Demonstration of structural polymorphism among MB3 light chains by two-dimensional gel electrophoresis

The heavy and light chain subunits of MB3 molecules were isolated from KT2 (DKT2, DR4, MB3 homozygous), ER (Dw4, DR4, MB3 homozygous), JMe (Dw5, DR5, MB3 homozygous), EBV-Sh (DSh, DRw6.2, MB3 homozygous), and EBV-Ky (DKy, DRw9, MB3 homozygous) cells and were compared with one another by two-dimensional gel electrophoresis. The MB3 light chains from KT2, ER, and EBV-Ky cells were clearly different in terms of their isoelectric points, whereas those from ER, JMe, and EBV-Sh cells were indistinguishable. No differences in charge or m.w. were noted for the MB3 heavy chains from the five cell lines. Thus, three out of the five MB3-positive, D/DR-disparate cell lines were found to express structurally distinct MB3 molecules, demonstrating that MB3 is a public serologic specificity shared by at least three structurally distinct MB (human I-A-like) molecules. Because the DR light chain subunits isolated from EBV-Wa, KT2, ER, JMe, EBV-Sh, and EBV-Ky cells differed from one another in their isoelectric points, the DR light chains were apparently more polymorphic than the MB3 light chains.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.     

Relative effectiveness and interaction of ultraviolet-B, red and blue light in anthocyanin synthesis of apple fruit

The effect of light on anthocyanin production in apple ( Malus pumila Mill. cv. Jonathan) skin disks was investigated, with prolonged irradiation from different light sources. High fluence rates of white light provided from a xenon lamp were unable to produce large amounts of anthocyanin, and anthocyanin production became saturated at about 30 W m⁻². When UV-B light, provided by a fluorescent lamp which had an emission peak at 312 nm, was combined with the white light, anthocyanin production was synergistically stimulated and increased up to the highest fluence rates of white light tested (44 W m⁻²). This UV-B light was more effective than red and blue light provided from fluorescent lamps, but anthocyanin production became saturated at about 1.7 W m⁻². However, simultaneous irradiation with red and UV-B light had a synergistic effect. UV-B light was also effective in increasing anthocyanin production in whole fruit. Therefore this synergism seemed to have an important role in the development of the desirable red skin color under field light conditions. The results of aminoethoxyvinylglycine treatment suggested that ethylene was not involved in the stimulative effect of UV-B light.     

Structures of sugar chains of the third component of human complement

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.     

Neighboring proteins in rat liver 60 S ribosomal subunits disulfide-linked by hydrogen peroxide oxidation or cross-linked with dithiobis(succinimidyl propionate)

Neighboring proteins in rat liver 60 S ribosomal subunits were investigated by two kinds of cross-linking techniques: treatment of 60 S subunits with 1) hydrogen peroxide, which promotes the formation of protein-protein disulfide linkages and 2) a disulfide-bridged bifunctional reagent dithiobis(succinimidyl propionate). The cross-linked protein complexes formed were separated by two-dimensional polyacrylamide gel electrophoresis in a basic-sodium dodecyl sulfate gel system under nonreducing conditions. Each complex in the gel was labeled with 125I and extracted under reducing conditions. The protein components of the complex were analyzed by two kinds of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography. Closely neighboring pairs disulfide-linked by hydrogen peroxide were identified as L4-L6, L4-L29, L6-L29, L18a-L29, and L29-L32; more distant pairs cross-linked with dithiobis(succinimidyl propionate) were identified as L3-L5, L3-L24, L3-L37a, L4-L14, L4-L18a, L5-L10, L5-L11, L7/L7a-L27, L7/L7a-L36, L13-L35, and L13a-L14.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

A novel cell-surface antigen expressed on most leukocytes and a minor cortisone-resistant population of thymocytes in rats: characterization by monoclonal antibodies

A cell-surface antigen on rat lympho-hemopoietic cells was determined by using a monoclonal antibody, R2-1B3 (1B3). The 1B3 antibody, when tested for its reactivity with different hemopoietic cells by cytofluorography with a FACS analyzer, labeled more than 80% of lymph node, spleen, and bone marrow cells and 10-20% of thymus cells. Cytofluorographic analysis performed on purified rat T cells, B cells, macrophages, and granulocytes demonstrated that the antigen defined by 1B3 was readily detectable on all of these cell types, with the greatest expression on B cells. A minor population of thymocytes that were labeled by 1B3 appeared to be cortisone-resistant and were located mainly in the thymic medulla. These 1B3 positive thymic cells seemed to be functionally more mature than 1B3-negative thymus cells as suggested by the fact that the cytotoxic treatment of thymus cells with 1B3 antibody and complement (C) resulted in significant reduction of their responsiveness to phytomitogens and lymphokines derived from concanavalin A (con A) activated rat spleen cell cultures. Immunochemical data showed that 1B3 antibody recognized the broad ill-defined band with a molecular weight of 32K to 47K daltons as estimated by SDS-polyacrylamide gel electrophoresis. These data indicate that the 1B3 defined antigen is distinct from other, previously reported, antigens on rat lymphoid cells including leukocyte-common (L-C) and MRC OX-22 antigens, and that this 1B3 antibody is a useful reagent for analyzing the intrathymic differentiation of T cells in rats.     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.     

Chemical modification of lipase with ferromagnetic modifier — A ferromagnetic-modified lipase

Summary A ferromagnetic modifier was prepared by reacting ferrous(Fe²⁺)- and ferric(Fe³⁺)-ions with polyethylene glycol having two carboxyl groups (MW:2000) at pH 8.0–8.5. Lipase fromPseudomonas fragi 22–39B was coupled with the modifier using water-soluble carbodiimide. The modified lipase, which was dispersed into buffered solutions in the size range of 30–70 nm, exerted the hydrolytic activity of 8.0 U/mg. In a magnetic field of 250 Oe, the ferromagnetic-modified lipase was readily recovered from the colloidal solution.