Anti-interferon-α neutralizing antibody is associated with nonresponse to pegylated interferon-α plus ribavirin in chronic hepatitis C.

Pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV) treatment fails to achieve a sustained virological response (SVR) in approximately 20-50% of patients with chronic hepatitis C virus (HCV) infection. We assessed the contribution of an anti-IFN-α neutralizing antibody (NAb) on the nonresponse to treatment. NAbs were detected using an antiviral assay that assessed the neutralizing effects of serum samples against IFN. Serum samples were obtained at the end of the treatment and evaluated for the presence of NAbs using recombinant IFN-α as a standard. We studied 129 PEG-IFN-α/RBV-treated patients. In the 82 end-of-treatment responders, no NAbs were detected. Of the 47 patients who did not respond, seven (15%) were positive for NAbs. We also examined an additional 83 patients who had not responded to PEG-IFN-α treatment, and detected 12 with NAbs. Patients with good IFN-responsive characteristics, including HCV genotype 2/3 and major allele homozygotes for interleukin-28B, were included in the 19 patients with NAbs. No NAbs interfered with the antiviral activity of natural human IFN-ß (nIFN-ß) and re-treatement of patients with NAbs with nIFN-ß/RBV achieved SVR. Our analyses revealed that the emergence of anti-IFN-α NAbs was a candidate causal factor of PEG-IFN-α-treatment failure. Therefore, these antibodies should be assayed in patients who do not respond to PEG-IFN-α therapy, and if detected, other effective treatments, i.e., medications that are not neutralized by anti-IFN-α NAbs, should be considered.

Transcription factor Sp3 activates the liver/bone/kidney-type alkaline phosphatase promoter in hematopoietic cells.

The promoter region of the liver/bone/ kidney-type alkaline phosphatase gene was examined to define the cis-acting regulatory sequences and transcription factors responsible for its expression in hematopoietic cells. Transient transfection experiments revealed that regions deleted up to -154 base pairs upstream from the transcription initiation site had significant activities to induce bacterial chloramphenicol acetyltransferase gene. The shortest DNA fragment was found to contain three GC boxes in addition to a TATA box. Electrophoretic mobility shift assay and Southwestern analysis showed that Sp3 could bind to the fragment. Western blot analysis also detected Sp3 protein in eluate from the DNA probe mixed with the nuclear extracts. Through the use of Drosophila Schneider cells that lack the Sp1 family of transcription factors, Sp3 was shown to activate the basal promoter in a dose-dependent manner. When the amount of Sp3 was limited, the most proximal GC box was found to be critical for the basal promoter activity.

A novel intracellular antigen in HL-60 cells that changes in molecular weight after granulocytic and monocytic differentiation.

The present study reports the identification and partial characterization of a novel antigen with M(r) 100,000 by a monoclonal antibody (D29A8) that was obtained by immunizing BALB/c mice with nuclei of HL-60 cells. D29A8 detected mainly a nucleolar macromolecule with M(r) 100,000 (p100). On the other hand, when HL-60 cells were induced to differentiate either into a granulocytic or monocytic pathway, the antibody detected mainly a cytoplasmic macromolecule with M(r) 95,000 (p95). Since two subtypes of the antigen (p100 and p95) appear to be present in the same cells that differ in the stage of cell differentiation, the antigen may play an important role in cellular differentiation.

The mode of binding of pyridoxal 5'-phosphate in rabbit muscle glycogen phosphorylase b: circular dichroism and absorption studies.

Large bands absorbing at 251 and 335 nm were observed in the difference circular dichroism and difference absorption spectra of the holo- versus the apo-enzyme of rabbit muscle phosphorylase b. In addition to the previously known band at 335 nm, the band at 251 nm is here assigned to the bound pyridoxal 5'-phosphate (PLP). These observed difference spectra are simulated by the optical properties of the enol-imine form of the Schiff base rather than those of the substituted aldimine. Pyridoxal and salicylaldehyde also bound to apophosphorylase in the same manner as PLP. Since analogs having no hydroxyl group at the ortho position to the aldehyde group did not bind to the apoenzyme, the hydroxyl group is thought to be important in stabilizing the interaction between apophosphorylase and PLP. The PLP bound at Site I of bovine serum albumin, which is known to be one of the PLP-binding proteins absorbing at 330-340 nm, showed two prominent bands at 253 and 335 nm. The circular dichroism and absorption spectra induced by the binding of PLP fit those of the enol-imine of the Schiff base, as in the case of phosphorylase.

