Koichiro Murai

Purification and properties of alkaline ribonuclease from human serum

1. Five alkaline ribonucleases (EC 3.1.4.22) were purified about 140- to 1900-fold from human serum by phosphocellulose and DEAE-cellulose chromatographies and Sephadex G-75 filtration, with a total recovery of 22%. These were designated as RNAases 1-5. 2. Optimum activities were observed at pH 8.5-8.7 for RNAases 1-4, and at pH 7.5 for RNAase 5. The molecular weights of these enzymes were estimated by gel filtration as 45 000, 32 000, 20 000, 13 000 and 8500, respectively. 3. These RNAases were found to be heat-labile proteins but are markedly stabilized with bovine plasma albumin. The reaction was activated by Na+, K+, Mg2+ and Ca2+, and inhibited by Co2+, Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of the reaction. Spermine caused 2- to 7-fold activation. 4. Among the substrates examined, these RNAases preferentially hydrolyzed pyrimidine bodies and except for RNAase 5 had a higher affinity for poly(C) than poly(U) as substrate. Each enzyme was free from other nucleolytic enzymes and hydrolyzed only RNA.

Thyroid function in Yusho patients exposed to polychlorinated biphenyls (PCB)

Thyroid function was investigated in 123 yusho patients who were exposed to toxic levels of polychlorinated biphenyls (PCBs) 16 years ago. In yusho patients, compared with the patients without evidence of yusho or normal controls, the serum triiodothyronine (T3) and thyroxine (T4) levels were significantly higher, while thyroid stimulating hormone (TSH) levels measured by sensitive assay were normal. There was no difference in serum levels of albumin, alkaline phosphatase, total cholesterol, and thyroxine binding globulin (TBG) between the two groups and the prevalence of positive antithyroid autoantibodies was almost the same, suggesting that hyperthyroxinemia in yusho patients was not due to increased TBG binding or abnormal autoimmune mechanism. Serum free T4 levels, however, were not elevated, although T4/TBG ratio was significantly higher. The thyroid hormone levels were higher than normal value in 4 of 123 yusho patients but only 1 case had clinical symptoms such as excessive perspiration. Despite higher serum PCBs in yusho patients, there was no correlation between PCB levels and levels of T3, T4, or TSH. The present results suggest hyperthyroxinemia without obvious clinical symptoms in yusho patients long after exposure to PCBs.

Familial primary biliary cirrhosis associated with impaired concanavalin A-induced lymphocyte transformation in relatives

The familial occurrences of biochemical and immunological abnormalities and histocompatibility antigens were studied in 18 healthy first-degree relatives of patients with primary biliary cirrhosis (PBC) in two families. In each of these two families, there were two members who suffered from PBC. All relatives had normal serum aspartate aminotransferase, alkaline phosphatase, bilirubin, total cholesterol, and immunoglobulins except the two, who had a mild elevation of alkaline phosphatase without cholestasis. Autoantibodies were present in some relatives; five (28%) for antithyroglobulin antibody and antithyroid microsomal antibody, one (6%) for antimitochondrial and antinuclear antibody, and one (6%) for rheumatoid factor. Abnormalities of T or B lymphocytes in peripheral blood were detected in two (11%) relatives. Impairment of concanavalin A-induced lymphocyte transformation determined by ethidium bromide fluoroassay was found in seven (39%) relatives, although an abnormal response for phytohemagglutinin was detected in none of the relatives. The HLA haplotypes were not necessarily associated with positive autoantibodies or impaired concanavalin A-induced lymphocyte transformation in these families. These findings suggest that impairment of concanavalin A-inducible lymphocytes (mainly suppressor T cells) is one of the contributing factors in the development of PBC.

Correction of serious iron overload in a chronic hemodialysis patient by recombinant human erythropoietin and removal of red blood cells: confirmation by follow-up liver biopsy.

A chronic hemodialysis case, a 46-year-old woman with secondary hemosiderosis induced by parenteral iron and blood transfusion due to a refractory anemia, was effectively treated with recombinant human erythropoietin and the removal of red blood cells. The cumulative dose of the iron removed was 5,712 mg. Plasma ferritin decreased from 8,290 to 2,203 micrograms/l during 18 months. Concomitantly, liver histology performed before and after the therapy revealed a prominent regression of the deposited iron.

