An IgM-secreting cell line derived from the mouse plasmacytoma 104E expresses a different VH gene

The BALB/c plasmacytoma 104E has been maintained in histocompatible mice for more than ten years. It secretes an IgM which binds α-1,3-dextran. The amino acid sequence of its λ light chain and μ heavy chain have been completely determined. Attempts to adapt 104E to continuous growth in culture led to the isolation of a ‘variant’ cell line (104-76), which produced IgM (μ,κ) and lacked dextran-binding specificity. We have purified the heavy chains from 104-76 and compared them with 104E μ. The 104-76 does secrete fully assembled IgM pentamers but the isolated μ chain has a different isoelectric focusing pattern from 104E μ. The cyanogen bromide fragments which orginate from the constant regions of 104E and 104-76 are the same size, but three of the four have a different charge, perhaps a result of the carbohydrate they bear. The cyanogen bromide fragments which originate all or in part from the variable region of 104-76 μ are completely different in both size and charge from the variable region fragments of 104E. These results indicate that 104E and 104-76 express completely different V_h genes. Therefore, 104-76 is unlikely to be a simple mutational derivative of 104E. Alternative explanations for the origin of 104-76 are considered.     

Ultrastructural immunocytochemical localization of enkephalin in the goldfish preoptic nucleus

The ability of membrane perturbing drugs to inhibit the specific binding of [3H]/B4101 (2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane) to the alpha 1-receptor of beef cortical membranes was determined. Except for quinidine and phenytoin, the IC50 for inhibition of specific [3H]WB4101 binding to the alpha 1-receptor correlated with the membrane/buffer partition coefficient of the drug. Almost all of the drugs inhibited specific ligand binding at concentrations approaching those reported necessary to stabilize membranes although both activities correlate well with reported membrane/buffer partition coefficients. The results suggest that specific receptor function can be modified by drug perturbation of the lipid microenvironment of the receptor macromolecule.     

Polypeptide profile of HBSAg excreted by a human hepatoma cell line.

In this study the polypeptide composition of HB_sAg particles secreted by a human hepatoma cell line (Alexander) was analysed. Five major components were identified with molecular weights of 22,000, 28,000, 33,000, 38,000, and 49,000. The two smaller species (MW 22,000 and 28,000) were found to be the major components. The polypeptide profile is similar to that described for HB_sAg derived from sera of acute hepatitis patients and of chronic carriers. Data are also presented suggesting that the various HB_sAg polypeptide components are aggregates and glycosylated derivatives of the 22,000 MW polypeptide subunit.     

Amino acid sequence studies of human C4b-binding protein: N-terminal sequence analysis and alignment of the fragments produced by limited proteolysis with chymotrypsin and the peptides produced by cyanogen bromide treatment

Treatment of human C4b-binding protein (C4BP) with cyanogen bromide gave five major peptides and limited proteolysis with chymotrypsin yielded two fragments. The yields, apparent mol. wts and N-terminal amino acid sequences of these peptides and fragments indicates that in dissociating conditions, after reduction of disulphide bonds, C4BP is composed of only one type of polypeptide chain of approx. 70,000 mol. wt. The amino acid sequence data obtained, which accounts for over 55% of the total sequence, allows an alignment of the cyanogen bromide peptides. In addition the amino acid sequence data indicates that the 70,000-dalton polypeptide chain of C4BP contains nine internal homology regions, each 60 amino acids long, which would account for 540 of the expected 600 amino acids in C4BP. Similar internal homology regions are found within the Ba region of factor B [Morley and Campbell, EMBO J. 3, 153-157 (1984)] and it is of interest that the regions found in C4BP are homologous to those found in Ba.     

