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.     

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.     

The entrapment of [14C]ascorbic acid in human erythrocytes

Radioactively labelled ascorbic acid and dehydroascorbic acid, when incubated with human blood, migrate irreversibly into human red blood cells. Isolation and characterization of the moieties trapped within the cells via infrared spectroscopy established both their identities as L-ascorbic acid. Evidence in the form of the degree of in vitro entrapment of ascorbic acid as a function of the times of incubation and the effect of incubation temperature, anion recognition site inhibitor, and active transport inhibitor on the rate of entrapment support the hypothesis that ascorbic acid is oxidized on or near the surface of the red blood cell to dehydroascorbic acid which migrates through the lipid portion of the cell wall and is reduced back to ascorbic acid within the cell. The resulting L-ascorbic acid can not pass through the cell wall and is therefore entrapped.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Determination of the serological sex-specific (Sxs) antigen (“H-Y antigen”) in birds and mammals using high-titer antisera and a sensitive urease ELISA

Summary A rapid, sensitive, and reproducible enzyme-linked immunosorbent assay (ELISA), for the detection of the serological sex-specific (Sxs) antigen (formerly termed H-Y antigen; see Introduction), is described. This assay uses bovine lestes extract as the solid phase antigen, and high-titer anti-Sxs antisera and a urease-conjugated anti rat-IgG as the first and second antibody respectively. The urea containing substrate causes a pH shift in a positive reaction, which in turn is visualized by the use of bromocresol purple as a pH indicator. The method, and some representative applications of it, are described in detail.     

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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Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.