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Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

<P>Reed-Sternberg cells (RS cells) of Hodgkin's disease (HD) are frequently infected with Epstein-Barr virus (EBV) and express EBV-encoded nonpolyadenylated RNA transcripts (EBER)-1. EBV latency has been classified into three distinct forms: Latency I, expressing only one of the latent proteins, EBV nuclear antigen (EBNA)-1, latency II, coexpressing EBNA-1 and LMPs, and latency III, expressing all latent viral proteins. RS cells express LMP-1 in addition to EBNA-1 and are considered to be EBV latency II frequently encountered in nasopharyngeal carcinoma. We examined 13 cases of EBV-infected HD by combined EBER-1 in situ hybridization and immunostaining for LMP-1. All of the RS cells expressed EBER-1, but a substantial number of EBER-1+ RS, cells were negative for LMP-1. The percentage of LMP-1+ RS cells out of EBER-1+ RS cells varied from 7% to 100% (average 69%). In this study, we showed that all EBV-infected RS cells were not restricted to latency II, and some belonged to latency I.</P>

To elucidate the latent state and reactivation of Epstein-Barr virus (EBV) in non-neoplastic lymphoid lesions, we investigated 144 non-neoplastic lymphoid lesions by in situ hybridization (ISH) to detect the expression of EBV-encoded small RNAs (EBER)-1 and BCRF-1 and by immunostaining for latent membrane protein (LMP)-1 and ZEBRA. ISH for EBER-1 detected EBER-1-positive cells (EPC) in 31 of the 144 examined lesions (22%). EPC were detected in 4 of 49 cases of nonspecific lymphoid hyperplasia, in 16 of 20 abscess-forming granulomatous lymphadenitis (AFGL), 5 of 25 Kikuchi's disease, and in 3 of 3 infectious mononucleosis. LMP-1 was expressed in 6 of 124 non-neoplastic lymphoid lesions (4.8%). LMP-1-positive cells were observed in 6 of the 31 EBER-1-positive cases (19%). EPC were detected significantly more frequently in LMP-1- and ZEBRA-positive specimens than in the LMP-1- and ZEBRA-negative specimens. BCRF-1 was expressed in 4 of 11 cases examined: 2 of 3 AFGL, 1 of 2 Kikuchi's disease, and in the 1 case of atypical lymphoid hyperplasia. This study suggests that Epstein-Barr virus is prevalent and can be reactivated in the lymph nodes effaced by destructive inflammation, such as AFGL. Such inflammation may provide a local milieu that is conducive for EBV to enter the lytic cycle.

We studied the brains of two cases of amyotrophic lateral sclerosis with dementia. Bunina bodies were found in the motor neurons of cranial nerve nuclei (trigeminal, facial and hypoglossal nerves) as well as in the spinal motoneurons. They appeared mostly in the cytoplasm and occasionally in the neuronal processes. However, the present electron microscopic study disclosed clearly that Bunina bodies were present not only in the cell body but also in the dendrites. No Bunina bodies were observed in the axons. It is inferred that the Bunina bodies were degenerative products formed as a result of a protein metabolism disorder.

Progenitor cells in the bone marrow and the spleen of mice, whether infected with Schistosoma japonicum or not, formed cell clusters and colonies when incubated with culture supernatant fluid of spleen cells incubated with soluble egg antigen (SEA). The egg extract, up to a concentration of 250 micrograms/ml protein, did not directly stimulate progenitor cell proliferation in the bone marrow. Eosinophilia in mice infected with S. japonicum may be mediated indirectly by egg antigen-stimulated immune lymphocytes and not directly by the egg antigen.

The influence of physical exercise on the urinary excretion of proteins was examined in 17 male high school baseball players. Their urine was collected before and after exercise to determine the concentrations of total protein, albumin, beta 2-microglobulin and creatinine along with the activity of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30). Concentrations of total protein, albumin, beta 2-microglobulin and creatinine increased significantly (p less than 0.01) after exercise, while N-acetyl-beta-D-glucosaminidase activity did not increase. Similar results were obtained when the concentrations of these urinary components were calculated on the basis of a urinary density of 1.024, and when they were expressed relative to the amount of creatinine. Positive correlations were seen among total protein, albumin, beta 2-microglobulin and creatinine concentrations, but not between the beta 2-microglobulin concentration and N-acetyl-beta-D-glucosaminidase activity. Isoenzyme activities of N-acetyl-beta-D-glucosaminidase in the urine were determined by electrophoresis on cellulose acetate plates. After exercise, the A-form increased slightly, and the B-form decreased slightly, but these changes were not statistically significant.

