Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion

Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml ascorbic acid, with or without 10 mM beta-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of beta-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of beta-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of beta-glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo.     

Plant indicators in Iraq

Summary Native plants of Iraq have shown considerable variation in their ability to accumulate mineral elements. Halophytes such asSeidlitzia rosmarinus andHalocnemum strobilaceum indicate very high soil sodium contents, and others high magnesium and sulphate contents. Selectivity in the uptake and accumulation of mineral elements was exhibited by many native plants. Surprisingly high or low concentrations of particular elements were found in certain species in comparison with other species collected from the same site and at the same time. Magnesium and sulphate concentrations were as high as 5.80 and 12.16 per cent, respectively, when the concentrations in other species growing on the same site were as low as 0.59 and 1.29 per cent respectively. Selectivity was also indicated by the ability of certain species to accumulate a specific element from soils with high or low concentration of that element.     

Reactions of the adult female of the soft scale Kilifia acuminata to some physical factors in laboratory experiments (Homoptera: Coccoidea: Coccidae)

The soft scale insect, Kilifia acuminata (Signoret) reacts positively to light. The simple eyes are the photoreceptors by means of which the insect can sum up the light from various directions and steers in a path towards the high intensity beam. The insect reacts skototactically towards dark objects.
The linear velocity of the insect significantly increases with the increase of temperature. It has a zone of thermopreferendum extending between 22° and 32°C. Above 37°C, the insect is sensitive to small temperature differences of 2°C.
At different humidity combinations below 75% R.H., the insect prefers the moist side, while in the upper range above 83% R.H., it usually prefers the dry side. The preferred zone of humidity extends between 75 and 83%.
Orthokinesis, klinokinesis and klinotaxis are the main mechanisms regulating the insect reaction towards different physical factors.
The insect behaviour and its correlation to the normal environment is also discussed.     

Stimulation of heterotrophic and autotrophic growth ofSphaerotilus discophorus by manganous ions

Growth ofS. discophorus in a casamino acids-mineral salts medium was stimulated 3-fold on addition of 0.05% MnSO₄·H₂O to the medium. Growth was measured by determinations of total nitrogen, protein and DNA on the washed cellular material.Autotrophic growth ofS. discophorus strain 43-R was obtained in an inorganic mineral salts medium supplemented with trace amounts of the essential vitamins, thiamin, biotin and cyanocobalamin and with Mn⁺⁺ as the sole available source of energy. A gas mixture of 5% CO₂-95% air was bubbled continuously through the cultures during incubation. Concomitant with growth, Mn⁺⁺ disappeared from the cultures and MnO₂ was formed.     

Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

Summary We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.     

Purification and characterization of secreted acid phosphatase under phosphate-deficient condition inPholiota nameko

Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu²⁺, Fe²⁺, Hg²⁺, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe³⁺ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.     

Syncytia formation in HIV-1 infected cells is associated with an increase in cellular oleic acid

Infection of cells with the human immunodeficiency virus type-1 (HIV-1) usually results in the formation of giant multinuclear cells (syncytia) [(1986) Nature 322, 470-474; (1986) Nature 322, 725-728; (1985) Hum. Pathol. 18, 760-765; (1987) Arm. Neurol. 21, 490-496]. The appearance of syncytia is associated with an increase in the monounsaturated oleic acid content. This report describes experiments which compare the activity of known antiviral agents with that of saturated fatty acid derivatives in inhibiting oleic acid and syncytia formation. A concept is introduced which proposes that infection of cells with the human immunodeficiency virus causes a rise in cellular oleic acid which leads to increased membrane fluidity.     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.