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Ecological patterns of micro-organisms in desert soils.

The Atacama Desert's hardy aerobic microbial populations have been found to occupy ecological niches substantially different from those occupied by aerobic micro-organisms in the California and Valley of 10 000 Smokes deserts. Two specialized groups of microflora, the actinomycetes and micro-aerophiles, appear to be abundant in some portions of the Atacama Desert, though many of the bacterial and mold types and species common in the California deserts are absent. The factors defining the ecological niches and relationships of microbial populations in these soils are largely unknown. In several respects, moreover, micro-environments in these soils are similar to some of those expected on Mars. For the purpose of establishing the engineering constraints on the design of life detection instruments and their sampling and processing components, the nature and diversity of species of micro-organisms prevalent in the soils of extremely dry climates of the earth have not been adequately investigated. Useful ecological information about these soils will include an estimate of the likely lower limit in numbers or mass of viable aggregates in the autochthonous populations and the survivability of these populations under mechanical processing and isolated storage. Knowledge of the artificial niches, growth media for example, which members of these populations accept, will be useful in designing life detection instruments themselves. The range of practical designs for soil sampling devices will be determined by the nature of the particles and surfaces, and their depths, on which various physiological groups or largest populations of micro-organisms reside in the extremely dry deserts of the earth. The results of several exploratory experiments are used to provide some understanding of the environment and the ecological patterns of micro-organisms in soils of the earth's driest deserts.

Actinomycetales↗

Differentiation of chimeric bone marrow in vivo reveals genotype-restricted contributions to hematopoiesis.

Bone marrow transplantation is of increasing utility in cancer treatment and is an important component of gene therapy protocols. Understanding the functional identities of progenitor cells involved in repopulation is important for the optimal application of this procedure. We have simultaneously used two types of genetic markers to study engraftment of mice after irradiation. The first involves intrinsic genetic differences, including a cellular marker, between two mouse strains used to construct chimeric mice by aggregating embryos. To produce a second marking system, bone marrow from these allophenic mice was subsequently infected with retrovirus. Individual progenitor cells, including primitive lympho-hematopoietic stem cells, participating in repopulation were identified by virtue of their uniquely marked clonal progeny. In this way numbers and genotypic identities of clones contributing to repopulation were determined. Engraftment could be divided into two distinguishable temporal phases. The first comprised roughly the first 3-4 months following transplant and was characterized by numerous clones, many of which apparently had limited lineage potencies. The subsequent phase was characterized by few, often a single, clones represented in all lympho-hematopoietic tissues. These findings are consistent with the notion that different classes of progenitor cells are differentially responsible for temporal progression. More differentiated, perhaps lineage restricted, progenitors transiently dominate the first few months before the emergence of pluripotent stem cell clones. Senescence of progenitors of the first phase may reflect their limited lifespans. A clear genotypic difference was obvious in engraftment. Cells of one strain, DBA/2, completely dominated the first temporal phase, whereas the C57BL/6 partner strain dominated the second phase. The genotype-restricted dominance of different stages of repopulation suggests important differences in the organization and regulation of stem and progenitor cell populations. Inherent differences in seeding, proliferation, and differentiation of progenitors of the two inbred strains may account for the differences. This in vivo model of competitive repopulation provides the opportunity to explore potentially important loci in the process of engraftment. We propose that DBA/2 progenitor cells, due to a proliferative and/or numerical advantage, account for their superiority immediately after engraftment. C57BL/6 stem cells, with long-term repopulating potential, predominate later, perhaps because of subtle numerical or proliferative advantages.

Animals↗

Adhesion and migration of marrow-derived osteoblasts on injectable in situ crosslinkable poly(propylene fumarate-co-ethylene glycol)-based hydrogels with a covalently linked RGDS peptide.

