Breath sample collection through the nostril reduces false-positive results of 13C-urea breath test for the diagnosis of helicobacter pylori infection.

BACKGROUND: One of the disadvantages of '3C-urea breath test is possible interference by urease activity not related to Helicobacterpylori. AIMS: We design the simple and non-invasive modification to avoid the contamination of 13CO(2) produced in the mouth. PATIENTS AND METHODS: One hundred and twenty-nine patients who underwent diagnostic upper endoscopy were enrolled. Within 1 week of the endoscopic procedure, each patient received the modified 13C-urea breath test. Breath samples were collected at baseline and at 1, 3, 5, 10, 15, 20 and 30 min after ingestion of 100 mg 13C-urea solution through the mouth and the nostril at each time point. RESULTS: The breath delta13CO2 value through the nostril at 1 min was already higher in H. pylori-positive patients than in H. pylori-negative patients. Using 2.5% as the cut-off value, the sensitivity and specificity of the modified 13C-urea breath test at 20 min were both 100%, whereas the sensitivity and specificity of the standard 13C-urea breath test were 97.7 and 94%, respectively, using 3% as the cut-off value. CONCLUSIONS: The modified 13C-urea breath test in which breath samples are collected through the nostril provides an easy way of avoiding false-positive results for the detection of H. pylori infection.

X-deficient woodchuck hepatitis virus mutants behave like attenuated viruses and induce protective immunity in vivo.

The X protein (HBX) of the hepatitis B virus (HBV) has been shown to be important for the establishment of HBV infection in vivo. Our previous studies suggested that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. In this study, we generated a series of woodchuck hepatitis virus (WHV) X mutants, including mutants of the domain interacting with the proteasome, and studied their infectivity in woodchucks. Many of the mutants were defective in transactivation but none of them were completely replication defective in vitro. In vivo, all the wild-type and some X mutant-transfected animals demonstrated evidence of infection with anti-WHc and/or anti-WHs seroconversion. Most of the wild-type- and X mutant-transfected animals had transient viremia. Some animals were later challenged with infectious WHV. Animals inoculated with X mutants, including those with no serologic evidence of infection, were protected from the challenge, suggesting previous infection with resulting protective immunity. Our study demonstrates that the previously described functional domains of HBX are biologically important and the X-defective mutants, possibly as attenuated viruses, are not completely replication defective in vivo.

Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex.

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.

Localization of glucokinase-like immunoreactivity in the rat lower brain stem: for possible location of brain glucose-sensing mechanisms.

Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic beta-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopositive cells comprise a part of a vast glucose-sensing mechanism in the brain.

[A case of glioblastoma manifesting 49 years after lobotomy].

We report a case of glioblastoma manifesting 49 years after a lobotomy. He was diagnosed as having schizophrenia at age 20 and was operated on with a standard lobotomy when he was 27 years old. He had led a useful life after 40 years old without medication. Because of hallucination and delusion, he was hospitalized at the end of 1996. CT showed a well enhanced tumor adjacent to the cavity made by the lobotomy in the left frontal lobe. This is the second case report of glioblastoma just beside the cavity formed by lobotomy. The relationship between glioblastoma and old lobotomy is discussed, especially in regard to morphology and CT findings.

Quantal analysis suggests presynaptic involvement in expression of neocortical short- and long-term depression.

Long-term depression together with long-term potentiation represent popular experimental models to study synaptic plasticity. However, analyses of the mechanisms underlying the expression of cortical long-term depression are in their infancy and have been confined to the hippocampus. Short- and long-term depression in neocortex is not well understood. Here we recorded small excitatory postsynaptic potentials intracellularly from rat visual cortex slices. The responses fluctuated between several amplitude levels suggesting a quantal nature of the synaptic transmission. Consistent changes in the quantal steps accompanied neither paired-pulse depression (50 ms interval within the pair) nor long-term depression (induced by 1 Hz, 5 min stimulation). The amplitude distributions shifted to smaller values suggesting decreases in the number of quanta released without essential changes in the postsynaptic quantal efficiency. Both the coefficient of variation of response amplitudes and the number of response failures increased; cases were encountered suggesting a very low release probability after depression. Changes in quantal content estimated from the deconvolution analysis were correlated with the magnitude of depression. The findings suggest predominantly presynaptic loci for expression of short- and long-term neocortical depressions. The likely underlying mechanism is a decrease in transmitter release probability. Long-term depression decreased the probability so strongly that some inputs became virtually silent.

A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B.

To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance.

Metabotropic glutamate receptors and visual cortical synaptic plasticity.

