Reactivity of glycoconjugate in membrane system II: Can a neutral glycolipid function as lectin receptor in the presence of gangliosides in plasma membrane?

To examine how surface Potential controls the reactivity of glycoconjugates at cell surface, the interaction of galactose-sPecific lectinse.g. peanut agglutinin,Ricinus cummunis agglutinin with liPosomes bearing asialo GM₁ were studied in the Presence of varying amount of ganglioside mixture, GM_n. The Presence of 5% GM_n causes comPlete slowing down of PreciPitin reaction and thereby make carbohydrate moiety of asialo GM₁ comPletely inaccessiblei.e. ‘cryPtic’. In contrast the Presence of 1–2% GM_n enhances the aPParent rate and amPlitude of the PreciPitin reaction as surface Potential becomes more negative. The relevance of the findings has been discussed in relation to the exPression and involvement of the cell-surface sialic acid residues during develoPment and differentiation.     

Interrelationships among three derivative forms of the immigrans-Hirtodrosophila radiation: evidence from heterochromatin and polytene chromosomes

The phylogenetic relationships among three derivative forms of the immigrans-Hirtodrosophila radiation viz. Chaetodrosophilella, Zaprionus and the immigrans species group are examined, by comparing the banding patterns of their polytene chromosomes and by analysing the nature of their heterochromatin. Based on the results of these studies it is concluded that D. quadrilineata has a very strong affinity with the members of the immigrans species group, while the genus Zaprionus represents a very distinct evolutionary lineage. This study further indicates that D. quadrilineata is a karyotypically primitive species having five pairs of rods and one pair of dots, while the karyotype of other members of the immigrans species group appears to have undergone modifications through fusions, fissions, inversions and the addition or deletion of heterochromatin to dots and other chromosomes.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

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.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.     

HLA-DP and HLA-DO genes in presumptive HLA-identical siblings: structural and functional identification of allelic variation

We analyzed HLA class II genomic polymorphisms in three families in which bone marrow transplantation was performed between individuals presumed to be HLA identical, but in which unexplained mixed lymphocyte culture reactivity was observed. These families were characterized by classical HLA serology, MLC, and DP typing. In each family, a pair of "HLA-identical" siblings demonstrated a small proliferative response in bidirectional MLC. Southern blotting analysis performed with cDNA probes for DQ alpha, DP alpha, and DP beta identified DP genomic differences in each case. Hybridization of Bgl II-digested genomic DNA with a DP alpha cDNA probe revealed three prominent polymorphic fragments (7.7, 5.8, and 3.7 kb), which discriminated between presumptive identical siblings and indicated crossover events within HLA. Similarly, hybridization of SstI-digested genomic DNA with a DP beta cDNA probe, although resulting in a more complex pattern, identified DP genomic disparity between the presumed HLA identical siblings. Hybridization of SstI-digested DNA from two families with evidence of DP recombination was performed by using an oligonucleotide probe specific for the newly described HLA class II gene DO beta. Two major polymorphic fragments, at 6.2 and 3.3 kb, segregated in these families and localized the crossovers flanking the DO beta gene between the DQ and DP loci. The contribution of the antigenic differences marked by these HLA DP and DO DNA polymorphisms to allorecognition in MLR and in graft-vs-host disease are discussed.     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

Relationship between Sodium Influx and Salt Tolerance of Nitrogen-Fixing Cyanobacteria

The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na⁺ than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K⁺ (>25 mM) in the medium causes membrane depolarization and inhibits Na⁺ influx in both cyanobacteria (S. K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na⁺ influx accompanied by membrane depolarization. These experimental manipulations affecting Na⁺ uptake demonstrated a good negative correlation between Na⁺ influx and salt tolerance. All treatments which inhibited Na⁺ influx (such as alkaline pH, K⁺ above 25 mM, NO₃⁻, and NH₄⁺), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na⁺ influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria.     

Propeptide of human protein C is necessary for gamma-carboxylation

Protein C is one of a family of vitamin K dependent proteins, including blood coagulation factors and bone proteins, that contains gamma-carboxyglutamic acid. Sequence analysis of the cDNAs for these proteins has revealed the presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from the growing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. In the present study, deletion mutants have been constructed in the propeptide region of the cDNA for human protein C, and the cDNAs were then expressed in mammalian cell culture. These deletions included the removal of 4, 9, 12, 15, 16, or 17 amino acids comprising the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These experiments have shown that protein C is readily synthesized in mammalian cell cultures, processed, and secreted as a two-chain molecule with biological activity. Furthermore, the pre portion or signal sequence in human protein C is 18 amino acids in length, and the pro portion of the leader sequence is 24 amino acids in length. Also, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) is important for gamma-carboxylation of protein C, while the present data and those of others indicate that the carboxyl-terminal portion of the propeptide (residues -1 through -4) is important for the removal of the pro leader sequence by proteolytic processing.