Marrow-derived osteoblasts were cultured on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) based hydrogels modified in bulk with a covalently linked RGDS model peptide. A poly(ethylene glycol) spacer arm was utilized to covalently link the peptide to the hydrogel. Three P(PF-co-EG) block copolymers were synthesized with varying poly(ethylene glycol) block lengths relative to poly(ethylene glycol) spacer arm. A poly(ethylene glycol) block length of nominal molecular weight 2000 and spacer arm of nominal molecular weight 3400 were found to reduce nonspecific cell adhesion and show RGDS concentration dependent marrow-derived osteoblast adhesion. A concentration of 100 nmol/mL RGDS was sufficient to promote adhesion of 84 +/- 17% of the initial seeded marrow-derived osteoblasts compared with 9 +/- 1% for the unmodified hydrogel after 12 h. Cell spreading was quantified as a method for evaluating adhesivity of cells to the hydrogel. A megacolony migration assay was utilized to assess the migration characteristics of the marrow-derived osteoblasts on RGDS modified hydrogels. Marrow-stromal osteoblasts migration was greater on hydrogels modified with 100 nmol/mL linked RGDS when compared with hydrogels modified with 1000 nmol/mL linked RGDS, while proliferation was not affected. These P(PF-co-EG) hydrogels modified in the bulk with RGDS peptide are potential candidates as in situ forming scaffolds for bone tissue engineering applications.

Animals↗

[Isolation and characterization of brain tumor stem cells in human medulloblastoma].

BACKGROUND & OBJECTIVE: Tumor stem cells have been isolated from several kinds of solid tumors, including primary brain tumors such as glioma and medulloblastoma. This investigation was to establish a simplified culture system to isolate and passage brain tumor stem cells (BTSCs) from human medulloblastoma, observe their proliferation and differentiation, and determine the expression of normal neural stem cell antigens, CD133 and Nestin, in BTSCs. METHODS: Eleven specimens of medulloblastoma were acutely dissociated and triturated into single cells without trypsin digestion. The tumor cells were seeded into serum-free DMEM/F12 medium (200,000 viable cells/ml) containing B27 (1:50), basic fibroblast growth factor (bFGF, 20 microg/L), epidermal growth factor (EGF, 20 microg /L), insulin (4 u/L), L-glutamine, and antibiotics. After generation, the primary brain tumor spheres (BTSs) were mechanically dissociated and passaged in the above serum-free medium. The monoclonal formation experiment was performed to determine the proportion of BTSCs in medulloblastoma and to observe the formation of BTSs. The differentiation of BTSCs was induced in mitogen-free DMEM/F12 medium supplemented with 10% fetal bovine serum. The expression of CD133 and Nestin in BTSs was observed with immunofluorescence staining; the distribution of CD133-positive cells in tumor sections was assessed by immunohistochemistry. RESULTS: In each of the 11 specimens, only a minority of medulloblastoma cells showed the capacity of self-renew and proliferation. These BTSCs generated free-floating neurosphere-like BTSs in the simplified serum-free medium. The proportion of BTSCs with monoclonal formation capacity in primary tumor cells was (31.18+/-6.18)%. The BTSCs attached to poly-L-lysine-coated coverslips and differentiated when the serum-supplemented medium was added. The expression of CD133 and Nestin was detected in BTSCs. CD133-positive cells scattered or formed nest-like aggregations in tumor masses, and accounted for (33.06+/-8.57)% of all tumor cells. CONCLUSIONS: BTSCs, with the capacity of self-renew and proliferation and express CD133 and Nestin, are exist in human medulloblastoma. They could be isolated and cultured in the simplified serum-free medium, and their differentiation could be induced in serum-supplemented medium.

AC133 Antigen↗

Spatial distribution and sampling plans for Mesoplatys ochroptera (Coleoptera: Chrysomelidae) on sesbania.