Effects of recombinant leukocyte interferon on serum immunoglobulin concentrations and lymphocyte subpopulations in chronic hepatitis B

To investigate immune effects of interferon (IFN) therapy in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, serum immunoglobulin concentrations and peripheral lymphocyte subpopulations were sequentially studied before, during, and after therapy in nine patients who were treated with recombinant human α-IFN in doses ranging from 3 to 10 million units per day for 28 days. Serum immunoglobulin A levels decreased significantly, from 414±23 mg/dl (mean ± SE) to 379±28 mg/dl (P<0.05), after the first week of therapy and to a bottom value of 323±20 mg/dl (P<0.001) at the fourth week. Immunoglobulin G levels decreased significantly, from 2603±175 to 2328±169 mg/dl (P<0.005), after the first week of therapy and to a bottom value of 2005±199 mg/dl (P<0.001) at the fourth week. Immunoglobulin M levels were also reduced significantly after 3 weeks of therapy (from 229±23 to 188±15 mg/dl;P<0.01). These reductions in immunoglobulins A, G, and M returned to pretreatment levels by 4 months after the end of the therapy. In lymphocyte subpopulations, significant depressions were found in CD3-, CD4-, CD8-, and B1-positive cells in peripheral blood after the first week of therapy (CD3, from 1700±114 to 1234±114/mm3,P<0.005; CD4, from 1036±88 to 780±64/mm3,P<0.005; CD8, from 620±57 to 426±60/mm3,P<0.05; and B1, from 519±84 to 276±48/mm3,P<0.01) followed during therapy, while Leul la-positive cells did not change significantly. During the 6-month follow-up period, three patients had a sustained clinical remission in which HBeAg disappeared from serum. Disappearance of HBeAg was unassociated with initial levels or percentage changes of serum immunoglobulins and peripheral lymphocytes expressing each of the test markers in these patients. These findings suggest that immune effects of IFN therapy are independent from its antiviral effects.

Purification and properties of urinary alkaline ribonucleases from patients with nephrotic syndrome

Four urinary alkaline ribonucleases (RNase, EC 3.1.4.22) were purified from patients with nephrotic syndrome using phosphocellulose, DEAE-cellulose and Sephadex G-75 chromatographiy. These enzymes were designated as RNases 1--4, respectively, in order of elution on phosphocellulose chromatography. The respective purification of each fraction was 41-, 23-, 34- and 27-fold with a total recovery of 25%. The pH optima of these RNases were around 8.5 with Tris/HCl buffer and the reaction was activated by mono- and divalent cations, such as Na+, K+, Mg2+ and Ca2+, but inhibited by Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of reaction. The molecular weights of RNases 1--4 were estimated by gel filtration as 45 000, 32 000, 20 000, and 13 000, respectively. Each enzyme hydrolyzed pyrimidine nucleotides preferentially with higher affinity for poly(C) than poly (U) as determined with synthetic polymers and was free from other nucleolytic enzymes. The patients with renal disorders excreted one to four RNases in urine and the number of enzymes increased as the concentration of urinary protein increased. On the other hand, normal subjects excreted a single fraction essentially identical to RNase 1.

Low activity of gamma-glutamyl transpeptidase in serum of acute intrahepatic cholestasis

Low gamma-glutamyl transpeptidase (gamma-GTP) activity in serum was observed in 11 patients with acute intrahepatic cholestasis (cholestatic hepatitis and fulminant hepatitis), despite a marked increase in bilirubin levels. Inhibitors of gamma-GTP were not detected in sera of these patients. Their gamma-GTP levels in the liver were significantly higher than those in chronic liver diseases. An electrophoretic study of liver gamma-GTP in acute intrahepatic cholestasis showed the same mobility as in chronic liver diseases. These results suggest that the low serum gamma-GTP activity in acute intrahepatic cholestasis is due to factors inhibiting the release of the enzyme from the liver.

Disseminated Intravascular Coagulation in a Patient with Progressive Systemic Sclerosis Associated with Necrotizing Angiitis and Generalized Lymphadenopathy

A 47-year-old woman with progressive systemic sclerosis developed disseminated intravascular coagulation in the course of her terminal illness. She also had complicating necrotizing angiitis and generalized lymphadenopathy. The likely relationship between disseminated intravascular coagulation and vasculitis is discussed.