Formation of sulfate in rat liver mitochondria was studied. About 0.1 mumol of sulfate was formed in mitochondria from 1 g of liver in 60 min when 10 mM L-cysteine was used as the substrate. Addition of either 10 mM 2-oxoglutarate or 10 mM glutathione to this system increased sulfate formation 3 to 4 times. The addition of both 2-oxoglutarate and glutathione resulted in a 20-fold increase in sulfate formation. Sulfate formation in the presence of 5 mM L-cysteine was 58% of that with 10 mM L-cysteine. L-Cysteine-glutathione mixed disulfide was not a good substrate, indicating that this mixed disulfide was not an intermediate of sulfate formation in the present system. Incubation of 3-mercaptopyruvate with rat liver mitochondria also resulted in sulfate formation, and the addition of glutathione accelerated it. Formation of sulfite and thiosulfate was also detected. These results indicate that sulfate is produced in mitochondria, at least in part, from L-cysteine through the transamination pathway (3-mercaptopyruvate pathway).

Twenty-four patients with rheumatoid arthritis (RA) and 20 normal controls were examined for the ability of their peripheral blood B cells to produce interleukin 1 (IL-1) with or without lipopolysaccharide (LPS). B cells were purified from peripheral blood by negative selection methods (i.e., removal of adherent cells and sheep red blood cell rosette-forming cells, followed by treatment with monoclonal antibodies (OKT3 and OKM1) and complement). The amount of IL-1 in B cell culture supernatants (SN) was measured by thymocyte and fibroblast proliferation assays and an enzyme-linked immunosorbent assay for IL-1 alpha and beta. As a group, cultured B cells from patients with RA, both spontaneously and when stimulated with LPS, produced higher levels of IL-1 than those from normal controls. IL-1 production by RA B cells with LPS had a weak but positive correlation with disease activity. Moreover, RA B cell culture SN with elevated levels of IL-1 had a synergistic effect on the growth of anti-human IgM (anti-mu) stimulated B cells. In separate experiments, the growth of RA B cells was significantly promoted by IL-1 beta both with and without anti-mu stimulation. These results suggest that B cell-derived IL-1 may be involved in the B cell clonal expansion of RA through its own activity as a B cell stimulatory factor.

Etoposide (VP-16), one of the topoisomerase II (TopoII) inhibitors, interferes with TopoII by inducing the formation of and stabilizing a cleavable enzyme-DNA complex. VP-16 has been demonstrated to induce apoptosis in murine thymocytes. To clarify the mechanism of action of VP-16, we examined the in vitro effect of a non-cleavable-complex-forming type TopoII inhibitor, ICRF-193 which inhibits the DNA strand breakage induced by VP-16, on murine thymocytes in which apoptosis had been induced with VP-16. DNA fragmentation is characteristic of apoptosis. In the early stages, ICRF-193 decreased DNA fragmentation induced by VP-16, although this inhibitory effect decreased in the later. These data suggest that TopoII inhibitors induce apoptosis in murine thymocytes in two ways: with DNA-strand breaks in the early stage or without them. ICRF-193 itself induced apoptosis in murine thymocytes. The time course of DNA fragmentation caused by ICRF-193 was different from that of VP-16.

It has previously been confirmed that the guinea pig has aggregations of 10-20 lymphoid follicles at the junction of the nasal cavity and the nasopharyngeal duct. The vascular architecture of this nasal-associated lymphoid tissue (NALT) was studied by the corrosion cast/scanning electron microscope method. The NALT was supplied by branches of the inferior nasal artery. These afferent arterial branches gave off arterioles to the follicles and the interfollicular regions, where the arterioles ramified into capillaries. Some of these arterioles reached the subepithelial region to form a single-layer dense capillary network. The subepithelial capillaries gathered into short collecting venules, which in turn drained into high endothelial venules (HEV) in the interfollicular region. The HEV, which also receives tributaries from the follicular and interfollicular capillary plexuses, descended in the interfollicular regions and finally flowed into the efferent veins at the bottom of the NALT. Indentations impressed by high endothelial cells (HEC) were prominent on the surface of the HEV casts, and their frequency was larger in the upper course or segments than in the lower. This suggests that the incidence of HEC in the upper segments is higher than in the lower segments, and these findings are consistent with the hypothesis that some substances which are taken up into the subepithelial capillaries and transported to the venules induce differentiation and maintain of HEVs.