With the widespread introduction of the nitrogen-fixing legume sesbania, Sesbania sesban (L.) Merril, in agroforestry systems, the defoliating beetle Mesoplatys ochroptera Stål has become a serious pest of the trees in Africa. To determine within-field and within-plant spatial distribution of M. ochroptera on both seedlings and trees of sesbania, distribution statistics were computed using Iwao's mean crowding regression model. In 1- to 3-mo-old seedlings, the model accounted for 29.8, 32.2, and 61.0% of the variation observed in mean crowding to mean relationships in egg masses, larvae and adults, respectively. The model slopes of the regression were greater than unity for all stages indicating aggregated spatial distribution. Values of the intercept were greater than zero for egg masses, larvae and adults indicating that the basic components of the population are groups of individuals. The highest density (>80%) of mating and feeding adults was found in the upper third of 1- to 2-mo-old seedlings, while most of the egg masses were found in the lower half of seedlings. In trees, >60% of the individuals of all stages were found in the lower third of the foliage canopy, while <10% were found in the upper third. Sampling adults was found to be easier and gave better density estimates of M. ochroptera population than egg masses and larvae. Therefore, sampling plans useful for population studies and decision-making in pest management were developed for adults.

Animals↗

Phosphatidylinositol and inositol involvement in Alzheimer amyloid-beta fibril growth and arrest.

A key pathological feature of Alzheimer's disease is the formation and accumulation of amyloid fibres. The major component is the 39 to 42 residue amyloid-beta peptide (Abeta) which is an internal proteolytic fragment of the integral membrane amyloid precursor protein. Aggregation of Abeta into insoluble amyloid fibres is a nucleation-dependent event that may be modulated by the presence of amyloid-associated molecules. Fibril formation is also associated with neurotoxicity which may be the result of specific Abeta interactions with membrane proteins and/or lipids. Using circular dichroism spectroscopy, tyrosine fluorescence spectroscopy and electron microscopy, we have examined the binding of Abeta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to the glycolipid, phosphatidylinositol (PI), and different inositol headgroups. At pH 6.0 and in the presence of PI vesicles, both Abeta40 and Abeta42 adopted an amyloidogenic beta-structure. In contrast, at neutral pH only Abeta42 folded into a beta-structure in the presence of PI vesicles. To determine whether the induction of beta-structure stemmed from interactions with the headgroup of PI, the effects of inositol derivatives on Abeta were also examined. At pH 7.0, myo-inositol was sufficient to induce beta-structure in Abeta42 but had no effect on the conformation of Abeta40. Myo-inositol may promote beta-structure as a result of its ability to be both a hydrogen-bond donor and acceptor. Mono-, di- and triphosphorylated forms of inositol had reduced ability to induce beta-structure in both peptides. The results from this study indicate that interaction of Abeta40 and Abeta42 with PI acts as a seed for fibril formation while myo-inositol stabilizes a soluble Abeta42 micelle.

Alzheimer Disease↗

The spreading of human normal glial and malignant glioma cells in culture. Studies on standard culture conditions.

Using three lines of human normal glial cells and four established lines of human malignant glioma cells we have studied cell spreading following seeding onto glass and plastic substrata. The cells were detached with EDTA and trypsin, suspended in EMEM with 10% calf serum and studied with time-lapse, phase-contrast cinematography in suspension and during attachment and spreading. Cells were fixed and prepared for light microscopy while in suspension and during the spreading process. They were also prepared for scanning and transmission electron microscopy at different times during spreading. The projected areas of stained cells, in suspension and at different stages of spreading, were measured morphometrically and the results compared statistically. The glial cells in suspension were often found to retain somewhat their shape from the previous monolayer. They spread radially outwards with even lamellar cytoplasm and peripheral ruffling, as a group more quickly than the malignant glioma cells. They also became polarized and started to translocate in a shorter time. The glioma cells were spherical in suspension and characterized by pronounced blebbing of the cell surface. Blebbing continued during spreading and was finally replaced by ruffling at the edge. The cells spread like the glial cells radially outwards but the lamellar cytoplasm was occasionally somewhat irregular. Cells from the glioma lines spread as groups slower than the glial cells but with individual rates for the different lines. One of the glioma lines appeared to spread more thinly than the glial cells. Cells which sedimented on top of other cells could not spread. Aggregations of cells spread and became polarized more quickly than single cells in all cases.

Cell Adhesion↗

Electrochemical protoplast fusion in citrus.

