Acquisition of antigens characteristic of adult pericentral hepatocytes by differentiating fetal hepatoblasts in vitro

Antigens specific to pericentral hepatocytes have been studied in adult mouse liver, during fetal development, and in cultured fetal hepatoblasts. Antibody reactive with glutamine synthetase stained all fetal liver cells but almost all cells lost this antigen after birth; only a single layer of pericentral cells retained it in adulthood. In contrast, monoclonal antibodies to major urinary protein (MUP) did not detect the antigen until approximately 3 wk after birth, after which time the cells within 6-10 cell diameters of the central veins were positive. Cultured fetal liver cells from embryos at 13 +/- 1 d of gestation were capable of differentiating in vitro to mimic events that would occur had the cells remained in the animal. About 10-20% of the explanted cells grew into clusters of hepatocyte-like cells, all of which stained with albumin antibodies. MUP monoclonals were reactive with one-half of the differentiated fetal hepatocytes. Glutamine synthetase was present in all hepatocytes after several days in culture and gradually decreased and remained in only occasional cells, all of which also contained the MUP antigen. These findings suggest that a sequence of gene controls characterizes expression of specific genes in developing liver, and that differentiating fetal hepatoblasts are capable of undergoing similar patterns of gene activity in culture.     

Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy

Purified human milk beta-N-acetylglucosaminide beta 1, 4 galactosyltransferase (EC 2.4.1.38) was used to galactosylate N-acetylglucosamine (GlcNAc) residues present in ultra-thin sections of Lowicryl K4M-embedded rat and pig liver. Both endogenous galactose and galactosylated transferase products could be revealed by Ricinus communis lectin I-gold complexes (RcL I-g15). Without galactosyltransferase (GT) treatment, labeling for galactose (gal) was limited to the trans region of rat and pig hepatocyte Golgi apparatus. After exposure to GT, additional labeling was found over cis Golgi apparatus cisternae. RcL I-g15 labeling was sensitive to a purified preparation of endoglucosaminidase F/peptide N-glycosidase F (at pH 9). This indicates that endogenous gal and gal transferred by GT to terminal GlcNAc residues are present N-linked oligosaccharides. The RcL I-g15 labeling produced by GT was insensitive to extensive washing with solutions containing either EDTA and urea or SDS and 2-mercaptoethanol or 0.1 M GlcNAc. Substrate inhibition studies showed that 50 mM GlcNAc specifically inhibited the additional RcL I-g15 labeling produced by GT. The use of purified glycosyltransferases therefore appears to allow specific detection of oligosaccharide substrates and their high resolution localization in thin sections by electron microscopy.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Barbiturate effects on hippocampal excitatory synaptic responses are selective and pathway specific

Barbiturate actions on excitatory synaptic responses in CA 1 and dentate regions of hippocampal slices were studied to determine whether different effects occur on anatomically distinct synaptic pathways. Pentobarbital facilitated transmission between stratum radiatum inputs and CA 1 neurons at low concentrations (0.02-0.08 mM) and produced postsynaptic depression at higher concentrations. Only depression was observed for stratum oriens inputs to CA 1 and perforant path inputs to dentate granulae neurons. The (+) isomer of pentobarbital was approximately four times more potent than the (-) isomer of racemic mixture. Phenobarbital (0.04-0.12 mM) produced only depression of synaptic responses in CA 1 and dentate pathways. Comparison of effect on field excitatory postsynaptic potentials and population spike responses indicated that the barbiturates act at selective and pathway-specific sites. The results provide further evidence for specific cellular and membrane recognition sites for barbiturate action.     

Biochemical characterization of polypeptide components involved in neurite fasciculation and elongation

Polypeptide components and carbohydrate linkage types of F11 antigen and G4 antigen, two chick cell-surface glycoproteins implicated in neurite fasciculation and elongation [Rathjen, F.G., Wolff, J.M., Bonhoeffer, F. and Rutishauser, U. (1987) J. Cell Biol. 104, 343-353], have been studied in comparison to mouse L1 antigen. Tryptic fingerprint analysis does not reveal any relation of the 130-kDa components of G4 or F11 antigens to each other or to neural cell-adhesion molecules. The 180/190-kDa component of G4 antigen comprises parts of the 130-kDa and 80/65-kDa components and shares a sequence corresponding to the amino terminus of the G4 130-kDa component as shown serologically with anti-peptide sera. This closely parallels the relationship found for mouse L1 antigen components. In contrast, the F11 170-kDa component is different from the F11 130-kDa component, as shown serologically and by fingerprint analysis. A combination of chemical and enzymatic deglycosylation methods reveals that while O-glycosylation cannot be detected F11 130-kDa, G4 130-kDa and L1 140-kDa components contain N-linked carbohydrates. Endoglycosidase H treatment shows that the oligosaccharides present in the G4 130-kDa component and mouse L1 are mostly of the complex type, while the F11 130-kDa component consists of two populations, one containing mainly complex-type carbohydrates and a second containing high-mannose/hybrid-type carbohydrates.     

Comparison of population dynamics between slow- and fast-growing strains of the rotifer Brachionus calyciflorus pallas in continuous culture

Summary The population dynamics of a slow- and a fast-growing strain of the rotifer Brachionus calyciflorus are compared. Rotifers were grown in steady-states, at various specific growth rates (), in both two-stage chemostat and turbidostat cultures on the green alga Chlorella pyrenoidosa. Population variables, including specific ingestion (I), loss (L) and filtration (F) rates, yield (Y), production (P) and half-saturation coefficient of growth (K _g), were calculated using a growth model based on saturation kinetics. I, L, F and K _g were shown to be higher and Y and P lower for the fast-growing strain. Differences between the two strains with regard to these variables may represent tradeoffs associated with the faster potential growth rate. Steady-state relationships between these values and for the fast-growing strain, however, deviated from model predictions which suggest a possible shift from carbon to non-carbon growth limitation.     

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