Comparative glucan specificities of two types of spinach leaf phosphorylase.

Two types of alpha-glucan phosphorylase [EC 2.4.1.1] from spinach leaves have been separately purified to near homogeneity. Type I enzyme shows a subunit molecular weight of 92,000 and Km values for amylopectin, glycogen and amylose much smaller than that for maltopentose. Cyclodextrin is a normal competitive inhibitor with respect to maltopentose, while it is a multi-site competitive type inhibitor with respect to amylopectin, glycogen or amylose. Type II enzyme shows a subunit molecular weight of 108,000, and utilizes amylopectin, amylose and maltopentose well, but glycogen very poorly. On affinity electrophoresis, Type II enzyme shows no affinity for glycogen and the dissociation constant for amylopectin is more than a thousand-fold greater than that of Type I enzyme. Type II enzyme has similar characteristics (subunit size, glucan specificity, and mode of cyclodextrin inhibition) to potato phosphorylase, for which cyclodextrin is a normal competitive inhibitor with respect to either maltopentose or amylopectin. Enzyme-glucan binding models have been proposed to explain these different kinetic properties. In spinach Type I phosphorylase, multiple glucan binding sites are located on the same face of the enzyme molecule to enable a single large substrate to be bound by riding on all the site; in spinach Type II and potato phosphorylases, two glucan binding sites are three-dimensionally arranged in a manner that excludes the possibility of the binding of amylopectin by riding on the two sites.

The role of pyridoxal 5'-phosphate in plant phosphorylase.

The removal of pyridoxal 5'-phosphate from potato phosphorylase [EC 2.4.1.1] was achieved by incubation in an acidic ammonium sulfate solution containing hydroxylamine. Potato apophosphorylase is catalytically inactive, but reactivated by incubation with pyridoxal phosphate. Upon titration, the degree of recovery of activity agreed well with the degree of incorporation of pyridoxal phosphate. Therefore, it can be concluded that pyridoxal phosphate in the plant enzyme is the cofactor required for the enzyme activity, as it has been shown to be in the animal enzyme. The reconstitution of the apoenzyme using six pyridoxal phosphate analogues modified at the 5' position indicates that the structural properties of the cofactor binding site is similar in potato and rabbit muscle phosphorylases. This suggests that the plant enzyme has also a substrate binding site neighboring with the 5'-phosphate moiety of the bound cofactor similar to that shown to exist in the animal enzyme. On the other hand, the analysis of the far-ultraviolet circular dichroism spectra of these two enzymes shows that the secondary structures are rather different from each other; the potato enzyme is estimated to have 16% alpha-helix and 37% beta-structure, and the muscle enzyme b 41% alpha-helix and 21% beta-structure. This indicates that the cofactor binding locus must have been more strictly conserved than other regions.

A monomeric intermediate in the reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate.

The process of reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate (PLP) has been investigated to elucidate the structure-function relationship in phosphorylase [EC 2.4.1.1]. In time-course studies, the recovery of enzyme activity was found to be delayed, especially at low temperatures, compared with the binding of PLP to protein. On polyacrylamide gel electrophoresis, an intermediary enzyme species was detected which bound PLP in the same binding mode as the native holoenzyme does, but which had neither enzyme activity nor affinity for the substrate amylopectin. The intermediate is monomeric and is converted to the active holoenzyme with concomitant dimerization. NaBH4-treatment of the monomeric intermediate produced the reduced monomeric enzyme, which could be converted into the reduced dimeric enzyme with considerable enzyme activity. The findings support the view of the catalytic activity of phosphorylase requires the dimeric structure of protein. As in the animal enzyme, the aldimine bond between the PLP and the epsilon-amino group of the lysyl residue is also not essential for enzyme activity in plant phosphorylase.