Alteration in the membrane fatty acid composition of human lymphocytes and cultured transformed cells induced by interferon

The effects of interferon (IFN) treatment on the lipid composition of human peripheral blood lymphocytes or transformed cell line cells were investigated. The major phospholipid classes of lymphocytes as analyzed by 2-dimensional TLC and quantified by phosphorous content were phosphatidylcholine (PC, 43%) and phosphatidylethanolamine (PE, 28%), along with phosphatidylserine (9%) and phosphatidylinositol (8%). The membrane-impermeant reagent, trinitrobenzenesulfonate was used to covalently label cell surface PE. Fatty acid (FA) composition, determined by gas-liquid chromatography, showed a distinct pattern in each lipid class, with a predominance of 16 and 18 carbon fatty acids (FA) in PC and PE respectively. Arachidonic acid (20:4) and, to a lesser extent, docosahexanoic acid (22:6) were predominant in PE. The degree of unsaturation in each class, expressed as the ratio between unsaturated and saturated FA (U/S), was higher in PE (1.72) than in derivatized trinitrophenyl cell surface PE (TNP-PE, 0.57) or PC (0.64). Treatment with IFN resulted in an increased U/S ratio in cell surface PE (1.10) but not in other PE species (1.46). A small increase in unsaturation (0.88) was also observed in PC. Most of the increase in TNP-PE U/S was accounted for by an increase in 20:4 and a concomitant decrease in 18:0. These alterations were observed in the absence of quantitative change in the principal phospholipid classes or in the FA composition of the total lipid extract. In K562, a transformed cell line with characteristics of the erythromyeloid lineage, PE was found to be the most saturated lipid class with a predominance of 18:0. In PC, 16:0 was most abundant. Among unsaturated FA, 18:1 predominated in all lipid classes studied. Treatment with natural IFN alpha for 30 hr generally resulted in a decrease in saturated FA and an increase in unsaturated FA, which was most marked in PE. The U/S ratio in PE was highest in K562 cells during the time of maximal cell proliferation as assessed by tritiated thymidine incorporation. TNP-PE simultaneously decreased. Daudi cells, a B-lymphoblastoid cell line, demonstrated changes in FA composition of lipids with decreased saturated and monoenoic FA after IFN treatment, whereas DIF3 (a clone selected for lack of sensitivity to IFN) showed no change. These studies document changes in membrane FA composition of lymphocytes treated with IFN and correlate IFN-induced changes in transformed cell line FA with effects on proliferation. They further show the existence of a transverse molecular species asymmetry of PE in the plasma membrane of these cells which is altered after IFN treatment.     

Salmonid spleen and anterior kidney harbor populations of lymphocytes with different B cell repertoires

An O-antigen extract of the fish pathogen Vibrio anguillarum was used to stimulate plaque-forming cells (PFC) in coho salmon. The kinetics of the response demonstrates peak PFC per organ on day 16 post immunization for the spleen and anterior kidney. Significant PFC were also seen in thymic tissue. PFC responses were shown to be immunoglobulin mediated and antigen specific. Inhibition profiles of responding PFC populations demonstrate that the cells of the anterior kidney are more restricted in their recognition of antigen than those of the spleen. These data lend functional support to the concept of the hematopoietic nature of the anterior kidney of teleosts.     

Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor

Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events.     

Topographic and functional assay of antigenic determinants of human prolactin with monoclonal antibodies

Six distinct antigenic determinants were identified on human prolactin (hPRL) by competition assays with murine monoclonal antibodies (MABs). The affinity of binding and the cross-reactivity of the antibodies with two non-primate prolactins was also determined. Binding of 125I-hPRL to MAB-coated microtitre plates in the presence of a second MAB resulted in either inhibition or enhancement of antigen binding to the plate. These results were interpreted in terms of conformational changes to epitopes, induced allosterically by the binding of a second MAB to the antigen. The topographic relationship of epitopes to the biologically active regions on the hormone was examined on the basis of the neutralizing potency of MABs in the proliferation of the prolactin-dependent cell line NB2. The NB2 growth-inhibitory activity was restricted to three distinct epitopes (NE02/6, 1208 and NE03) but absent from three other MABs tested (QB01, WC01/3 and 1200). On the basis of the competition and functional studies, an elemental scheme of the topographic localization of epitopes is presented. The experimental approach employed may contribute to studies on the allocation of biologically active sites on protein hormones.     

Studies on an epithelial/Burkitt hybrid cell line prepared from a lymphoblastoid cell line with one EBV genome equivalent per cell.

