The effects of growth factors on Tenon''s capsule fibroblasts in serum-free culture

Recent studies from our laboratory have shown that in the mouse and rat nephron Ca2+ and Mg2+ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca2+ and Mg2+ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PDte.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca2+ and Mg2+ net fluxes in the mTAL are not driven by the transepithelial PDte. At zero voltage, a small but significant net secretion of Ca2+ into the tubular lumen was observed. With a high lumen-positive PDte generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca2+ and Mg2+ reabsorption was noted; instead significant and sustained Ca2+ and Mg2+ net secretion occurred. When a lumen-positive PDte was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca2+ net secretion and a lesser Mg2+ net secretion, not modified by ADH, were observed. Replacement of Na+ by K+ in the lumen perfusate induced, in the absence of PDte changes, important but reversible net secretions of Ca2+ and Mg2+. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca2+ and Mg2+ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca2+ and Mg2+, the secretory processes would appear to be of cellular origin.     

Effects of serum-free culture media on human liver and fibroblastic cells

The morphological aspects and amino acid variations of human fibroblast and liver cell monolayer cultures were studied in serum free media. Under these conditions, the behaviour of the two cell types differed greatly. The morphological changes for the liver cells, as compared with the fibroblasts, appear more quickly and some of these changes are particular. Furthermore, extracellular amino acid variations are of lesser importance for the liver cells, during the 10 days following the suppression of serum, except for serine.     

Erythropoietin production by human renal carcinoma cells in culture

Cells from human renal tumors were grown in monolayer cultures, and the media obtained at each medium change were assayed for erythropoietin activity. The medium from carcinoma I (a granular cell tumor) contained a high level of activity initially. The concentration of erythropoietin activity decreased with time in culture, but was significantly higher than that in controls after four months in vitro. There was , in addition, evidence of an inhibitory material present in the culture media. The activity formed by the tumor cells could be neutralized by an antibody to human urinary erythropoietin. The difference between activity measured in marrow cell cultures and that found by in vitro assay, and the chromatographic properties of the active preparation, suggest that the tumor-derived activity may be largely asialoerthropoietin. Two other renal carcinomas, of a different cellular type, produced significant erythropoietic activity.     

In vitro isolation and cultivation of Cowdria ruminantium under serum-free culture conditions

Imagined halitosis is poorly documented in the psychiatric literature. It is perhaps best thought of as a symptom rather than as a specific syndrome (a collection of symptoms that co-vary together). Many of the cases with imagined halitosis described in the literature resemble the psychiatric syndrome of social phobia.     

Nisin production by Lactococcus lactis using two-phase batch culture

This prospective study investigates the effect of injury and surgery of cartilage and bone on serum group II phospholipase A2 (PLA2-II) and C-reactive protein (CRP) levels. Serum concentrations of PLA2-II and CRP were measured before and after the operation in nine patients with closed tibial shaft fractures treated by nailing, 11 patients with fractures of lateral tibial plateau treated by bone grafting, and 19 patients with ruptured anterior cruciate ligament treated by reconstruction. The postoperative PLA2-II and CRP values were statistically significantly higher than the pre-operative values in the tibial plateau fracture and ligament rupture groups, whereas the increase in the PLA2-II values in the tibial fracture group was not statistically significant. The highest values of both parameters were found on the second postoperative day. The changes in the PLA2-II and CRP values were parallel in the lateral condyle fracture and in anterior cruciate ligament rupture groups. PLA2-II behaves as an acute phase reactant in the serum of patients undergoing acute and elective knee surgery.     

Substratum-dependent and region-specific control of attachment and proliferation of gastrointestinal epithelial cells in primary serum-free culture

A system for the primary serum-free culture of fetal rat gastrointestinal epithelial cells was used to examine the role of the extracellular matrix (ECM) in the attachment and proliferation of these epithelial cells. Forestomach epithelial cells (FSEC) were able to attach to and proliferate on plastic dishes without a substratum, while glandular stomach epithelial cells (GSEC) and duodenal epithelial cells (DEC) were unable to do so. The presence of a substratum promoted the attachment and proliferation of these epithelial cells. The effects of various components of the ECM differed depending on the type of cell. FSEC attached most efficiently to a substratum of fibronectin, while GSEC did so to laminin. DEC attached more efficiently to type I collagen and fibronectin than to any other substratum. FSEC proliferated most rapidly on laminin, while GSEC and DEC did so on collagen gels. These substrata induced the most efficient attachment and proliferation of FSEC, and they were effective in promoting the attachment and proliferation of GSEC and DEC in decreasing order of efficiency, indicating the existence of a head-to-tail gradient in the response of epithelial cells to substrata. The expression of c-myc mRNA in these cells differed depending upon the substratum on which they were cultured and the mRNA level was well correlated with the extent of the cell proliferation, indicating that the cell proliferation is mediated by c-myc gene expression, which is regulated by cell-ECM interactions. The results of the present study demonstrate that proliferation of gastrointestinal epithelial cells is regulated region-specifically not only by soluble factors but also by insoluble components of the ECM.     

Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture

Chondrocytes propagated in monolayer culture proliferate and change into 'fibroblastoid'-like cells. This change is characterized by a shift in production of collagen type II to I and from high- to low-molecular-weight proteoglycans. When propagated in three-dimensional culture, chondrocytes have limited ability to divide but re-express their original characteristics. The goal of the present study was to determine whether a microcarrier suspension culture system would support chondrocyte proliferation and phenotype expression. Our experiments indicate that a collagen type I microcarrier (cellagen) best supported chondrocyte proliferation and phenotype expression. Cells in cellagen microcarriers multiplied at least twentyfold within 2 weeks and had doubling times of 2 to 3 d. Viable and metabolically active cells were retrieved with ease. The harvested chondrocytes had no detectable staining for collagen type I and stained intensely for collagen type II. Our studies demonstrate that the microcarrier suspension culture system supports growth and enhances expression of the 'chondrocytic' phenotype. Attachment to a constrained surface and the fluid shear forces on the microcarriers during suspension culture may have helped chondrocytes to reacquire their rounded shape and produce cartilage matrix components.     

Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo

A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations. Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher. Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher. However, nondividing vascular endothelium in situ was very poorly transduced. Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture. These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml. Kidney perfusion studies in rats and pigs gave similar results. The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml. Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation.     

The effects of insulin, transferrin and androgens on rat prostate explants in serum-free organ culture

A model previously developed in our laboratory to culture rat prostate explants in serum-free chemically-defined medium was used to evaluate the direct influence of potential regulators. The aim of the present work was to verify the effects of insulin (I) and transferrin (Tr), two hormones considered as essential in other serum-free culture systems, and three androgenic hormones, since the prostate is known to be androgen-dependent. Explants of rat prostate were cultured for five days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air-5% CO2). The addition of Tr (50 micrograms/ml) had no effect, but I (5 micrograms/ml) significantly increased DNA synthesis. This influence was amplified by combination of the two hormones. However, protein synthesis was only slightly stimulated. Testosterone (T) or androstanediol significantly increased DNA synthesis when compared to corresponding control values at five days. In combination with I plus Tr, each hormone showed potentiated effects, particularly T with a twofold increase over day 0 values. When dihydrotestosterone was added singly, the incorporation of 3H-thymidine was stimulated by 300% over control values at five days, and by 100% over values in uncultured explants. This influence was maximal since it was not improved by I plus Tr. Protein synthesis was increased significantly by the triple combination. In addition, each androgen as well as the combination of I plus Tr had a positive influence on explant morphology. The above conditions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors in prostate metabolism and to eventually identify putative cancer markers.     

Characterization of the production of acetaldehyde by astrocytes in culture after ethanol exposure

Mice selected for large and small brain weights (LB and SB strains, respectively) were tested for audiogenic seizure sensitivity. We have examined 150 LB and 120 SB mice of the 13th and 14th generations. In mice of the 12th generation brain and body weight values were 498.6 +/- +/- 2.1 mg and 26.2 +/- 0.4 g, respectively, for the LB and 415.35 +/- 1.9 mg and 23.8 +/- 0.3 g for the SB, the difference being significant for both values (p < 0.001). The SB mice revealed higher audiogenic sensitivity (larger proportion of seizures and deaths) at the age of 22-27 days. Adult LB mice were more sensitive than SB of the same age (irrespective of whether they received priming stimulation or not). The obtained evidence is discussed in the context of differences between audiogenic fits and other seizure states.     

Lack of effect of hematopoietic growth factors on human breast epithelial cell growth in serum-free primary culture

A number of recombinant cytokines believed to regulate normal hematopoiesis are now being used in cancer treatment protocols to reduce the myelosuppressive toxicity of intensive chemoradiotherapy regimens. It is widely assumed that such cytokines are relatively specific for hematopoietic cells, although some cell lines derived from a variety of non-hematopoietic human tumors can respond to some of these factors. However, relatively little is known about their ability to stimulate (or inhibit) the proliferation of freshly isolated normal or malignant non-hematopoietic cells. We have used a serum-free culture medium that selectively supports the growth of human breast epithelial cells (HBEC) obtained directly from normal or malignant tissue samples to evaluate potential stimulatory or inhibitory effects of eight cytokines: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Steel factor, interleukin-2, interleukin-3, interleukin-6, transforming growth factor-beta and macrophage inflammatory protein-1 alpha, on these cells cultured both in the presence of epidermal growth factor, a potent stimulator of HBEC growth, and in its absence. HBEC growth was assessed after 7 and 14 days using the tetrazolium-dye reduction assay. Potential effects on the well studied MCF-7 breast cancer cell line, cultured under the same conditions, were also investigated. None of the cytokines (which were tested over a wide range of concentrations) had any modulating effect on the growth of normal or malignant HBEC under the conditions used with the exception of transforming growth factor-beta, which was consistently and significantly inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor II may constitute one of these steps. The TGF-alpha transgenic mouse hepatocyte line TAMH appears to undergo transformation in a similar manner to that of hepatocytes overexpressing TGF-alpha in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.     

