Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

Decrease of palmitoyl-CoA elongation in platelets and leukocytes in the patient of hereditary methemoglobinemia associated with mental retardation

Effect of the deficiency of NADH-cytochrome b5 reductase on fatty acid elongation was studied in the platelets and leukocytes taken from a patient of hereditary methemoglobinemia associated with mental retardation. The activity of fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA. The de novo biosynthesis of fatty acids was blocked by the addition of phosphotransacetylase, and the elongation system could be assayed in the homogenates separated from de novo biosynthesis. As compared to normal subjects approximately 40% decrease of fatty acid elongation was observed both in the platelets and leukocytes from the patient.     

Identifying the fluorescent body (F-body) with quinacrine mustard staining of canine

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.     

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.     

cDNA cloning of a novel heterogeneous nuclear ribonucleoprotein gene homologue in Caenorhabditis elegans using hamster prion protein cDNA as a hybridization probe.

The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.     

Human corticotropin-releasing hormone test in normal subjects and patients with hypothalamic, pituitary or adrenocortical disorders

Human corticotropin-releasing hormone (hCRH) test was performed in 57 normal volunteers and 102 patients with hypothalamic, pituitary and adrenocortical diseases. Intravenous bolus injection of synthetic hCRH, 100 micrograms for adults or 1.5 micrograms/kg for children, increased plasma ACTH and cortisol levels in about 90% of normal subjects. In 47 patients with Cushing's disease, plasma ACTH tended to show an exaggerated response to hCRH and peak ACTH was the most frequent abnormal component among the several reaction parameters. Poor responders among normal subjects and patients with Cushing's disease had significantly higher plasma cortisol levels before CRH administration. Patients with hypothalamic hypopituitarism showed exaggerated response, whereas patients with primary pituitary lesion, isolated ACTH deficiency or adrenal Cushing's syndrome showed no ACTH response. These differences in the response of patients suggest the value of the hCRH test in their differential diagnosis.     

Characterization of dehydropeptidase I in the rat lung

The activity of dehydropeptidase I in rat tissues decreases in the order of lung greater than kidney greater than liver-spleen greater than other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and high-performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150,000 by gel filtration, comprising a homodimer of two 80,000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.     

Effect of polyamines on in vitro reconstitution of ribosomal subunits

The effect of polyamines on in vitro reconstitution of Escherichia coli 30S and 50S ribosomal subunits has been studied. Spermidine stimulated the reconstitution of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits at least 1.6-fold. The reconstitution of 30S particles from normal 16S rRNA and total proteins of 30S subunits exhibited only slight spermidine stimulation. However, the optimal Mg2+ concentration of the reconstitution was decreased from 20 mM to 16 mM in the presence of 3 mM spermidine. In the absence of spermidine the assembly of 30S particles from normal 16S rRNA was more rapid than the assembly from 16S rRNA lacking the methyl groups on two neighboring adenines. The reconstitution of 50S particles from 23S and 5S rRNA and total proteins of 50S subunits was not influenced greatly by spermidine. Gel electrophoresis results, from reconstitution experiments of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits, showed that the assembly of S1 and S9 proteins to 23S core particles was stimulated by spermidine during reconstitution. The relationship of polyamine effects on in vitro ribosome assembly from its constituents to in vivo ribosome assembly is discussed. The reconstitution of Bacillus subtilis 30S particles from 16S rRNA and total proteins of 30S subunits was also stimulated approximately 1.3-fold by 3 mM spermidine.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.