A segment of rye chromosome 1 enhances growth and embryogenesis of calli derived from immature embryos of wheat

Summary The influence of the short arm of rye chromosome 1 (1RS) from Secale cereale var. Imperial on the growth and differentiation of callus cultures from wheat Triticum aestivum var. Chinese Spring immature embryos was analysed. This chromosome arm was found to stimulate both embryogenesis and the rate of growth of calli. Recombinant lines carrying segments of 1RS were used to delineate the regions of 1RS responsible for the tissue culture effects. The enhancement of embryogenesis and the stimulation of growth were shown to be associated with two distinct genetic regions of the chromosome arm; the former is located between the centromere and the Sec 1 locus, while the latter is situated in the immediate vicinity of the Sec 1 locus.     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels

Summary A new low shear stress microcarrier culture system has been developed at NASA’s Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.     

Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates

Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.     

The fine structure of the terminal segment of the bovine seminiferous tubule

Summary The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Theoretical basis of single breath gas absorption tests

Absorption of gas from alveoli is examined in a simplified model of the respiratory system during a stylized single breath consisting of constant inspiratory flow, constant expiratory flow, and breathholding. The equations describing gas behavior are general since they are based upon conservation of mass. The equations simplify considerably when gases that are not soluble in pulmonary tissue and/or blood are utilized. In a three-compartment model, diffusing capacity of the lung for carbon monoxide (D _CO ) will be underestimated except when both uneven distribution of lung volume andD _CO are present; under most circumstances, the standard clinical 10-s method [9] is at least as accurate as any other. When pulmonary capillary blood flow is calculated by the one point method [2] in a one-compartment lung, it is underestimated; in the three-compartment model, it is underestimated except when both uneven distribution of . and lung volume are present. The multiple single breath method [2] accurately measuresD _CO and . Measurement of pulmonary tissue volume is improved by correcting the value of the intercept of acetylene absorption to the time when carbon monoxide apparently began rather than utilizing the beginning of inspiration.Nomenclature D _CO diffusing capacity of the lung for CO (ml CO, STPD/min/mm Hg) - pulmonary capillary blood flow rate (L/min) - V _t pulmonary tissue volume (L) - V _A alveolar compartment volume (L) - V _Ao alveolar compartment volume at conclusion of inspiratory flow (L) - inspiratory flow rate (L/sec) - expiratory flow rate (L/sec) - Bunsen coefficient of pulmonary tissue for test gas (ml test gas/ml tissue/atm) - Bunsen coefficient of pulmonary tissue for test gas (ml test gas/ml blood/atm) - F _A fractional pressure of test gas in the alveolar compartment (atm)