A statistical comparison of various factors on embryogenic proliferation,morphogenesis and regeneration in Lolium temulentum cell suspension colonies

Cell suspension colonies from four embryogenic Lolium temulentum lines were selected and plated individually in 25 embryoid maturation treatments which varied in various factors reported to stimulate embryogenesis or improve regeneration. Using a numerical scoring system to compare the cultures against a control, treatments were identified which increased growth, suppressed morphogenesis or encouraged premature shoot formation.No treatment significantly improved the proportion of colonies with globular or mature embryoids, but some prevented maturation and increased the proportion with translucent embryogenic proliferation. Other treatments accelerated maturation causing increased de-differentiation of embryogenic tissues. These treatments also tended to discourage the differentiation of discreet embryoids.Colonies were later transferred en masse to a regeneration medium and scored using another numerical system. Embryoid maturation conditions were then identified which increased or suppressed subsequent shoot regeneration. The two scoring systems enabled cultures of the four lines to be characterised in detail and identified somatic variation in embryogenic development, morphogenesis and de-differentiation.     

Hepatocyte culture on biodegradable polymeric substrates

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 mug/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.     

A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.     

Killer polyamines?

Mammalian cells can rapidly make large changes in their rate of polyamine biosynthesis in response to mitogenic and trophic signals. However, cultured cells seem to grow adequately as long as they are supplied a steady but unregulated supply of polyamines. This implies that complex and rapid changes in polyamine synthesis serve a function in a special rather than a general biological context. We suggest that the appropriate context in which regulation of polyamines mediates crucial functions is the mammalian embryo and that one function of polyamines is to act as substrate in an oxidative pathway that arbitrates programmed cell death.     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

Breast Carcinoma With Osteoclast-Like Giant Cells

The cytological and histological findings of a case of breast carcinoma with osteoclast-like giant cells are presented. A fine needle aspiration specimen demonstrated the characteristic combination of malignant epithelium and reactive multinucleated cells and enabled identification of this rare variant of breast cancer. Immunocytochemical studies using the monoclonal antibody KP1[CD68] support a histiocytic origin for the osteoclast-like cells.     

Role of endothelial cells in the proliferative response of cultured pulmonary vascular smooth muscle cells to reduced oxygen tension

Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O₂ for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.