Kunihiko Kobayashi

Functional casein-polysaccharide conjugates prepared by controlled dry heating

Casein was conjugated with dextran and galactomannan in a controlled dry state at a relative humidity of 79% and at 60°C for 24 hr. The covalent attachment of polysaccharides to casein was confirmed by SDS-PAGE and HPLC. The emulsifying activity of the casein-dextran and casein-galactomannan conjugates was 1.5 times higher than that of casein. The emulsion stability of the casein-dextran and casein-galactomannan conjugates was 10 times higher than that of casein. The improvement in these emulsifying properties reached a steady state when the conjugation of casein with polysaccharide was done for 24 hr in a controlled dry state, suggesting the rapid formation of conjugates through a Maillard reaction in the case of casein. Compared to commercial emulsifiers, the casein-polysaccharide conjugates showed better emulsifying properties in acidic and high-salt concentration systems.

Jacalin, a jackfruit lectin, precipitates IgA1 but not IgA2 subclass on gel diffusion reaction

Precipitation reaction in agarose gel between jacalin, a lectin from jackfruit seeds, and purified immunoglobulins of various classes and their components was studied. Single precipitation arcs were observed with monomeric and dimeric IgA1 subclass as well as secretory IgA1, but not with IgA2 of both allotypes, IgG, IgM, IgD, IgE, free secretory component or with the J chain. Thus, jacalin can be used for differentiating IgA1 from IgA2 subclass by a simple agarose gel diffusion.

Jacalin: isolation, characterization, and influence of various factors on its interaction with human IgA1, as assessed by precipitation and latex agglutination.

An IgA1-specific lectin, Jacalin, was isolated from dried seeds of the jackfruit, Artocarpus integrifolia, by affinity binding to IgA1-Sepharose and elution with D-galactose. Jacalin is a glycoprotein with two non-covalently bound subunits (15 and 18 K). Interactions between Jacalin and human Igs were studied by precipitation in gel and in solution, and by agglutination of IgA1-coated latex by Jacalin. Jacalin precipitated only with IgA1-containing samples, including monomers, polymers, monoclonal, polyclonal and secretory IgA1, but not IgA2 of both A2m(1) and A2m(2) allotypes, nor with IgG1, 2, 3 and 4, IgM, IgD, and IgE; after neuraminidase treatment, only IgA1 and IgD were precipitated. Jacalin had a relatively broad pH range of activity in both precipitation and agglutination of IgA1-latex. Bivalent metal cations (Ca, Mg, Mn, Cu, Zn, Co, Cd), EDTA, Triton X-100, Tween-20, Na deoxycholate and ionic strength did not influence these reactions. Na dodecylsulphate, guanidine and urea inhibited the reactions whereas NP-40 rather enhanced them. Among 39 types of sugar tested, 10 displayed inhibitory activity, decreasing in the following order: p-nitrophenyl-alpha-D-galactopyranoside, 1-O-methyl-alpha-D-galactopyranoside, D-melibiose, p-nitrophenyl-beta-D-galactopyranoside, GalNAc, stachyose, 1-O-methyl-alpha-D-mannopyranoside, D-galactose, D-galactosamine and 1-O-methyl-alpha-D-glucopyranoside. IgA1, treated with neuraminidase or not, but not the other human Igs, was also an excellent inhibitor of agglutination, being more powerful than the best sugars studied. Only neuraminidase-treated IgD was also inhibitory, but less so than IgA1. Jacalin preferentially bound to alpha-linked non-reducing D-galactose. The configuration of OH-groups at C-2, C-4 and C-6 of D-galactose was important for the reaction. Jacalin recognizes terminal Gal beta 1-3GalNac-, as in the IgA1-hinge, and/or GalNAc-, but not Gal beta 1-4GlcNAc-, nor Gal beta 1-6GlcNAc-, nor their sialylayted extensions. Latex agglutination and its inhibition assay are particularly well suited for the study of these lectin-glycoprotein interactions.

Mesangial deposition of J chain-linked polymeric IgA in IgA nephropathy.

Renal biopsy specimens from 8 patients with IgA nephropathy (Bergers disease) were examined immunocytochemically at the light and electron microscopic levels with peroxidase-labeled antibodies to IgA, IgM, J chain and secretory component. In 2 of the 8 specimens heavy deposits of IgA, but no IgM, were found in the mesangium. After acid-urea treatment of these tissue sections, J chain, a subunit of polymeric immunoglobulins, was identified in a distribution similar to that of IgA. In the remaining 6 specimens, small amounts of IgM in addition to denser deposits of IgA and J chains were found. We conclude that the IgA deposits in at least some patients with Bergers disease consist of IgA polymers linked by J chain.

Two populations of immunoglobulin-forming cells in the skate, Raja kenojei: Their distribution and characterization

We recently reported the presence in the skate, Raja kenojei, of two immunoglobulins, a high molecular weight immunoglobulin analogous to mammalian IgM, and a low molecular weight immunoglobulin belonging to a hitherto undescribed class. In view of these findings, we studied lymphoid organs of the skate using morphological and immunocytochemical means. The spleen and the intestinal mucosa had numerous plasma cells and lymphocytes, while the Leydig organ of the esophagus, the epigonal organ and the liver contained these cells in a much lesser frequency. Immunocytochemical studies proved these plasma cells and some of the lymphocytes to be immunoglobulin-forming cells. Double immunofluorescence staining of the spleen and other lymphoid tissues demonstrated the occurrence at a 1: 1 ratio of two distinct populations of cells, one forming the high molecular weight immunoglobulin and the other producing the low molecular weight immunoglobulin.

