Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Adverse immune reactions to gold. I. Chronic treatment with an Au(I) drug sensitizes mouse T cells not to Au(I), but to Au(III) and induces autoantibody formation

Upon weekly i.m. injections of disodium gold thiomalate (Na2AuTM) 100% of A.SW mice produced IgG autoantibodies to antinuclear Ag and nucleolar Ag, respectively; about 70% of C57BL/6 mice produced IgG antinuclear Ag, whereas DBA/2 mice were resistant. Moreover, C57BL/6 mice, but not DBA/2 mice, showed increased mesangial deposits of IgG. These alterations were due not to disodium thiomalate, but to the gold ion of Na2AuTM. An assumed T cell reactivity of susceptible mouse strains to Na2AuTM was tested by means of the direct popliteal lymph node (PLN) assay. However, no distinct PLN reaction to Na2AuTM was detectable. Likewise, AuCl did not induce a PLN reaction. Both Na2AuTM and AuCl contain gold in the Au(I) state. The poor PLN responses to Au(I) contrasted with the strong PLN responses to Au(III) compounds. PLN reactions to Au(III) were dose dependent, T cell dependent, and specific. When Au(III) was reduced to Au(I) by addition of Na2TM or methionine before testing in the PLN assay its sensitizing capacity was significantly decreased. Thus, the oxidation state of gold, i.e., Au(III) vs Au(I), plays a major role for its sensitizing capacity. Therefore, we propose that the Au(I) of Na2AuTM is oxidized to Au(III) before T cells are sensitized and adverse immunologic reactions develop. Results obtained with the adoptive transfer PLN assay indicated that, indeed, repeated i.m. injections of Na2AuTM sensitized A.SW and C57BL/6 splenic T cells to Au(III).     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression

The human T lymphocyte Ag CD28 (Tp44) is a homodimeric glycoprotein expressed on the surface of a majority of human peripheral T cells and thymocytes. Although exposure of T cells to anti-CD28 mAb does not activate T cells, stimulation of CD28 can synergize with signals transmitted through the TCR or other stimuli to augment proliferation and lymphokine production. We have used a portion of the human CD28 cDNA to isolate a homologous murine cDNA from an EL4 T lymphoma library. The murine clone has 61% nucleotide identity with the human cDNA. Both human and murine sequences exhibit homology with members of the Ig supergene family and CTLA-4, a T cell specific murine gene. Many characteristics of the human CD28 molecule are conserved within the putative murine CD28 polypeptide. The murine cDNA sequence encodes a polypeptide of 218 amino acids that has 68% identity with the human sequence. Both the murine and human molecules are integral membrane glycoproteins with hydrophobic signal peptide sequences and transmembrane region. All five potential N-linked glycosylation sites are conserved and six of the seven cysteine residues of the mouse protein are found in the human CD28 polypeptide. The murine cDNA is encoded by a single copy nonrearranging gene whose expression at the mRNA level is restricted to T cells. A rabbit antiserum was raised against a synthetic peptide corresponding to a hydrophilic portion of the translated murine cDNA sequence. This antiserum identifies an 80-kDa homodimer consisting of disulfide-bonded subunits of 40 kDa that is expressed on splenic T cells, thymocytes, and several T cell tumors, but not on B cells. deglycosylation studies indicate that four of the five N-linked glycosylation sites are used and that the mature core protein has a molecular mass of 25 kDa, close to that predicted by the cDNA sequence. Transfection of the murine cDNA into Chinese hamster ovary cells resulted in the expression of an 80-kDa dimeric molecule that was immunoprecipitated by the antipeptide antiserum. Taken together, these data provide strong support that we have identified the murine homologue of CD28.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.     

Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity

A soluble form of homologous restriction factor (HRF-U) was isolated from normal human urine. With respect to m.w. (65,000) and immunoblotting characteristics, it resembled membrane HRF (HRF-M) that had been isolated from human E membranes. The protein exhibited limited cross-reactivity with the channel-forming proteins of C and cytotoxic lymphocytes. It inhibited reactive lysis of E by human C5b-9. Inhibition occurred at the attachment stage of C5b-7 to target cells, rather than at the C8 or C9 stage of membrane attack complex assembly which is inhibited by HRF-M. In this respect, HRF-U acts analogously to S protein of serum, but no immunochemical relationship between these two proteins was detected. HRF-U might be derived from the soluble HRF detected in cytoplasmic granules of killer lymphocytes.     

Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes

The aim of the present study was to characterize the acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity in human liver microsomes. Liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In 34 patients the enzyme activity of the microsomal fraction averaged 6.6 +/- 0.7 (mean +/- SEM) pmol.min-1.mg protein-1 in the absence of exogenous cholesterol. Freezing of the liver biopsy in liquid nitrogen increased the enzyme activity five- to sixfold. Similarly, freezing of the microsomal fraction prepared from unfrozen liver tissue increased the enzyme activity about twofold. These results may help to explain previous disparate results reported in the literature. The enhanced ACAT activity obtained by freezing was at least partly explained by a transfer of unesterified cholesterol to the microsomal fraction and possibly also by making the substrate(s) more available to the enzyme. Preincubation of the microsomal fraction, prepared from unfrozen liver tissue, with unlabeled cholesterol increased the enzyme activity about fivefold. This finding indicates that hepatic ACAT in humans can also utilize exogenous cholesterol as substrate. Addition of cholesterol to frozen microsomes prepared from unfrozen liver tissue increased the ACAT activity two- to threefold, whereas addition of cholesterol to microsomes prepared from frozen liver tissue did not further increase the enzyme activity. No evidence supporting the concept that ACAT is activated-inactivated by phosphorylation-dephosphorylation could be obtained by assaying the enzyme under conditions similar to those during which the human HMG-CoA reductase is inactivated-activated.     

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.