Influence of serum-free culture conditions on subcellular localization of steroid 5alpha-reductase in rat C6 glioma cells

Rat C6 glioma cells are considered to be well characterized, and therefore commonly used as a model system to investigate the function of glial cells. However, recent study has shown that an alteration in the expression of their phenotypic antigens is observed when the cells are maintained under the serum-free conditions, proposing the possibility that various properties of glioma cells can be altered by the growth conditions. To test this possibility, the effects of serum-free culture conditions on the expression of steroid 5alpha-reductase (5alpha-R) type 1 isozyme in glioma cells were examined using immunocytochemical technique. Immunoreactivity of 5alpha-R type 1 was confined to the perinuclear region of glioma cells cultured in serum-containing medium, and observed in the cytoplasmic space as well as the perinuclear region of the cells cultured in serum-free medium. In contrast, serum deprivation failed to affect the expression of phenotypic antigens, glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Further studies showed that the expression of cytoplasmic 5alpha-R immunoreactivity induced by serum deprivation was reversible, and might be attributed to removal of serum proteins rather than biologically active small molecules from culture medium. This alteration in the expression of 5alpha-R immunoreactivity is therefore considered to reflect the translocation of the enzyme from the perinuclear region to the cell cytoplasm rather than the induction of cytoplasmic enzyme, and suggest that the culture conditions cause an alteration in the subcellular localization of 5alpha-R type 1 isozyme without phenotypic change of the glioma cell.     

The production of cricket paralysis virus in suspension cultures of insect cell lines

We have observed seven initially obese individuals who, during the course of a strenuous weight-reduction program, developed diabetes mellitus: non-insulin-dependent diabetes mellitus in five cases and insulin-dependent diabetes mellitus in two cases. None had any sign of prior diabetic symptoms. Although weight reduction is encouraged in obesity, crash diets without proper medical surveillance may have deleterious effects. This sequence of induction of diabetes has not previously been reported in the medical literature. The metabolic situation in extremely low-calorie diets may be comparable to that in starvation. An attempt is made to explain our observation concerning the induction of a diabetic state during such diets, on the basis of increased insulin resistance in states of starvation and anorexia nervosa, with a concomitant role in stress hormones.     

构建安全文化促进安全生产

安全生产是矿山企业生产经营管理的主题.文中介绍了安全文化的概念和构成、安全文化的地位和作用以及构建安全文化的途径.     

Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture

Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.     

Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions

A single Plasmodium falciparum isolate was adapted for growth in serum-free culture medium. The parasitemia increased from 0.5% to 20% on day 7 after thawing. The asexual forms of the parasites appeared morphologically normal and pigment formation was comparable with that seen under standard conditions with serum present. Parasites were coincubated in 96-well plates with serum, peripheral blood mononuclear cells (PBMC), and PBMC in the presence of autologous serum from healthy non-immune individuals (n = 12), healthy semi-immune individuals (n = 12), and malaria patients (n = 7). Growth was monitored for six days. The concentration of interleukin-6 and interferon-gamma (IFN-gamma) in supernatants from the continuous cultures were measured by a bioassay and an enzyme-amplified sensitivity immunoassay. The results of this study showed that parasites cultured in serum-free medium in the presence of PBMC develop more rapidly, particularly with cells from malaria patients, compared with parasites cultured alone. The growth of parasites was different if 10% autologous serum was added to the culture. Parasite growth with sera from acutely infected individuals was similar with that with sera from aparasitemic, nonimmune individuals, and both supported significantly higher parasite growth over the six-day culture period compared with sera from the uninfected semi-immune individuals. Production of IFN-gamma by cells from nonimmune individuals and malaria patients was higher when cultures did not contain autologous serum. Nonimmune donor cells produced high amounts of IFN-gamma, but cells from the semi-immune donors produced little of this cytokine. There was no marked inhibition of parasite growth with any combination of serum and cells over six days of culture. A difference between the groups was observed after two days of culture, when growth with cells and serum from the uninfected, semi-immune group was significantly lower than that from the nonimmune group, but this was not subsequently sustained. The results of the study show that continuous cultivation of P. falciparum in serum-free medium provides a novel in vitro model to study mechanisms of the interplay between components of the human immune system and the malarial parasite, in which any possible influence of human serum is removed.