the Kupffer cells. Kupffer cells are reported to affect the physiological functions of stellate cells. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.

Percoll density gradient-enriched populations of rat pituitary cells: Interleukin 6 secretion, proliferative activity, and nitric oxide synthase expression

We used a discontinuous Percoll density gradient centrifugation to prepare enriched populations of prolactin (PRL), growth hormone (GH), and folliculo-stellate (FS) cells from rat anterior pituitaries in order to characterize these various cell populations. After cell dissociation and centrifugation, enriched PRL cells (55% of total cells as determined by immunocytochemistry [ICC] were present in Fraction 1 (Fr1) (density ([d]) = 1.059). Fr2 (d=1.071) had enriched S100-positive FS cells (31% of total cells), but enriched GH cell (60% of total cells) were present in Fr3 (d=1.094). Interleukin 6 (IL-6) was secreted mainly by enriched PRL cells in Fr1 (350 pg/mL/106 cells) and Fr2 (194 pg/mL/106 cells), and much less by the enriched GH cells inFr3 (16 pg/mL/106). Proliferation studies with combined 3H-thymidine and ICC for pituitary hormones showed that only the PRL cell had significant prolifereative activity. Immunostaining showed that immediately after separation, all three isoforms of nitric oxide synthase (NOS) were expressed anterior pituitary cells. After 3 d of culture, there was a marked increase in nuclear staining for neuronal NOS (nNOS) in all three fractions, whereas inducible NOS (iNOS) and endothelial NOS (eNOS) rexpression did not change significantly. These results indicate that: 1. Enriched populations of PRL, FS, and GH pituitary cells can be readily obtained with a rapid discontinuous percoll density separation procedure. 2. PRL cells from different fractions of the gradient show differenet proliferation rates and IL-6 secretion varied in different enriched cell populations. 3. Although all three isoforms of NOS were expressed in rat pituitary cells, nNOS is the prindipal isoform in anterior pituitary cells, and its expression was icreased after 3 d of culture of anterior pitutuitary cells.

Does age-associated reduced Leydig cell testosterone production in Brown Norway rats result from under-stimulation by luteinizing hormone?

Previous studies have shown that reductions in Leydig cell testosterone production occur with aging in the Brown Norway rat. The recent observation that changes in luteinizing hormone (LH) pulse interval and amplitude also occur with aging suggests the possibility that age-related reduced Leydig cell steroidogenesis might be related to changes in LH. We reasoned that, if this is the case, exogenously administered LH should restore testosterone production by aged rat Leydig cells to the higher levels produced by Leydig cells of young rats. To test this hypothesis, young (4-month-old) and aged (21-month-old) rats received testosterone- and estradiol-containing Silastic implants designed to suppress LH and, thus, endogenous Leydig cell testosterone production. At the same time, the rats received miniosmotic pumps programmed to deliver pulsatile ovine LH at a predetermined daily dose. In some experiments, treatment effects were determined by measuring testosterone production by testes perfused in vitro with maximally stimulating ovine LH. In others, Leydig cells were isolated by centrifugal elutriation and Percoll density gradient centrifugation, and their in vitro ability to produce testosterone in response to maximally stimulating LH was determined. Testes or isolated Leydig cells from untreated young rats produced about twice as much testosterone as that produced by Leydig cells from aged rats. The administration of testosterone- and estradiol-filled implants for 5 days reduced testosterone production significantly at both ages. In young rats administered 24 microg LH/day for 5 days, along with the implants, testosterone production was maintained at the high level of the young controls. Comparable treatment of aged rats resulted in testosterone production only at the low level of the aged controls. Indeed, even with higher LH doses (36 microg/day), testosterone production by the aged rat Leydig cells did not rise above the aged-control level. The inability of exogenously administered LH to increase testosterone production by testes and Leydig cells of aged rats suggests that Leydig cell steroidogenic deficits in the aged Brown Norway rat are unlikely to be the result of age-related changes in LH.