An epithelial hybrid cell line was prepared by fusing D98 cells to an HR-1 cell line previously shown to contain only one EBV genome equivalent per cell. At an early passage level, p 11, the EBV genome could not be induced to synthesize EBV antigens after treatment with iododeoxyuridine (IUdR). When the cells were retested for this property at passage 34, full induction of the EBV genome was observed. The ability of induction of EBV antigens and virus DNA depended on a spontaneous increase in the level of EBV DNA in the cells as they were passaged.     

Effect of 2'5'-oligoadenylic acid on a mouse cell line partially resistant to interferon

We have investigated the sensitivity of a mouse cell line, NIH 3T3 (clone 1), that was found to be resistant to the anticellular and certain antiviral activities of interferon, to 2′,5′-oligoadenylate (2′5′A). The 2′5′A was introduced into the cells by coprecipitation with calcium phosphate and by increasing cell permeability with lysolecithin. In both cases neither cellular protein nor DNA synthesis was inhibited by 2′5′A in the NIH 3T3 cells. In contrast, the synthesis of proteins and DNA in L cells, an IFN sensitive line, was inhibited by low concentrations of 2′5′A. Furthermore, treatment of the infected cell cultures with 2′5′A resulted in the inhibition of vesicular stomatitis virus (VSV) replication in L cells but not in NIH 3T3 cells. The production of MuLV in NIH 3T3 cells was also not affected by 2′5′A. These observations, together with our previous finding that NIH 3T3 (clone 1) cells are deficient in RNase F activity suggest that activation of the RNase F by 2′5′A is responsible for the inhibition of both protein and DNA synthesis in sensitive cells.     

Induction of megamitochondria in the mouse liver by isonicotinic acid derivatives

Megamitochondria were induced in mouse hepatocytes simply by feeding the animal for 7 to 10 days with a diet containing isonicotinic acid derivatives, namely, nialamide, isoniazid, and iproniazid. Monoamine oxidase activities of megamitochondria were invariably decreased significantly. Isonicotinic acid, however, did not induce megamitochondria and had no effect on monoamine oxidase activity of mitochondria. Coupling efficiencies of megamitochondria induced by the reagents, specified above, were not affected. Cytochrome c oxidase and ATPase activities of megamitochondria were also unchanged compared to the control. Isoniazid, iproniazid, and nialamide affected monoamine oxidase activity of mitochondria not only in vivo, as described above, but also in vitro: the activity was inhibited by 50% at concentrations of 5.8 mM, 215 and 140 μM for isoniazid, iproniazid, and nialamide, respectively. Rotenone-insensitive NADH-cytochrome c reductase of megamitochondria, another marker enzyme for mitochondrial outer membrane besides monoamine oxidase, was not affected so much: the lowest activity was 88.6% of the control in the case of nialamide-induced megamitochondria. These reagents had no effect on the enzyme activity in vitro. These data seem to indicate that the reagents examined in the present study affected monoamine oxidase specifically. Mechanism of the formation of megamitochondria is discussed with respect to membrane fusion phenomenon.     

Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III

A filamentous phage derivative, fKN16, has been constructed from the tetracyclineresistance transducing phage fd-tet by deleting a 507-base-pair (bp) segment of phage gene III. In accord with the importance of the gene III protein in infection, the infectivity of fKN16 phage is less than 10^?8 that of fd-tet phage. In contrast to most gene III amber mutants, which are polar on the downstream phage genes VI and I, fKN16 should be a nonpolar mutant since its 507-bp deletion spans an integral number of coding triplets. And indeed, two phage traits that may depend on gene VI and I function—the level of phage production and packaging into unit-length virions—appear to be normal in fKN16. High phage production coupled with very low infectivity make fKN16 suitable as a vector for DNA cloning experiments requiring a high level of biological containment. The characteristics of fKN16 as a vector were investigated in detail, using HindIII fragments of phage λ DNA as model foreign DNA. fKN16 may also be useful in studying the role of the gene III protein in the filamentous phage life cycle.     