We report here the development of a novel protoplast fusion method for citrus somatic hybridization. This new procedure, which we have named electrochemical protoplast fusion, is based on chemically induced protoplast aggregation, using a low concentration of polyethylene glycol, and DC pulse-promoted membrane fusion. Based on the results of nucleus and mitochondria molecular analyses, we were successful in using this method to regenerate both symmetric somatic hybrids and cybrids. Various parameters, including pulse intensity, pulse length, and composition of the fusion media, were tested, and the optimum fusion condition selected consisted of two 100-micros pulses of 1,500 V cm(-1). Our conclusion is that electrochemical fusion is a reliable and reproducible method that combines the best features of both the chemical and electrical methods, thereby promoting cell division and high embryogenesis rates of the fused cells. It represents a new approach to citrus somatic hybridization. Various interesting features of this new approach are presented and discussed.

Agriculture↗

Endogenous Nod-factor-like signal molecules promote early somatic embryo development in Norway spruce.

Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages. Auxin is important for PEM formation and proliferation. In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death. Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures. Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues. On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO). The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures. LCO is not present in nonembryogenic cultures. Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp. NGR234. The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus. Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce.

2,4-Dichlorophenoxyacetic Acid↗

Evaluation and preparation of controlled release lipid micropheres of sulphamethizole by a congealable disperse phase encapsulation method.

A congealable disperse phase encapsulation method was used to prepare controlled release lipid microspheres of sulphamethizole as a model drug. Hydrogenated cotton seed oil (HCSO) and stearic acid were employed as the lipid matrix materials. Tween 60 was the droplet stabilizer used to form microspheres. In in vitro dissolution tests, the drug release was found to be affected by the type of lipid material depending on hydrophilicity. Generally, an initial rapid release followed by a slower release of the drug from the lipid microspheres was observed. Lipid microspheres were also compressed in the tablet form to prevent the initial rapid release of the drug. But the drug release drastically decreased. To achieve a controlled release of the drug. Eudragit L as a channeling agent was added internally to HCSO-microspheres. Although the drug release increased, the controlled release pattern was not achieved. The external addition of polyethyleneglycole 4000 to HCSO-microspheres before compressing tablets, did not produce an affirmative change in the release profile. The lipid microspheres prepared by stearic acid released all of the drug within 1 h. Upon compression, the drug release was very low. Therefore, stearic acid-microspheres were compressed in the tablet form adding disintegrating agents, sodium alginate and Ac-Di-Sol (cross-linked sodium carboxymethylcellulose). A pH-dependent drug release was obtained from the tablets containing sodium alginate. With the tablets of stearic acid-microspheres containing Ac-Di-Sol, the controlled release could be achieved due to gradual disintegration from the tablet to aggregates, and to individual microspheres. Furthermore, in vivo study on 6 healthy volunteers confirmed the controlled release pattern of this dosage form.

Adult↗

In vitro study of the comparative behaviour of a human acute lymphoblastic leukemia cell line and a reference normal cell line towards the effects of a mitogenic lectin.

An in vitro study of the behaviour of a human acute lymphoblastoid leukemia cell line (REH) towards the action of a mitogenic lectin of Robinia pseudoacacia was carried out. The results were compared with those a reference cell line (LHN13) established from normal human lymphocytes. In both cell lines, the lectin induces agglutination (measured by counting the number of aggregates as well as the number of cells in each aggregate) and decrease of growth (measured by counting the number of cells and the incorporation of tritiated thymidine into TCA-precipitable material per 10(6) cells). The agglutination and the decrease of growth are produced at the doses of 0.5 and 1 microgram/ml of culture medium and after 4 h of exposure of cells to the lectin, respectively. These effects increase progressively with higher doses of lectin and continues throughout the culture. However, the REH line is less sensitive than the LHN13 line to the effects of lectin. Both agglutination and growth decrease of REH as well as LHN13 cell lines by the lectin are reversible; this is confirmed by the fact that the monospecific anti-Robinia lectin serum suppresses these effects.

Agglutination↗

Thermodynamics of A beta(1-40) amyloid fibril elongation.