A progesterone receptor (PR) in human uterine cervical nuclei was demonstrated by a nuclear exchange assay using a synthetic progestin, promegestone (R5020) as a radio-labeled ligand. Total exchange of previously bound progesterone with R5020 was achieved by incubation at 0 degree C for 3 h. A 0.6 M KCl solution was used to extract the nuclear PR in uterine cervical tissue, and the dextran coated charcoal (DCC) method was used to separate the free [3H] R5020 from the bound form. Scatchard plots of nuclear PR binding showed two components with dissociation constants of Kd = 2.3 X 10(-10) and 4.6 X 10(-9) M. Three histological regions of the uterine cervix was studied as to their nuclear PR contents throughout the menstrual cycle. In the follicular phase, the connective tissue (CT) had the highest PR concentration (658.9 fmole/mg DNA), followed by the columnar epithelium (CE) (253.6 fmole/mg DNA), and the squamous epithelium (SE) (184.7 fmole/mg DNA). In the luteal phase, there was no significant difference among the three regions. Comparing these phases of cycle revealed that the CT had higher PR contents in the follicular phase than in the luteal phase, but no such difference was found in the CE or SE. These three regions had the same Kd value in both phases.

Cepharanthine, a biscoclaurine alkaloids which interact with biomembranes, has been found to inhibit platelet aggregation. The effects of this drug on morphological and physiochemical phenomena following collagen-induced platelet stimulation were investigated. In the presence of cepharanthine, stimulated platelets became spherical, but did not form pseudopoda , nor did they become aggregated. Physiochemical reactions such as accelerated oxygen consumption, release of membrane-bound Ca2+, release of Ca2+ into the extracellular medium and deporalization of the membrane potential were all inhibited by cepharanthine. Using D,L-dipalmitoyl phosphatidylcholine liposomes as the substrate, cepharanthine was shown to inhibit phospholipase A2 activity. These results suggest that the changes in the membrane following the interaction of collagen with its receptor are important for platelet activation. Cepharanthine may inhibits these membrane state changes, thus blocking all subsequent reactions.

<p>A combination of agarose gel electrophoresis and a newly developed technique of electro-affinity transfer was applied to the detection of circulating immune complexes of human alpha-fetoprotein (AFP) and anti-AFP. After electrophoretic transfer to nitrocellulose membrane, to which affinity-purified polyclonal horse antibodies to human AFP were bound, the membranes were treated with or without rabbit immunoglobulins to human AFP, followed by overlaying with horseradish peroxidase-labeled goat anti-rabbit IgG for color development. Artificial complexes formed in vitro from human AFP and rabbit anti-AFP were clearly separated from free AFP by the agarose electrophoresis. The complexes were stained 20-40% as dark as the equivalent amount of free AFP by treatment with rabbit anti-AFP, and 10-20% as dark without the antibody treatment over a wide range of antigen-antibody ratios.</p>

Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3-mercaptopyruvate and certain amino acids was catalyzed actively by both liver and kidney homogenates in the presence of L-glutamate. However, more half-cysteine was formed by liver than kidney, and more HCETC was produced by kidney than liver. L-Glutamate was the most potent amino donor, and L-aspartate strongly inhibited the reaction. Results indicate that L-cysteine can be transaminated both in liver and kidney of the guinea pig, and that kidney is more active than liver. 2-Oxoglutarate is the most active 2-oxo acid for cysteine transamination. Oxaloacetate (and aspartate in the reverse reaction) is inhibitory to the reaction. These results are in agreement with the previous conclusion that cysteine aminotransferase is identical with aspartate aminotransferase.

1. Absorption maxima of hydrochloric biliverdins derived from the natural indirect bilirubin existed at 680 mμ and 375 mμ, but the maxima of biliverdins purified on the column of silica gel existed at 640 mμ and 390 mμ. 2. The natural salt-form bilirubin was oxidized by hydrochloric acid to biliverdin, of which absorption maxima existed at 685 mμ and 370 mμ in a methanolic solution as well as in 5% hydrochloric methanol, but the purified biliverdin in chloroform solution showed the maxima at 640 mμ and 390 mμ. 3. The natural ester-form bilirubin could be transformed into biliverdin by oxidation of its alcoholic solution in the presence of hydrochloric acid. The crude biliverdin had absorption maxima at 645 to 655 mμ, 600 mμ and 320 mμ, and the crude hydrochloric biliverdin had the maxima at 665 to 675 mμ, 620 mμ and in the near ultra-violet range, while the purified biliverdin in chloroform solution had the maxima at 640 mμ and 380 mμ. 4. The biliverdins derived from the indirect, salt-form and ester-form bilirubin had quite similar absorption maxima after purifications by adsorption chromatography.

