Suspension culture ofAedes albopictus cells for flavivirus mass production

Summary Aedes albopictus clone C6/36 cells were adapted to suspension culture in spinner flasks, and largescale cell cultures were achieved. Suspension cultured cells, attached on microcarriers, were inoculated with Japanese encephalitis virus. Virus-infected fluid with 10⁷ to 10⁸ plaque-forming units/ml was harvested daily from Days 2 to 8 postinfection. This method makes it possible to prepare large amounts of purified virions with less culture space and labor.     

A rapid colorimetric assay to evaluate the effects ofBacillus thuringiensis toxins on cultured insect cells

Summary A new cytotoxicity assay has been developed to assess the activity of the toxins produced byBacillus thuringiensis kurstaki on CFl cells. The assay is performed in a 96-well plate using a tetrazolium salt, MTT, as the substrate. The compound diffuses through the cell membrane and is reduced by mitochondrial dehydrogenases of live cells to a purple color compound, which precipitates within the cells. The precipitate is dissolved by isopropanol and the absorbance at 570 nm is measured with a 96-well plate reader. The amount of dye converted is proportional to the number of liver cells and the length of incubation with substrate. The LC₅₀ of the 60 kDa toxin, an activated form of the 130 kDaB. t. kurstaki toxin has been determined and found to be comparable to the data obtained by the trypan blue staining of dead cells.     

Methods for determining the survival of cultured insect cells

Summary Described are feeder cell layer and conditioned medium techniques for assessing the long-term survival ability of TN-368 lepidopteran insect cells after virtually any experimental treatment. The procedures have been used successfully to determine cellular survival after exposure to x and gamma radiation, ultraviolet light, heat, and a variety of mutagenic and hazardous chemical agents. These methods, with minor variations, have also been used to assay for the survival of the following lepidopteran and dipteran insect cell lines: IPLB-SF-1254 (Spodoptera frugiperda), WR69-DM-1 and WR69-DM-2 (Drosophila melanogaster), ATC-10 (Aedes aegypti), RU-TAE-14 (Toxorhynchites amboinensis), and RU-ASE-2A (Anopheles stephensi). These techniques may also be used to clone cells.     

Transfection of Lepidopteran insect cells with Baculovirus DNA

Summary Several protocols are presented for preparation and transfection of Baculovirus and plasmid DNAs into Lepidopteran insect cells using the calcium-phosphate co-precipitation technique. Important parameters for optimum efficiency include the inherent susceptibility of the recipient cell line for transfection, and the method of preparation of viral and plasmid DNAs. The protocols presented provide reproducible high efficiencies for transfection of several Lepidopteran cell lines.     

Biological and mechanical quality of red blood cells cultured from human umbilical cord blood stem cells

Human umbilical cord blood (CB) has moved from the status of biological waste to that of a valuable source of haematopoietic stem (HS) cells. There are potentially three major clinical applications for HS cells andex vivo-expanded HS cells: reconstitution of haematopoiesis in patients undergoing chemotherapy; gene therapy (e.g. in thalassaemia, sickle cell anaemia); and large-scale production of mature blood cells. Erythropoiesis is accomplished by highly complex interactions of haematopoietic progenitor cells, stromal cells and cytokines in the bone marrow. Among them, erythropoietin is the principal regulator.Ex vivo cell culture experiments to obtain mature red blood cells were the focus of this study. Attempts to elucidate appropriate medium components and amounts of haematopoietic growth factors were successful: enucleated and haemoglobin-filled erythroid cells were obtained from primitive HS cells. Dimethylsulphoxide (DMSO) was found to be of particular importance as an efficient differentiation inducer. The differentiation process was followed microscopically and by fluorescence-activated cell sorting (FACS). Using the micropipette aspiration technique, the elastic properties of erythroid cells were evaluated as erythropoiesis progressed. Discocyte-like cells, comprising reticulocytes and finally differentiated red blood cells, showed an about ten-fold higher membrane shear modulus compared with control cells.     

人N端脂多糖结合蛋白基因在sf21昆虫细胞中的表达

目的 表达重组人N端脂多糖结合蛋白（truncated lipopolysaccharide binding proten,tIBP）。方法 通过病毒鉴定、SDS-PAGE、western blot、毛细管电泳、体外结合实验、细胞活性实验对重组病毒及重组蛋白tLBP的分子量、纯度和生物学活性进行分析。结果 重组病毒空斑分析确定MOI(multiplicity of infection)为8～10,SDS-PAGE显示感染时间65～72h为最佳表达分析;凝胶扫描显示特异表达蛋白占总产物的9.8%,纯化蛋白的分子量约27000u,纯度达95%以上;毛细管电泳显示表达产物呈单一峰型;Lowry法测定约1L上清液可获9mg纯化蛋白;Western blot间接显示表达产物反应带与预期值相符;酶联体外结合实验证实该蛋白可特异结合LPS。应用U937细胞,经LPS(1ng/mL）刺激与LPS（1mg/mL）刺激加纯化蛋白细胞中获得高效表达,并观察到它在体外具有结合或中和内毒素的活性作用。     

