Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Ultrastructure of the theca interna of ovarian follicles in sheep

Summary The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy.In follicles <3.0mm in diameter, the theca interna consisted of about 8–12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure.In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages.It was concluded that: (1) the theca interna of 2.0–2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.     

Effects of acute hypoxia on incorporation of [1-14C]arachidonic acid into glycerolipids of rat brain

The effects of 1 min of acute hypoxic treatment (1% O₂ in N₂) on incorporation of [1-¹⁴C]arachidonic acid into brain lipids of 16-day-old rats were investigated at 3, 6, and 12 min after intracerebral injection of the labeled fatty acid. The hypoxic-hypoxia condition associated with convulsive seizures caused a decrease in the conversion of labeled arachidonate to its acyl-CoA as well as incorporation of the label into the brain phospholipids. Among the phospholipids, there was a specific decrease in the labeling of diacylglycerophosphoinositol (GPI), and this change was accompanied by an increase in labeling of the diacylglycerols. These results indicate that metabolism of the long-chain fatty acids and some glycero-lipids in brain are vulnerable to acute hypoxic treatment.     

A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides

The polysaccharide secreted by Klebsiella aerogenes type 54 strain A3 was isolated, methylated, the ester carboxyl-reduced, and the product partially hydrolyzed. The resulting, partially O-methylated oligosaccharides were reduced and ethylated, and the mixture of products was fractionated by l.c. The l.c. fractions containing per-O-alkylated oligosaccharide-alditols were analyzed by e.i.-m.s. Pure per-O-alkylated oligosaccharide-alditols were also analyzed by ¹H-n.m.r. spectroscopy. The products obtained by base-catalyzed degradation and subsequent ethylation of the per-O-methylated polysaccharide were fractionated by l.c. The main product isolated was analyzed by e.i.-m.s., c.i.-m.s., and ¹H-n.m.r. spectroscopy. The results of these studies, in conjunction with results of analytical methods commonly used in the elucidation of polysaccharide structures, unambiguously characterized the primary glycosyl structure of the polysaccharide. Base-labile substituents, previously reported to be present in the polysaccharide, were not studied. Structure 1 revises, and complements, previously reported structures.

     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.