Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells

It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w.     

Proteinase production and pathogenicity of Candida albicans. I. Invasion into chorioallantoic membrane by C. albicans strains of different proteinase activity

In order to investigate a role of proteinase in the pathogenesis of Candida infections, invasion of C. albicans strains of different proteinase activity into the chorioallantoic membrane (CAM) of developing chicks was studied. Eight strains were used after examining the inducible proteinase activity in the culture containing bovine serum albumin as the sole source of nitrogen. Six were proteinase-producing strains (type I) and two were proteinase-deficient ones (type II). Type I strains were subdivided into type Ia strains in which the proteinase activity persisted for a week in the in vitro culture and type Ib ones in which the enzyme activity was lost by the 7th day after inoculation. By inoculation onto CAM, the type I strains could invade the tissue in which secreted proteinase was detected on the periphery of the invading Candida cells by immunohistochemical method. At an early stage of the infection, proteinase secretion was detected on the surface of the yeast cells before their entry into the tissue. The type II strains remained on the surface of the CAM and did not invade the tissue where the secretion of the enzyme was not detected. The mortality rate of the chick embryo was not correlated with the degree of proteinase production of these strains. Two type Ib strains invaded the CAM tissue and elicited some tissue reactions by the host, yielding a low mortality rate of the chick embryos. These results suggested that the secretion of proteinase was an important factor for the invasion of CAM but other factors were also involved for the pathogenicity of C. albicans.     

Infant feeding practices: 1984-85 versus 1977-78.

In 1979 and 1980 the Canadian Paediatric Society''s Nutrition Committee published guidelines for professionals counselling mothers of infants on feeding practices. The practices in 1984-85 of mothers in Toronto were determined for comparison with the practices identified in a similar study conducted in Toronto and Montreal in 1977-78 to ascertain if practices had changed in favour of the recommendations. Between July 1984 and February 1985, 404 metropolitan Toronto mothers of infants were interviewed. Compared with the 1977-78 group of mothers, more of the 1984-85 mothers had chosen to breast-feed and fewer had stopped breast-feeding in the first month. As well, fewer of the 1984-85 infants had been fed unmodified cow''s milk in the first 6 months of life and introduced to solid foods before 4 months of age. We conclude that major changes in infant feeding practices had occurred since 1977-78 and that the 1984-85 practices corresponded closely to the infant feeding guidelines.     

Saturated and trans-unsaturated fatty acids elicit high levels of superoxide generation in intact and cell-free preparations of neutrophils

Saturated and trans-unsaturated fatty acids, such as laurate and elaidate, elicited O2- generation in intact porcine and human neutrophils and also in a cell-free preparation of porcine neutrophils. The activities thus induced were comparable to those induced by cis-unsaturated fatty acids. However, the activation by saturated or trans-unsaturated fatty acids was depressed almost completely in the presence of Ca2+ at around 1 mM, which is usually contained in the media for phagocytes. In contrast, the activation by cis-unsaturated fatty acids such as arachidonate was scarcely affected by Ca2+. These findings appear to demand reevaluation of the effects of long chain fatty acids on the respiratory burst system in phagocytes.     

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Serum free thyroxine and thyroxine-binding globulin concentrations in early phase of subacute thyroiditis

Serum total thyroxine (T4), total triiodothyronine (T3), T4-binding globulin (TBG), free T4(FT4) and free T3(FT3) concentrations and the T3-uptake(T3-U) value were estimated in 11 patients with subacute thyroiditis, and compared with the same parameters in 11 patients with Graves' disease, whose serum T4 concentrations were similar to the former group. Seven patients with subacute thyroiditis, who were treated with dicrofenac sodium alone, were investigated as to the sequential changes in serum parameters during their clinical courses. The mean serum T3-U value and FT4, T3 and FT3 concentrations in patients with subacute thyroiditis were increased, but all were significantly lower than those in patients with Graves' disease (p less than 0.01, p less than 0.001, p less than 0.001 and p less than 0.001, respectively). Three patients with subacute thyroiditis, who showed shorter duration of symptoms than 10 days, had serum TBG excess. Thus the mean (+/- SD) serum TBG concentration (26.5 +/- 8.4 micrograms/ml) was significantly higher than that (18.3 +/- 2.9 micrograms/ml) in patients with Graves' disease (p less than 0.02). The ratios of serum T3 to T4 and FT3 to FT4 in patients with subacute thyroiditis were also significantly lower than those in patients with Graves' disease (p less than 0.001 and p less than 0.001, respectively). The serum FT4 in 7 patients treated with dicrofenac sodium alone decreased to the normal range after 3 to 8 weeks from the onset of the illness. In 3 patients with TBG excess and one patient (TBG; 29.0 micrograms/ml), serum TBG declined in consequence of the serum FT4 normalization.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.