Neutron-scattering studies of the ribosome of Escherichia coli: a provisional map of the locations of proteins S3, S4, S5, S7, S8 and S9 in the 30 S subunit.

Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed.     

Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.     

Mechanism of insulin aggregation and stabilization in agitated aqueous solutions

Undesirable aggregation of aqueous insulin solutions remains a serious obstacle in the development of alternative methods of diabetes therapy. We investigated the fundamental nature of the aggregation mechanism and proposed stabilization strategies based on a mathematical model for the reaction scheme. Insulin aggregation kinetics in the presence of solid-liquid and air-liquid interfaces were monitored using UV spectroscopy and quasielastic light scattering (QELS). Experimental observations were consistent with our model of monomer denaturation at hydrophobic surfaces followed by the formation of stable intermediate species which facilitated subsequent macroaggregation. The model was used to predict qualitative trends in insulin aggregation behavior, to propose stabilization strategies, and to elucidate mechanisms of stabilization. In the absence of additives, insulin solutions aggregated completely (more than 95% of the soluble protein lost) within 24 h; with sugarbased nonionic detergents, no detectable loss occurred for more than 6 weeks. (c) 1992 John Wiley & Sons, Inc.     

Lipid-alginate interactions render changes in phospholipid bilayer permeability

Lipid vesicles, e.g. liposomes, generally release their contents in a continuous manner. However, when these vesicles are entrapped in Ca-alginate and coated with poly(L-lysine), they release their contents in an unusual fashion, in 'bursts'. Molecular-level studies indicated that lipid-alginate interactions are responsible for changes in the barrier properties of lipid vesicles. Differential scanning calorimetry revealed that exposure of liposomes to alginate resulted in a 4-fold reduction in the phase transition enthalpy, with no change in the melting temperature. Size-exclusion chromatography of liposomes-in-alginate gave an additional liposomal peak with a smaller elution volume. These studies suggested that alginate is inserted into the lipid bilayer of vesicles. Lipid-alginate interactions were highly dependent on phospholipid head group charge and the phase transition temperature of the phospholipid. Based on these interactions, a mechanism to explain the 'burst' from these entrapped liposomes is suggested.     

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.     

Structural reactions to polarized light of microvilli in photoreceptor cells of the moth Spodoptera

Summary Intact armyworm moths (Spodoptera exempta, Farn. Noctuidae) were illuminated by polarized monochromatic light to induce structural changes in the rhabdomeres of the compound eyes. The degree of distortion of their microvilli depends on the light energy absorbed per time unit. Under polarized light, the number of quanta absorbed varies with the position of the plane of polarization relative to the axis of the microvilli (intrinsic dichroism). Therefore, in Spodoptera, different degrees of deformations could be demonstrated in differently oriented rhabdomeres of both types of ommatidia. Moreover, in rhabdoms of the lobed type with fan-like arranged microvilli, different reactions were regularly seen in differently oriented microvilli of one rhabdomere. This indicates that microvilli may react to light individually.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

New, rapid methods for purifying alpha-actinin from chicken gizzard and chicken pectoral muscle

We introduce two new, rapid procedures. One is specifically designed for isolating alpha-actinin from skeletal and the other for isolating alpha-actinin from smooth muscle. Approximately 20 mg of greater than 95% pure alpha-actinin can be obtained/100 g of ground chicken pectoral muscle in just 4 days. The smooth muscle protocol yields 2.7 mg of greater than 99% pure alpha-actinin/100 g of ground gizzard after just 5 days. Differences in protein contaminants and in the extractability of alpha-actinin necessitated the development of separate isolation procedures for the two muscle types. Antibody prepared against the purified gizzard alpha-actinin reacted with alpha-actinin from skeletal, cardiac, and smooth muscle in immunodiffusion. Anti-alpha-actinin reacted only with alpha-actinin from crude extracts of skeletal and smooth muscle on Staph A gels. Anti-alpha-actinin stained Z-bands from skeletal muscle in indirect immunofluorescence microscopy and stress fibers from baby hamster kidney fibroblasts and mouse mammary epithelial cells in the characteristic punctate pattern observed by other workers (Lazarides, E., and Burridge, K. (1975) Cell 6, 289-298). These two methods for purifying alpha-actinin from skeletal and smooth muscle represent a significant improvement over that published previously.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Histologic study of the teleost liver. II. The blood vessel system

In the liver of the teleosts investigated in the present study the sinusoidal region shows a space of DISSE, which contains numerous microvilli originating from the hepatocytes. In some species, especially in Tetraodon leiurus, there are also bundles of collagenous fibrils. In the DISSE's space of larger sinusoids (transition sinusoids) sections of filament-rich cells are found. These are sometimes interconnected by desmosomes and can be interpreted as processes of smooth muscle cells from the region of the venae hepaticae. The endothelium of smaller sinusoids is fenestrated and shows micropinocytotic activity. The endothelia of the transition sinusoids and of the venae hepaticae are endowed with structures, which can be interpreted as macrovesicles. In the sinusoidal region true KUPFFER-cells and ITO-cells could not be observed. Nevertheless, the close location of granulocytes to the sinusoidal endothelium suggests that phagocytotic processes cannot be excluded for the sinusoidal region. Exceptionally, in Hemihaplochromis multicolor there were also signs of possible phagocytosis by sinusoidal endothelial cells. The chemomorphology of the sinusoidal region, above all the evidence of alkaline phosphatase, shows great differences between species. The examination of the larger blood-vessels in the liver of Haplochromis burtoni reveals venae portae with very thin walls and venae hepaticae with thick walls, which contain smooth muscle cells. Granulocytes and melanocytes are abundant in the wall of the venae hepaticae. This phenomenon indicates that the defence-functions, which in the liver of higher vertebrates are carried out by the sinusoids (KUPFFER-cells), are undertaken by the region of the venae hepaticae in the liver of Haplochromis burtoni, which is free of KUPFFER-cells. On their extrahepatic course the venae portae are surrounded by a sleeve of exocrine pancreatic tissue, which accompanies the vessels deep into the liver. The pancreatic cells bordering the thin-walled venae portae have sparse microvilli indicating a transfer of substances between venous blood and pancreas similar to the sinusoidal region of the liver. Furthermore, the investigation resulted in a clue to the innervation of the exocrine pancreas.