Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Effects of starvation on growth and reproductive apparatus of two immature freshwater snails Biomphalaria pfeifferi and Biomphalaria glabrata (Gastropoda: Planorbidae)

Starvation of immature snails of Biomphalaria pfeifferi and B. glabrata results in an arrest of growth and animals remain immature. Spermatogenesis is limited to spermatogonia (B.g.) or spermatocytes 1 (B.p.). The number and the size of oocytes remain inferior to that of controls. Animals show reduced genital tracts.Once feeding is restored, growth is resumed but wet weight and shell diameter do not reach the same level as in controls. Fecundity and gametogenesis do not differ from that in controls. Genital tracts weight is proportional to body weight.     

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

Relationships between macroalgal functional form groups and substrata stability in a subtropical rocky-intertidal system

The general hypothesis that morphological, physiological, and ecological adaptations of macro algal functional-form groups can be related to the level of disturbance encountered in a natural environment was examined. Two articulated calcareous coralline algae (Amphiroa van-bosseae Lemoine, 24% cover and Corallina frondescens Post. & Rupr. 20%) and one non-articulated coralline alga (Lithophyllum sp., 16%), all late-successional predation-tolerant strategists, comprise most of the community cover on stable bedrock substrata at Punta Las Cuevitas, Sonora, Mexico. Conversely, Ulva rigida C. ag. (26% cover) and a ralfsioid crust (23%), shows to be early-successional opportunistic strategists, cover more of the disturbed boulder habitat. Porolithon sonorense Daws., a stress-tolerant strategist, is uniquely abundant on both substratum types (13% cover on boulders, 10% on bedrock). The sheet-like and filamentous algae, prevalent in the temporally unstable habitat, generally show greater productivity (>2×) than the thicker and calcareous forms conspicuous in the more constant environment. It appears that selection for delicate thalli with high productivities, as well as selection for tougher morphologies having lower photosynthetic rates due to greater proportions of structural tissues, are widespread, divergent evolutionary forces among marine algae. Experiments with captive sea urchins (Echinometra vanbrunti Agassiz), in conjunction with fish-preference data published for some of the same algae studied here, offer strong support for the functional-form model. Parrotfishes, rudderfishes, surgeonfishes, damselfishes and E. vanbrunti, in the Gulf of California, preferentially feed on delicate, early-successional, sheet-like, and filamentous algae, while rejecting or ignoring the more structured, late-successional and calcareous algae. There is no significant (P > 0.05) gradation in calorific content between the first four of the six functional groups (i.e., Sheet-, Filamentous-, Coarsely Branched- and Thick Leathery-Groups), but the mean value for these fleshy forms (2.6 kcal · g ash-free dry wt^?1) is significantly greater than that for the last two groups (0.3 kcal, Jointed Calcareous- and Crustose-Groups). The approach used in this study demonstrates a realistic technique for predicting macrophyte community composition from knowledge of the disturbance levels in a given habitat or the reverse. The form group-disturbance relationship has important implications for future biological monitoring of rocky-inter-tidal and subtidal systems.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

Promotion of hyphal growth in Ustilago violacea by host factors from Silene alba

Ustilago violacea specifically parasitizes susceptible members of the Caryophyllaceae. We isolated water-soluble compounds from leaves of Silene alba which promoted hyphal development in the dimorphic pathogen. We also isolated hyphal growth promoting -tocopherol from S. alba. The water-soluble activity, which we term hyphal growth factor, or HGF, separated into four bands with gel filtration chromatography and represented over 40% of the total hyphal growth promoting activity isolated from S. alba. The water-soluble HGF activity may be host-specific and may function as a determinant of the host-parasite specificity between U. violacea and caryophyllaceous host plants.Abbreviations HGF Hyphal growth factor - BHT butylated hydroxytoluene     

Cell envelope phospholipid changes in a moderate halophile during phenotypic adaptation to altered salinity and osmotic stress

Abstract The increased content of negatively-charged phospholipids in membranes of Vibrio costicola grown at high salinities is mediated by increased phospholipid synthesis of phosphatidylglycerol relative to phosphatidylethanolamine. This phenomenon provides a system for investigating the factors involved in triggering and controlling haloadaptation in this moderately halophilic bacterium. We review recent experiments, which show that when subjected to sudden increases in external salinity, V. costicola senses both the absolute NaCl concentration and the magnitude of the salt shift. We show that the latter is sensed at least in part via osmotic pressure effects, since shift-up into sucrose-containing media triggers comparable changes in growth and in phospholipid composition and synthesis.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Skeletal growth in a medieval population from Sudanese Nubia

Long bone growth is analyzed for 180 children from a Medieval population at Kulubnarti in Sudanese Nubia (550–1450 A.D.). A regional interpopulation comparison is made with growth data from Wadi Halfa in Lower Nubia, and an intrapopulation analysis is undertaken to assess diachronic changes in growth at Kulubnarti. Changes in growth patterns are interpreted in the context of mortality and morbidity profiles and relationships between the three variables are discussed. It is suggested that changes in the sociopolitical environment may have been responsible in part for altering levels of biological stress and impacting growth.