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system.

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.

Purification and properties of an auxin-binding protein from maize shoot membranes.

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.

Structural similarities in the active-site region between potato and rabbit muscle phosphorylases: a lysyl residue located close to the pyridoxal 5'-phosphate.

P1,P2-bis(5'-pyridoxal)diphosphate crosslinks between the original cofactor (pyridoxal 5'-phosphate) linking residue and Lys-573 in rabbit muscle phosphorylase (Shimomura, S., Nakano, K., & Fukui, T. (1978) Biochem. Biophys. Res. Commun. 82, 462-468). We have applied the same technique to potato phosphorylase to compare the structures of the active-site regions of the two enzymes, which have different regulatory properties. The reagent was bound to the potato enzyme in the same binding mode as the rabbit muscle enzyme. A sequence study on the potato enzyme labeled with this reagent revealed that it crosslinks between the original cofactor-linking lysyl residue and another lysyl residue, respectively corresponding to Lys-679 and Lys-573 in the rabbit muscle enzyme, and that the sequence Lys-573 to Leu-577 in the rabbit muscle enzyme is conserved in the potato enzyme. These findings indicate structural similarities in the active-site region between the phosphorylases, and suggest the importance of a lysyl residue in the catalytic mechanism of the phosphorylase reaction.

The overdosage and/or poisoning information gap.

The availability of poisoning information from the pharmaceutical industry was evaluated. Manufacturers of the top 50 drugs were surveyed. Information that was received was classified by established criteria for evaluation. Compendia on poisoning information was evaluated based on 11 components ranging from toxicity information to information available in the PDR. Additional information on emergency telephone numbers for after-hours service was accumulated. The results showed that much of the information available is incomplete and/or outdated. A concerted effort to establish a central source for manufacturers' poison information needs to be begun.

Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis.

A 51-year-old woman who had been treated for primary biliary cirrhosis (PBC) was admitted to our hospital for evaluation of unexplained, isolated, persistently increased aspartate aminotransferase (AST) activity. Results of laboratory tests on admission showed: AST 171 KU, alanine aminotransferase 28 KU, and anti-mitochondrial titer 1/1280. Results of hepatitis B surface antigen (HBs Ag) and hepatitis C virus antibody (HCV Ab; C100-3) assays were negative. Histology of a liver biopsy specimen was compatible with a diagnosis of PBC (stage III of Scheuer's classification). The molecular size of serum AST was estimated to be more than 500,000 by high-performance size-exclusion liquid chromatography. Electrophoretic analysis showed an abnormal band of AST between supernatant AST (sAST) and mitochondrial AST (mAST), which band was characteristic of AST-immunoglobulin complexes (AST-Ig). Ouchterlony double-diffusion and immunoprecipitation tests identified the immunoglobulin component as IgM. The presence of AST-Ig appeared to be responsible for the elevated serum AST.

Serum pseudouridine as a biochemical marker in patients with hepatocellular carcinoma.

The concentration of serum pseudouridine, a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in patients with hepatocellular carcinoma, liver cirrhosis, other benign hepatobiliary diseases, and healthy controls. The serum pseudouridine concentration in patients with hepatocellular carcinoma was significantly higher than that in patients with cirrhosis or the controls. Twenty-seven (51.9%) of 52 patients with hepatocellular carcinoma had serum pseudouridine concentrations higher than the mean value for healthy controls plus 2 SD. Fourteen of the 36 patients who had serum alpha-fetoprotein levels below 400 ng/ml, had elevated serum pseudouridine concentration. In total, 36 of the 52 patients (69.2%) could be detected by combination of these two markers. Two patients who had developed hepatocellular carcinoma during the course of cirrhosis and were continuously negative for alpha-fetoprotein, had higher levels of the pseudouridine concentration when hepatocellular carcinoma occurred. Furthermore, 4 of the 7 patients who had a very small cancer and were negative for alpha-fetoprotein, had elevated serum pseudouridine concentration. These results indicate that serum pseudouridine is a useful biochemical marker and that serum pseudouridine and alpha-fetoprotein in combination are considered to serve as complementary markers, for the diagnosis of hepatocellular carcinoma.