We describe herein the characterization of the equilibrium point for A beta(1-40) amyloid fibril elongation in phosphate-buffered saline at 37 degrees C. Seeded fibril elongation progresses rapidly to a reproducible end point of 0.7-1.0 microM unpolymerized monomeric peptide. This remaining monomeric material is functional, since after concentration it supports fibril elongation. Incubation of fibrils in the same buffer results in dissociation to a final monomer concentration in the same range. This robust C(r) value is equivalent to the A beta(1-40) fibril dissociation constant, Kd. The fact that a similar value for Kd is obtained from a ratio of dissociation and elongation rate constants further supports the view that these values are associated with a position of dynamic equilibrium and therefore are related to free energies of amyloid fibril elongation. The C(r) value reported here for wild-type A beta(1-40) fibrils corresponds to a free energy of fibril elongation of about -9 kcal/mol, a value similar to free energies of folding for small globular proteins. Elongation and dissociation of amyloid fibrils from point mutants of A beta(1-40) also yield C(r) values, different for different mutants, that reflect stabilizing/destabilizing effects. Interestingly, assembly of A beta(1-40) fibrils in the presence of a saturating concentration of the amyloid dye thioflavin T does not measurably affect fibril stability, in contrast to the commonly observed stabilization of globular proteins by ligand binding. The ability to quantify and compare amyloid fibril thermodynamic stabilities makes it possible to include fibrils, and potentially other aggregates, in quantitative descriptions of protein folding landscapes.

Amyloid beta-Peptides↗

Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures of human breast tissue.

We report on the discrimination of vascular smooth muscle cells and myoepithelial cells in primary cultures of human breast tissue. Breast tissue was disaggregated enzymatically and the resulting organoids seeded in monolayer culture on collagen-coated plastic in serum-free medium CDM3a. Two main types of organoids were present after enzymatic digestion. One resembled small blood vessels and the other interlobular ducts or acini of the breast gland epithelium. Within 3 to 8 d after plating the organoids migrated into typical monolayer islets. These monolayer islets were evaluated using phase contrast microscopy and further tagged with monoclonal antibodies for immunocytochemical demonstration of Factor VIII-related antigen, muscle iso-forms of actin, type IV collagen, vimentin, desmin, and keratins. It is concluded that vascular smooth muscle cells resembled myoepithelial cells by expressing vimentin filaments, depositing type IV collagen, and showing immunoreactivity to muscle iso-forms of actin. However, whereas vascular smooth muscle cells were associated with endothelial cells and sometimes expressed desmin, myoepithelial cells appeared together with luminal epithelial cells and expressed cytokeratins.

Actins↗

Analytical criteria for predicting apparent digestibility of soybean protein in preruminant calves.

A series of experiments on the use of soybean as a protein source in milk replacers for veal calves was undertaken to determine the relationships between the physicochemical and antinutritional properties and apparent digestibilities of nine soybean products. Soybean provided between 58 and 71% of dietary CP, and skim milk or whey powder provided the remainder. Soybean products were analyzed for CP, native protein, aggregated protein, carbohydrate-linked protein, peptides, antitryptic activity, and immunoreactivity of lectin, glycinin, alpha-conglycinin, and beta-conglycinin. The apparent digestibility of N calculated for soybean protein varied between 59 and 84%. Simple linear correlations were significant between apparent digestibility of soybean N and concentrations of native protein, antitryptic activity, glycinin, alpha-conglycinin, and beta-conglycinin. However, only variation in antitryptic activity, alpha-conglycinin, and beta-conglycinin contributed significantly to an explanation for the variation in apparent digestibility of soybean N in a multiple linear equation. Under our experimental conditions, beta-conglycinin was the best predictor of digestibility of soybean N. Antitryptic activity became the best predictor when soybean products had no detectable beta-conglycinin.

Animal Nutritional Physiological Phenomena↗

Plasminogen activator secretion by granule neurons in cultures of developing cerebellum.