Stability of minisatellite loci in the genome of cultured hela cells

The stability of minisatellite loci representing DNA with the tandem organization of repeats is studied. DNA profiles of 194 HeLa clones are identical to the parent culture. They exhibit 36 bands detectable by the minisatellite probe Red4. Since the method ensures the record of changes in minisatellite loci appearing during the first two divisions of parent cell, mutations are assessed in 27,936 locus tests. Hence, the incidence of mutations in the culture is not higher than 3.5×10⁻⁷ per locus/per mitosis, providing that the number of generations in parental homogenous culture is taken into account. Translated fromByulleten' Eksperimetal'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 311–313, September, 1996     

Effect of thyroliberin on nonapeptidergic cells in cultured rat hypothalamic slices

The direct effect of thyroliberin on nonapeptidergic cells of the supraoptic and paraventricular nuclei was studied on cultured rat hypothalamic slices. Incorporation of labeled thyroliberin into vasopressinergic and oxytocinergic cells and a decrease in functional activity of these cells were observed in both hypothalamic nuclei. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 5, pp. 526–528, May, 1999     

Effect of laser radiation proliferative activity of cultured cells

Using morphometry and radioautography methods, it is found that a single laser irradiation (0.5 and 0.05 J/cm²) of cultured human fibroblasts stimulates their proliferationin vitro. The effect of the laser is realized via intensification of DNA synthesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, Nno 3, pp. 313–315, March, 1994 Presented by D. S. Sarkisov, Member of Russian Academy of Medical Sciences     

The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes

The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.     

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C₂C₁₂ murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and α-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p<0.01) and tropomyosin (63% decrease, p<0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentation. These results may have implications for muscle growth and repair during spaceflight.     

Secretory reactions of cultured hypophyseal growth hormone-producing adenoma cells to somatoliberin and somatostatin

Somatoliberin stimulates secretion of growth hormone and has no effect on secretion of prolactin in primary cultures of hypophyseal adenoma cells obtained from acromegalic patients. A short-term contact of the cells with somatostatin inhibits secretion of growth hormone, while a long-term contact with this hormone inhibits prolactin production. Somatoliberin abolishes the inhibitory effect of somatostatin on the growth hormone secretion and at high concentrations stimulates it. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 3, pp. 323–326, March, 1997     

Development of a gene transfer system for Penicillium chrysogenum

Summary We report here the development of an endogenous gene transfer system for the industrially-important Deuteromycete Penicillium chrysogenum, utilising a recombinant plasmid designated pPC-31 to complement a tryptophan — auxotrophic strain. Transformation frequencies in the order of 300–1800 transformants per g DNA have been obtained, and Southern hybridisation analysis has demonstrated that in the majority of cases, integration is mediated by homologous recombination between pPC-31 and the host genome at the site of the resident mutant allele.     

Role of the plasma membrane lipid matrix in information transfer by regulatory peptides

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 115, N ^o 5, pp. 477–478, May 1993     

Pathogenesis of leptospirosis: interaction of Leptospira interrogans with in vitro cultured mammalian cells

Interactions of virulent Leptospira interrogans with murine monocyte-macrophage-like J774A.1 cells and Vero (African green monkey kidney fibroblasts) cells from attachment to internalization were investigated by a series of morphological analysis. Fontana silver staining revealed that only the pathogenic leptospires were able to attach to host cells and the attachment pattern varied depending on cell types that they interacted with. Transmission electron microscopy (TEM) analysis confirmed the formation of the leptospires-containing phagosomes and their colocalization with lysosomes in macrophages were verified by confocal microscopic analysis. Results of F-actin rearrangements examination indicated that virulent leptospires invaded host cells via a microfilament-independent pathway.     

Age-related changes in the occurrence and characteristics of thymic CD4(+) CD25(+) T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.     

Proliferation characteristics of cultured human aortic endothelial cells and expression of adhesion molecules

Basal expression of the adhesion molecules P-selectin and ICAM-1, which mediate adhesion and transendothelial migration of leukocytes, by endothelial cells of human aorta and umbilical vein and its relationship with the proliferative behavior of these cells are studied in primary prolonged cultures. Inverse proportionality between the percentage of resting endothelial cells and those expressing the adhesion molecules in preconfluent and confluent monolayers, on the one hand, and the percentage of cells in the active cycle which do not express the adhesion molecules, on the other, is demonstrated. This relationship is one of the major causes of thein vitro functional heterogeneity of the endotheliocyte population in the expression of adhesion molecules and its adhesiveness for leukocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8, pp. 214–217, August, 1996     

Allergen gene transfer

DNA-based immunization induces a biased Th1 immune response, and offers a novel strategy for modulation of the Th2-associated response. Recent studies have provided evidence that antigen-induced allergic responses can be modulated in rodents immunized with plasmid DNAs encoding the sensitizing antigens. Further understanding of the regulatory mechanism involved and optimization of gene transfer/expression systems will assist greatly in proving the clinical utility of this process.     

Proliferative response of cultured Nb2 rat lymphoma cells to human serum prolactin and growth hormone

Human prolactin and growth hormone induce specific proliferative responses in a culture of Nb2 rat lymphoma cells. These responses can be employed for characterization of the activity of serum immunoreactive prolactin and growth hormone in patients with endocrine disorders. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 11, pp. 559–563, November, 1996