Investigation of TTV by in situ hybridization in patients with chronic hepatitis.

To clarify whether TT virus (TTV) was present in liver tissues, 12 liver tissue samples from patients with chronic hepatitis positive for TTV in their serum and 11 samples from serum-negative patients were obtained by needle biopsies and investigated using in situ hybridization. Positive staining was observed in nine (75%) of 12 cases positive for TTV (serum-positive group) and three (27.3%) of 11 cases negative for TTV (serum-negative group) (P=0.061). Three kinds of staining patterns were observed: nuclear, cytoplasmic and both. In 58.3% (7/12) of the patients positive for TTV staining, the stained areas were found in both the nucleus and cytoplasm. Only cytoplasmic staining was observed in three cases from the serum-positive group. Only nuclear staining was observed in two cases from the serum-negative group. No significant differences were found in the clinical background between the in situ hybridization-positive and -negative groups, and between the serum-positive and -negative groups. The present study shows that TTV exists in the liver tissue, especially in hepatocytes, of chronic hepatitis patients and that the localization of TTV in the cell is different from case to case, although why this is so remains to be clarified.

Relationships between structure of nitrated arenes and their mutagenicity in Salmonella typhimurium; 2- and 2,7-nitro substituted fluorene, phenanthrene and pyrene.

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we studied the relationships between the mutagenic potency and chemical structure of 2-nitro- and 2,7-dinitro-arenes including nitrated fluorene (Fl), dihydrophenanthrene (DHPh), phenanthrene (Ph), tetrahydropyrene (THPy), dihydropyrene (DHPy) and pyrene (Py) together with 9-NO2-Ph, 1-NO2-Py and 1.3-diNO2-Py. The mutagenicity tests were carried out on Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6 in the absence of S9 mix. The order of mutability of mononitro- and dinitro-arenes in TA98 is as given below: 2-NO2-THPy less than 2-NO2-Fl less than 2-NO2-DHPh less than 9-NO2-Ph less than 2-NO2-Ph less than 2-NO2-DHPy less than 1-NO2-Py less than 2-NO2-Py, and 2,7-diNO2-DHPh less than 2,7-diNO2-Fl less than 2,7-diNO2-THPy less than 2,7-diNO2-Ph less than 2,7-diNO2-DHPy less than 2,7-diNO2-Py less than 1,3-diNO2-Py. 9-NO2-Ph and 1-NO2-Py, which have been detected in environmental samples, are not as potent mutagens as 2-nitrated phenanthrene and pyrene, respectively. 2-NO2THPy (37.7 rev/nmole) was a weak mutagen, but 2,7-diNO2-THPy (3197 rev/nmole) was as potent a mutagen as 2,7-diNO2 (3925 rev/nmole). Tetrahydropyrene has a twisted form in its structure. 1,3-diNO2-Py (99660 rev/nmole) was more mutagenic than 2,7-diNO2-Py (37960.0 rev/nmole), and their mutagenicities were correlated with the behavior of the K-band in their UV spectra by the introduction of nitro groups on pyrene.

Age-related alterations in the auditory brainstem responses and the compound action potentials in guinea pigs.

The auditory brainstem response (ABR) and the eight nerve compound action potential (CAP) were measured using click click stimuli to investigate the age-related alteration in the auditory function in 66 guinea pigs consisting of four age groups. With advancing age, a gradual elevation of the thresholds in both the ABR and CAP was clearly seen, together with the prolonged latencies for waves I, II, III, and IV to clicks at 95 dBpeSPL in the ABR. There were some individual differences in either threshold elevation or latency prolongation of both the ABR and CAP in aged guinea pigs. These findings suggest that the effect of individual differences on degenerative aging processes of the auditory system should be considered in selected aged animals, although a significant elevation of the neural auditory threshold is clearly found with advancing age as a whole.