Dispersed cell cultures were established from 7- to 9-day postnatal mouse cerebellum. The fibrin slice method was used to obtain a localization of plasminogen activator production to specific cells. Fibrinolytically active cells were small (5- to 8-micrometer diameter), round, and occurred singly or in aggregates. Fibrinolysis was both plasminogen and time dependent, inhibitable by epsilon-aminocaproic acid and soybean trypsin inhibitor and did not occur when cells were fixed in formalin prior to the fibrin overlay. Strong fibrin degradation occurred only when granule neurons were abundant in the cultures. These plasminogen activator secreting cells were identified as granule neurons by cell separation methods, nuclear morphology, and their ability to bind tetanus toxin and rabbit antiserum against mouse cerebellum (anti-Cbl-1 antiserum). Plasminogen activator also could be quantified in fractionated tissue homogenates or in cell culture medium by the 125I-labeled fibrin plate assay. Fibrinolysis in cerebellar extracts was 95% dependent on the presence of added plasminogen; furthermore, the activity was greater in cerebellar extracts as compared to cerebral cortex of the same age. At the age examined, the cerebellum contains many migratory neurons, and plasminogen activator production may be involved in the process of cell movement.

Animals↗

Lineage-independent activation of immune system effector function by myeloid Fc receptors.

An emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain complexes in which the ligand recognition elements associate with one or more 'trigger molecules' whose aggregation initiates a signal transduction cascade. The sequence motifs constituting the active sites of these trigger molecules are found in the T cell and B cell antigen receptors, and some Fc receptors, and appear to be central to effector function activation. For example, of the many molecules that mimic or potentiate the action of the T cell antigen receptor (TCR), none have yet been found to initiate effector programs autonomously in cells lacking TCR. We have devised two strategies to study activation mediated by myeloid Fc receptors, which appear not to associate with trigger molecules: the use of primary human cytolytic T cells as surrogate effector cells for genetically delivered receptors, and the use of vaccinia virus vectors to introduce genetically modified receptors into primary human monocytes. Using these approaches, we have found that the cytoplasmic domains of two Fc receptors show comparable function to equivalent domains of the trigger molecule family, but are not homologous to members of that family.

Amino Acid Sequence↗

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.

Arabidopsis↗

[Cultivation and serial propagation of a new rotavirus causing adult diarrhea in primary human embryo kidney cells].

OBJECTIVE: To investigate the methodology of cultivation of the new rotavirus that causes adult diarrhea. METHODS: 10% suspension of new rotavirus positive stool specimens in DMEM with 100 microgram/ml trypsin was made and centrifuged at the speed of 3 000 rpm for 10 minutes. The supernatant was filtered with 0.45 micrometer-pore-sized membrane filter, and the filtrate was incubated at 37 degrees C for 60 minutes. Primary human embryo kidney (PHEK) cells were prepared and seeded into roller tubes at the concentration of 1 ml odf cell suspension per tube. At 1 hour prior to inoculation, the PHEK cells were rinsed and refed with serum-free DMEM. Immediately before inoculation, the DMEM was decanted, and 200 microliter of prepared filtrate was inoculated into each tube. The tubes were incubated at 37 degrees C for 1 hour. At the end of 1 hour 800 microliter of DMEM containing trypsin (100 microgram per ml) were added to each tube. The tubes were placed in a roller tube apparatus at 37 degrees C for 3 days, removed and frozen-thawed once. 200 microliter of the cell culture fluids (CCF) were used for inoculation of each tube for next virus passage. Detection of new rotavirus from CCF was carried out by PAGE, IF, EM and IEM. RESULTS: New rotavirus was successfully isolated in cultured cells from 1 of 10 specimens. The isolated virus, designated J19, was propagated in PHEK cells for 28 passages. RNA patterns of J19 strain were identical to that of original new rotavirus inoculum and different from that of group A, B, C rotavirus. PHEK cells infected with the J19 strain were detected by IF. Infected cells reacted with convalescent antisera to the new rotavirus, but did not react with convalescent antisera to ADRV and hyperimmune antisera against group A, B rotavirus. Rotavirus particles were detected by EM, IEM in infected CCF of J19 strain. Other viral particles were not observed. The particles were aggregated with convalescent antisera to new rotavirus. CONCLUSION: We were successful in adapting a new rotavirus to serial propagation in PHEK cells. The J19 strain is a kind of new rotavirus different from group A, B, C rotavirus. This is the first report on the cultivation and propagation of the new rotavirus in cultured cells.

Adult↗