Ultrastructural analysis of platelets in nonhuman primates. III. Stereo microscopy of microtubules during platelet adhesion and the release reaction

The role of microtubules in adhesion and in the adhesion-stimulated release reaction of platelets from African green monkeys has been studied using conventional (100 kV) and high voltage (1000 kV) stereo electron microscopy. Upon exposure in vitro to either glass or a carbon-stabilized formvar surface, platelets were rapidly activated and extended numerous filopodia. The adhesion process evolved over a period of 10–20 min with extension of a delicate hyalomere across the surface between adjacent filopodia. Microtubules, which form a circumferential ring in the resting cell, were dissociated upon cell activation and then reassembled in patterns which conformed to the general cell shape. In dendritic cells with numerous filopodia the microtubules were oriented radially. With subsequent hyalomere development, the microtubules paralleled major cell axes. This morphological transition of platelet adhesion was accompanied by the release of β-thromboglobulin (B-TG) which was maximal at 20 min, a time that corresponded to maximal hyalomere extension. Treatment of platelets with colchicine or vinblastine sulfate prior to adhesion dissociated the microtubules, but affected neither the morphological transition nor β-TG release. Our observations separate microtubules from adhesion-related events and suggest that during adhesion the microtubules, by conforming to cell shape, may simply provide cytoskeletal support.     

Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique

A modified centrifugal elutriation technique is described for the isolation of large numbers of lymphocytes and monocytes. Elutriation was carried out by lowering the rotor speed at a constant flow rate which was generated by hydrostatic pressure. The flow rate could be kept constant if the separation procedure was performed at high pressure and high systemic resistance.

Up to 2.3 × 10⁹ mononuclear cells derived from 2000 ml blood were separated in one single experiment in approximately 1 h. The lymphocytes and monocytes were isolated at purities of 98 ± 1% and 94 ± 1% respectively. The purity of the lymphocytes was increased to 99.8 ± 0.1% by a second elutriation run. Additional advantages of the elutriation procedures are that the choice of medium is free, and that relatively large numbers of cells may be separated with high recoveries.     

Differential release of D-[3H]aspartate and [14C]gamma-aminobutyric acid following activation of commissural fibres in a longitudinal slice preparation of guinea pig hippocampus

Low-stimulus frequencies released both D-[3H]aspartate and [14C]GABA in double labelling experiments after activation of the commissural fibres to stratum oriens in hippocampal slices. The stimulus-dependent release of [14C]GABA was highest at low stimulus frequencies (10 Hz) whereas at higher stimulus frequencies (30--50 Hz) it was almost abolished. All stimulus frequencies used released D-[3H]aspartate. The results indicate that activation of the commissural fibres in stratum oriens, using low stimulus frequencies, activates pyramidal cells which stimulate inhibitory neurones releasing GABA as a transmitter.     

Antibodies to synthetic polypeptides corresponding to hydrophilic regions of human interferon gamma

Synthetic polypeptides corresponding to hydrophilic regions of human interferon gamma (HuIFN gamma) based on the amino acid sequence of HuIFN gamma inferred from its cDNA sequence were used to produce antibodies in rabbits which reacted with the polypeptides and which might also be expected to recognise native HuIFN gamma. Groups of 3 or 4 rabbits were immunised with synthetic polypeptides corresponding to HuIFN gamma amino acid sequences 1-20, 1-59, 24-59, 36-59 and 87-96 which included major hydrophilic domains of the IFN gamma molecule. All the rabbits produced antibodies which recognised the polypeptide immunogen, but to date only 1 of 4 rabbits immunised with polypeptide 24-59 and 1 of 3 rabbits immunised with polypeptide 1-59 have produced antibodies which also recognise native HuIFN gamma. The positively reacting antiserum from the rabbit immunised with polypeptide 24-59 could only be shown to weakly bind to HuIFN gamma, whereas the positively reacting antiserum from the rabbit immunised with polypeptide 1-59 was shown to both weakly bind to HuIFN gamma and weakly neutralise its in vitro antiviral effect. The results so far obtained suggest that the amino acid sequences close to the N-terminus are important for biological activity.     

Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1

Monoclonal antibodies directed against glycoprotein D of herpes simplex virus completely inhibited fusion of Vero cells infected with type 1 virus. In contrast, several monoclonal antibodies directed against other viral glycoproteins, including B, were ineffective or were only minimally inhibitory at the highest concentrations tested.