Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.     

Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK _a of the nitrogen atom in the Lys258 ε-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.     

Associations between renal impairment and anemia in older,rural Japanese men: the Nagasaki Island study

Background

Renal impairment is known to be associated with atherosclerosis, which in turn is reported to be positively associated with hemoglobin levels. In addition, renal impairment is known to be associated with a form of anemia known as renal anemia.

Methods

To clarify the associations between renal impairment and anemia, we conducted a cross-sectional study of 1,105 60 to 89-year-old men, who were not taking medication for anemia and were undergoing general health check-ups.

Results

Compared with non-chronic kidney disease, chronic kidney disease (CKD) with a glomerular filtration rate (GFR) <60 mL/min/1.73 m² was found to constitute a significant risk of anemia. However, we noted that this risk was lower for mild renal impairment (60 mL/min/1.73 m² ≤ GFR <90 mL/min/1.73 m²). Compared with the non-CKD reference group, the classical cardiovascular risk factors adjusted odds ratio (OR) for anemia was 1.81 (1.23 to 2.68) and compared with the normal renal function (GFR ≥90 mL/min/1.73 m²) reference group, the ORs for mild renal impairment and CKD were 0.26 (0.15 to 0.47) and 0.60 (0.33 to 1.09).

Conclusions

Independent from classical cardiovascular risk factors, CKD, which was identified during general health check-ups, appeared to constitute a significant risk of anemia for older Japanese men. For mild renal impairment, however, this association was a reduced risk of anemia and thus possibly a higher risk of atherosclerosis.     

Thyrotropin-releasing hormone and insulin in chemically induced pancreatic islet cell tumors in rats

Thyrotropin-releasing hormone (TRH) and insulin were measured by radioimmunoassay in acetic-acid extracts of 19 pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. In addition, gel filtration properties of TRH-immunoreactivity and immunoreactive insulin (IRI) were examined in 5 and 14 tumors, respectively. TRH was demonstrated in 10 of 19 tumors, with a mean of 166 +/- 47 (SEM) pg/mg wet weight, whereas the concentration was less than 3 pg/mg wet weight in the other tumors. In contrast, all tumors contained IRI, with a mean of 11.0 +/- 1.6 micrograms/mg wet weight. Ten tumors in which TRH was demonstrated contained more IRI than those in which TRH was not detected (13.1 +/- 1.8 vs 6.5 +/- 1.7 micrograms/mg wet weight, P less than 0.02). After gel filtration, all TRH immunoreactivity was eluted at the same place as synthetic TRH in the 5 tumors. In addition, gel filtration elutes showed essentially the same pattern of IRI in the 14 tumors, with 3 peaks. The predominant IRI peak comigrated with marker insulin (95.7 +/- 0.8%), another prominent peak occurred coincident with proinsulin standard (3.3 +/- 0.5%), a third peak was present in the void volume (0.28 +/- 0.04%). These distributions of IRI were similar to those in extracts of normal pancreases. The present studies demonstrate TRH immunoreactivity in pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. Chemically induced insulinomas can serve as a model for insulin storage which is analogous to islet B cells.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Relationship between general or specific immunoreactivity and prognosis in postoperative patients with hepatocellular carcinoma

Summary The levels of a variety of immunological parameters were examined in 203 preoperative patients with hepatocellular carcinoma (HCC) at various stages (I–IV). The changes in the peripheral blood lymphocyte (PBL) count, the serum level of immunosuppressive acidic protein and the degree of the skin reaction to purified protein derivative were associated significantly with the stage of HCC progression. However, the percentages of lymphocyte subsets, mitogenic responsiveness of PBL and serum immunoglobulin concentration remained at the levels of stage I. Further study demonstrated that in patients undergoing hepatic artery ligation, there were statistically significant correlations between the PBL count, immunosuppressive acidic protein concentration and intensity of the skin reaction to purified protein derivative, assayed 1 month after surgery, and the prognosis. HCC-specific immunity was examined in 34 patients treated by hepatic resection or hepatic artery ligation using in vitro responses of PBL to HCC extracts (ATS test). This test was performed using culture medium containing added arginine. None of the PBL from the patients showed a positive response to allogeneic HCC extracts, but the PBL from 12 patients (9 hepatic resections, 3 hepatic artery ligations) were stimulated significantly (SI 2.5) with autologous HCC extracts. In 7 of 9 hepatic resection patients who were positive in the ATS test, tumor recurrence was identified. Statistical analysis indicated that the ATS test result was significantly correlated with tumor recurrence in hepatic resection patients. Autologous-PBL-stimulating activities were isolated in a fraction at pH 8.3 and in fractions at pH 6.7–7.0 by chromatofocusing of the crude extract. Although identification of the HCC-specific antigen remains to be done, use of the above fractions may simplify the ATS test procedure and improve its sensitivity.     

Characterization of 1-(2-[18F] fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo- isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole, a PET ligand for imaging the metabotropic glutamate receptor type 1 in rat and monkey brains

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.