Renal failure caused by long-term use of a germanium preparation as an elixir.

Two Japanese women and one Japanese man, who had been taking the same germanium preparation, mainly containing inorganic germanium, as an elixir for health almost every day at 90 mg of germanium per day for 6 to 20 months, suffered from chronic renal failure. Histological examination of the kidney in one patient showed marked interstitial changes with vacuolar degeneration of the renal tubules. High germanium concentrations were found in hair and nails of the three patients, but no germanium was detected in hair or nails of normal persons. These results suggest that long-term use of a germanium preparation at high dosage can cause serious renal tubular damage and renal failure due to germanium toxicity.

Adsorption mechanism of polylysine on hydroxyapatite and its effect on dissolution properties of hydroxyapatite.

Adsorbed amounts of poly-l-lysine (pLys) and bromide ion on hydroxyapatite (HAp) from aqueous solutions of poly-l-lysine hydrobromide, and amounts of calcium and phosphate ions liberated concurrently from HAp during the adsorption of pLys were determined at 25 degrees . The pLys was adsorbed on HAp by the mechanism of ion-exchange between its amino groups and calcium ions of HAp. The released amount of calcium ion increased, therefore, with the adsorbed amount of pLys. On the other hand, the released amount of phosphate ion first decreased and then increased after attaining a minimum with the equilibrium concentration of pLys. The analysis using an equilibrium dialysis method revealed that the released phosphate ions were mainly in the bound state to the amino groups of pLys remaining in the solution, and that the concentrations of calcium and phosphate ions free from both HAp and pLys were restricted by each other under the law of the solubility product of HAp. The first decrease in the released amount of phosphate ion was concluded to be attributed primarily to the increase in the released amount of calcium ion because pLys remaining in the solution was little in this region. When sodium hydroxide was added to the solution, the adsorbed amount of pLys increased and then slightly decreased with the equilibrium pH of the solution due to the increase or decrease of the electrostatic attractive force between the adsorbate and the adsorbent. However, conformational change in pLys around pH 10 seemed to have little effect on the adsorption.

Interrelationships between the concentration of magnesium, calcium, and strontium in the hair of Japanese school children.

Inductively coupled atomic emission spectrometry was used for the determination of magnesium, calcium and strontium in the hair of Japanese school children (7-15 years of age, 158 males and 184 females). Sex-related differences in the levels of the three alkaline earth metals were observed (T-test, p less than 0.001). Geometric means (SD) of the concentrations of magnesium, calcium and strontium were 30.40 (1.42), 326.3 (1.62) and 0.509 (2.04) micrograms g-1 for males and 61.21 (1.73), 643.4 (1.57) and 2.749 (2.14) micrograms g-1 for females. For both sexes, significant positive correlations were observed between all concentration pairs of the three elements. The concentration ratios between any two of the three elements were also determined. The geometric mean of the concentration ratio of calcium to magnesium for males [10.73 (1.54)] is not significantly different from that for females [10.51 (1.40)] (T-test, p greater than 0.05). On the other hand, concentration ratios of magnesium to strontium [59.71 (1.99)] and calcium to strontium [640.9 (1.74)] for males are significantly higher (T-test, p less than 0.01) than those for females [22.27 (1.60) and 235.9 (1.65)]. The logarithmic-transformed ratio of magnesium to strontium has a strong positive correlation with the transformed ratio of calcium to strontium [r = 0.9529 (p less than 0.001) for males and r = 0.7284 (p less than 0.001) for females].

Determination of germanium and some other elements in hair, nail, and toenail from persons exposed and unexposed to germanium.

Inductively coupled atomic emission spectrometry was used for the determination of germanium in hair, nail, and toenail. The levels of germanium in three individuals administered a high concentration of a germanium preparation daily for about 12-16 months were very high: 56.4-173.7; 5.4-35.0; and 14.0-15.8 micrograms g-1 in hair, nail, and toenail, respectively. The levels for normal or unexposed persons are very low and were not detected by the method.