An epidemic of a pertussis-like illness caused by Chlamydia pneumoniae.

BACKGROUND Between June and July, 1994, we encountered an epidemic of a pertussis-like illness in adolescents in a junior high school located in a rural area of Japan. The purposes of this study were to record the clinical manifestations and to identify an etiology. PATIENTS AND METHODS We interviewed patients and parents and we performed physical examinations on patients with cough during the epidemic. The chest radiographs were also reviewed by us. To identify an etiology we performed culture and serologic studies for a variety of bacteria, Mycoplasma, chlamydiae and viruses. Polymerase chain reaction (PCR) for Chlamydia pneumoniae was carried out on throat swab specimens. RESULTS Of a total of 230 students 136 (59%) had severe cough illnesses. One developed pneumonia, 9 had bronchitis and the remaining 126 (93%) presented upper respiratory tract infections (URI). The mean duration of cough in cases with URI was 17.4 days and that in cases with bronchitis and pneumonia was 30.4 days. Serology and/or cultures for Bordetella pertussis, Bordetella parapertussis, Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia psittaci or viruses were negative. Detection of C. pneumoniae infection was carried out in 46 patients with pneumonia, bronchitis or URI by serology and PCR. The patient with pneumonia, 7 of 7 patients with bronchitis and 32 (84%) of 38 patients with URI were documented to be infected by C. pneumoniae either by serology, PCR or both tests. CONCLUSION An epidemic of a pertussis-like illness in a junior high school population was caused by C. pneumoniae.

A case of acute cerebellar ataxia with an MRI abnormality

A 5-year-old boy with acute cerebellar ataxia was examined by means of magnetic resonance imaging (MRI) and was found to have a lesion showing low and high signal intensity in T1- and T2-weighted images, respectively, in the left cerebellar peduncle in the acute phase. The lesion disappeared in the convalescent phase.

Clostridium ramosum, an IgA Protease-Producing Species and Its Ecology in the Human Intestinal Tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (proprion‐ate‐rifampicin‐gentamicin‐colimycin‐polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease‐positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease‐positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.

Complexes of Albumin and Alpha-1-antitrypsin With Fc-fragment of Iga Monomer Are Disulfide-bound To Penultimate C-terminal Cysteine in the C-alpha-3-domain

Six IgA myeloma sera, containing albumin (ALB)-IgA and alpha 1-antitrypsin (alpha 1-AT)-IgA complexes (CXs), were gel-filtered on Ultrogel AcA22. The CXs were eluted between monomeric and dimeric IgA. The CX-containing fractions were digested with three IgA-proteases: ALB and alpha 1-AT remained bound to Fc alpha-, but not to Fab alpha-fragments. Short (less than 2 h) peptic digestion entirely released free ALB and alpha 1-AT from the CXs, indicating that these proteins are linked to the third constant domain of the alpha-chain. Identical results were obtained with anti-ALB immunosorbent-purified ALB-IgA CXs. Reduction-alkylation of ALB or alpha 1-AT bound to IgA or Fc alpha confirmed disulfide binding. The data support the binding of ALB and alpha 1-AT to the same penultimate C-terminal cysteine of the alpha-chain as that which binds the J-chain.

Isolation and characterization of immunoglobulin of hagfish, Eptatretus burgeri, a primitive vertebrate

The immunoglobulin of the hagfish, Eptatretus burgeri, one of the most primitive vertebrates extant, was isolated from the serum of non-immune normal adult hagfish in a pure form. Analysis of the immunoglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition indicated that the immunoglobulin was composed of heavy (H) and light (L) chains. The mol. wt of the H-chain was 68,000, slightly smaller than that of the human mu-chain. The L-chain of the immunoglobulin appeared as 2 bands on SDS-PAGE, with mol. wts of 25,000 and 22,000. These findings were confirmed by gel filtration of reduced-alkylated immunoglobulin in 5 M guanidine-HCl. The H:L molar ratio of the immunoglobulin was roughly 1:1. Gel filtration of the immunoglobulin in non-dissociating buffer indicated that the mol. wt of the intact immunoglobulin was 150,000-160,000. Thus, the subunit chain composition of the immunoglobulin was assumed to be H2L2, identical with the fundamental structure of immunoglobulins. The instability of the hagfish immunoglobulin was ascertained by the fact that it dissociated into heterogeneous mol. wt components ranging from approx. 90,000 to 160,000 upon SDS-PAGE under non-reducing conditions. However, almost no free or monomeric H- or L-chains were dissociated from the immunoglobulin by this procedure and also by gel filtration in 5 M guanidine-HCl. Theses results indicated that the hagfish immunoglobulin is unusually labile in its tertiary structure but has disulfide binding between at least more than 2 subunit chains.