Separation of both forms of the direct bilirubin were carried out from the dog's gallbladder bile, and further isolations of them were also done. 1. The natural salt-form bilirubin was isolated after separation on the column of aluminium oxide with a n-propanolic aqueous solution. 2. The natural salt-form bilirubin was obtained in amorphous yellow powders which were strongly hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of these powders showed both the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger reaction. The salt-form bilirubin was transferred into chloroform only when some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it. 3. The absorption maxima of the natural salt-form bilirubin existed at 420 to 430 mμ in a methanolic solution and at 425 or 435mμ in 50% or 10% n-propanol. 4. The natural ester-form bilirubin was isolated after separating on the column of silica gel with a chloroformethanolic mixture. 5. The natural ester-form bilirubin was obtained in amorphous greenish yellow powders. It was further hygroscopic and easily soluble in water and methanol but not in chloroform or carbon tetrachloride. An aqueous solution of it showed the direct diazo and Gmelin reaction, but neither Ehrlich's aldehyde nor Schlesinger's reaction. No pigment was transferred into chloroform even if some quantities of hydrochloric acid were added to a mixture of chloroform and an aqueous solution of it, but did by saponification with 5% methanolic potash. 6. The absorption maxima of the natural ester-form bilirubin existed at 415 mμ in both methanolic and aqueous solutions.

Descriptions are carried on the method how to separate the indirect bilirubin from the chloroform extracts of the dried dog's gallbeadder bile by adsorption chromatography. 1. The optimal concentrations of the bilirubin content were 2 to 4 mg/100 ml when 1 ml of the sample was adsorbed on the Tswett tube of about 10 mm diameter. 2. Though several zones of the indirect bilirubin were separated on the column of silica gel when developed with various solvents, these zones were proved to be mingled with some oxidized or other intermediate products and the separation like this was thought to owe to the activity of the adsorbents. 3. The chromatogram of the crystalline bilirubin resembled to the one formed by the indirect bilirubin in the chloroform extracts. 4. The chromatogram of the chromatographically separated indirect bilirubin was similar to the former. 5. The absorption maxima of a chloroform solution of the natural indirect bilirubin existed at 450 mμ in the visible range, and it was the same as the maxima of the crystalline bilirubins.

HST virus which was isolated in 1950 from the roshida ascites tumor by Hamazaki and his associates is a pantropic virus which creates a unique inflammatory granulation in mice. When virus of an acute infections disease was inoculated on embryonated eggs, not only the egg membrane but also the chick embryo were infected more or less, and when the number of virus increased the chick embryo died, terminating the development of the egg. However, the tumor inducing virus which represents the Rous virus does not cause heavy disturbances in the embryo and it is well known that chick hatched from this egg can long maintain health unless it is subjected to a provocative factor. HST virus is no exception to this example and though it is inoculated on an embryonated egg it does not cause any serious disturbance on the embryo. The tissue changes of the chorio allantois infected by the "Virus were the focal proliferation and necrosis of ectodermal epithelium, the proliferation of the mesenchymal cells of the mesodermal layer adjacent to these foci, accompaning infiltration of lymphoid cells and leukocytes with edema, especially eosinophilic leukocytes. By these tissue changes a terrace-shaped thickening of the membrane was the result. In the viscera of the chick embryo a special change in the liver was seen, i. e., along the edge of the liver greyish white nodules submacroscopic to miliary in size appeared. The principal pathologic change of the foci is the coagulation necrosis of the liver parenchyma and only a slight infiltration about the periphery of the foci was observed. Moreover, proliferation of mesenchymal cells occurred next to the walls of the large blood vessels of the liver (principally, the portal veins) and with the added infiltration of a small number of lymphoid cells and leukocytes sharply defined nodular foci were formed. Though this was a rare instnace, similar pathologic changes were seen also in the walls of the blood vessels of the cerebrum stem of the embryo and along the periphery local gliosis was observed.

For the purpose of obtaining the dibasic acid indirect bilirubin in a pure state from the dried canine cholecystic bile, an optimal developing solvent was selected by paper partition chromatography as a preliminary experiment, and it was isolated on cellulose column as an applied experiment. 1. The dibasic acid indirect bilirubin was separable at the starting point in a pure state by paper chromatography under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5). 2. The dibasic acid indirect bilirubin formed a fixed band at the upper starting place on cellulose column under development with the top layer of a n-butanol, acetic acid, water mixture (4:1:5), and no other substance could be detected there. 3. The dibasic acid indirect bilirubin existing in the fixed band could be eluted out into chloroform with a 1% acetic acid solution. An orange yellow powder was obtained from the eluate by evaporating the solvent in vacuo. 4. Thus separated orange yellow powder agreed well with the crystalline bilirubin in the solubility into organic or inorganic solvents and in the spectrochemical characteristics as well as in the chemical properties.