Two forms of use-dependent synaptic plasticity, called long-term potentiation (LTP) and long-term depression (LTD), can be elicited in the visual cortex following different paradigms of electrophysiological stimulation. These neurobiological phenomena often are considered as necessary components of models for the alteration in function of the nervous system that must occur at some level for the establishment and (or) maintenance of memory engrams, for learning processes, or for the consolidation of active neural connections and regression of inactive contacts in the developing brain. It has been postulated that for LTP and LTD to be produced in the hippocampus, activation of a particular subtype of excitatory amino acid receptor, the metabotropic receptor, is a critical requirement. Only recently has it become possible to test this hypothesis directly, as a new compound, (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), has been introduced and the suggestion made that it selectively antagonizes the metabotropic receptor. This substance has been tested in the present study on responses recorded from slices of rat visual cortex and has been found both to block the activation of the metabotropic receptor and to interfere selectively with the form of synaptic plasticity called LTD. It thus appears from the experiments reported in this paper as though the metabotropic receptor subtype that is blocked by MCPG is required for the expression of LTD but not for the expression of LTP, in the visual cortex of adult rats.

An inhibitor for calcineurin, FK506, blocks induction of long-term depression in rat visual cortex.

Long-term depression (LTD) of synaptic transmission, often used as an essential component in synaptic models for learning, memory and forgetting, can be produced in layer II/III of the visual cortex by a prolonged, low-frequency stimulation (LFS) of layer IV. The activation of Ca2+/calmodulin-dependent protein phosphatase, calcineurin, has been postulated to play a role in the induction of LTD. The recent introduction of a specific inhibitor for calcineurin, FK506, prompted the investigation of the involvement of this phosphatase in the induction of LTD in visual cortex. Thus, we administered FK506 at 1 microM to visual cortical slices of young rats, and found that it did not significantly affect field responses of layer II/III evoked by test stimulation of layer IV at 0.1 Hz, but prevented LTD of the responses from being induced by LFS (1 Hz for 15 min) in all the 10 slices tested. Without FK506, significant LTD was induced by the same parameters of LFS in 8 of the 12 slices. These results suggest the critical involvement of calcineurin in producing LTD in visual cortex.

Polyamine oxidase from water hyacinth: purification and properties.

Polyamine oxidase was purified to homogeneity from leaves of water hyacinth by the criterion of sodium dodecyl sulfate gel electrophoresis (SDS disc PAGE). The enzyme showed a high specificity for spermidine and spermine (K(m) values 28 micromolar and 20 micromolar, respectively). The optimal pH of the enzyme for both spermidine and spermine was 6.5. The molecular weight of the enzyme estimated by Sephadex G-200 gel filtration was 87,000, while SDS disc PAGE gave a single band at the molecular weight of 60,000. Octamethylenediamine and quinacrine were strong inhibitors of the enzyme, but p-chloromercuribenzoate was without effect. A prosthetic group in the enzyme was identified as flavin adenine dinucleotide.

[The role of Ca ions and cAMP in ACTH lipolysis].

Ca ions and cAMP are known to be involved in the action of hormones. In this study, the actions of Ca ions and cAMP in ACTH lipolysis were investigated by comparing ACTH1-24, ACTH1-10 and ACTH11-24 with regard to: lipolytic activity, binding affinity to the fat cell membrane, changes in Ca ion binding to the fat cell membrane and cAMP formation. Rat adipocytes were used. Lipolytic activity was measured in terms of the amount of FFA released from fat cells into the medium after incubation with the ACTH analogs at 37 degrees C for 30 minutes. Binding affinity to the fat cell membrane was expressed as competitive binding affinity, determined by simultaneously incubating fat cell ghosts with 125I-ACTH1-24 and unlabelled ACTH analogs at 4 degrees for 40 minutes. Changes in Ca ion binding to the fat cell membrane was expressed as Ca binding capacity, measured by simultaneously incubating fat cell ghosts with 45Ca and ACTH analogs at 24 degrees C for 20 minutes. CAMP formation was measured by RIA as the amount of cAMP formed in fat cells incubated with 10(-6)M ACTH analogs at 37 degrees C for 0, 3, 5 and 10 minutes. FFA release after incubation with each of the ACTH analogs at 10(-6) M was 1.17 mEq/L/tube for ACTH1-24, 0.26 mEq/L/tube for ACTH1-10 and 0.25 mEq/L/tube for ACTH11-24, showing a high value only for ACTH1-24. The binding affinity to the fat cell membrane (high affinity constant) was 4.4 X 10(-8) M for ACTH1-24, 4.6 X 10(-8) M for ACTH1-10 and 4.6 X 10(-8) M for ACTH11-24. There were no significant differences among the binding affinities of these ACTH analogs. The binding capacity of 45Ca++ to the fat cell membrane (high affinity constant) was 3.64 X 10(-6) M for ACTH1-24, 4.49 X 10(-6) M for ACTH1-10 and 5.46 X 10(-6) M for ACTH11-24. There were no significant differences among these ACTH analogs. The increase in cAMP formation during the first 3 minutes of incubation was 35.5 fmol/mg dry weight of fat cells for ACTH1-24, 2.2 fmol/mg for ACTH1-10 and 1.8 fmol/mg for ACTH11-24. An increase was only observed for ACTH1-24. The fact that FFA release and cAMP formation were high only with ACTH1-24 among the ACTH analogs tested indicates that cAMP formation plays an important role in the lipolytic activity of ACTH in fat cells and that the three-dimensional structure of ACTH is important for cAMP formation.