Changes in high K+-evoked serotonin release and serotonin 2A/2C receptor binding in the frontal cortex of rats with thioacetamide-induced hepatic encephalopathy

Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.     

Growth hormone and insulinlike growth factor 1 promote intestinal uptake and hepatic release of glutamine in sepsis

The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric-gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric-gonadal complex, mesonephros (days 35-45 only) and gonad (days 35-45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric-gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric-ovarian complex contained mainly oestradiol and the mesonephric-testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric-gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.     

Management of hepatic encephalopathy

Among the general principles of the therapy of hepatic encephalopathy the authors discuss the intensive care of patients, maintenance of their volume and electrolyte balance, treatment of coagulation defect, therapy of gastrointestinal bleeding and portal hypertension, provision of central renal catheter, infection prophylaxis, monitoring of intracranial pressure, and if necessary, respiration and intubation of patients. The study also deals with possibilities of treatment based on the toxic hypothesis and on the theory of neurotransmitters. Attention is paid to the therapy of brain oedema and to the significance of hemodialysis, hemoperfusion, plasmapheresis and hybrid bioartificial liver cell treatment. The authors deal with the indications of glucagon-insulin therapy and emphasize the importance of liver transplantation in the treatment of hepatic encephalopathy.     

Cerebral changes in hepatic encephalopathy

Two subgroups of mitogen-activated protein kinases, c-jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), are thought to be involved in cultured cardiac myocyte hypertrophy and gene expression. To examine the in vivo activation of these kinases, we measured cardiac JNK and ERK activities in conscious rats subjected to acute or chronic angiotensin II (Ang II) infusion, by using in-gel kinase methods. About 50 mm Hg rise in blood pressure by Ang II (1000 ng . kg-1 . min-1) infusion caused larger activation of left ventricular JNK than ERK, via the AT1 receptor. In spite of short duration (about 30 minutes) of maximal blood pressure elevation by Ang II, JNK sustained the peak value (more than 5-fold increase) from 15 minutes up to at least 3 hours. Similar activation of JNK was seen in the right ventricle. Thus, cardiac JNK activation by Ang II seems to be in part mediated by its direct action via the AT1 receptor. The dose-response relationships for Ang II-induced rises in blood pressure and cardiac JNK and ERK activation indicated that cardiac JNK or ERK was not activated by a mild increase in blood pressure and that cardiac JNK was activated by Ang II-mediated hypertension in a more sensitive manner than ERK. Cardiac hypertrophy, induced by chronic Ang II infusion, was preceded by JNK activation without ERK activation. Furthermore, gel mobility shift analysis showed that cardiac JNK activation was followed by increased activator protein-1 DNA binding activity due to c-Fos and c-Jun. These results provided the first evidence for the preferential activation of cardiac JNK in Ang II-induced hypertension and suggested that JNK might play some role in Ang II-induced cardiac hypertrophic response in vivo. However, further study is needed to elucidate the role of JNK in cardiac hypertrophy in vivo.     

The neurobiology of hepatic encephalopathy

We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.     

Interpretations of uptake defects in hepatic scintiscanning

The absence of precise parameters for the interpretation of defective uptake means that personal experience is of great assistance in the examination of scintiscans. Classification of uptake defects in 208 cases was carried out by two experts in accordance with the following parameters: 1) irregular uptake or distinct defects; 2) central or peripheral defects; 3) increased size of the liver picture; 4) uptake by the spleen. Irregular uptake or central or peripheral uptake defects (with or without spleen uptake in each case) were observed as categories. The series as a whole showed that peripheral defects are associated with a greater frequency of false positives, and diagnosis must therefore be checked, spleen uptake is an accurate pointer to diffuse chronic hepatopathy, and central defects are indicative of substitutive pathology.     

Role of tryptophan in the elevated serotonin-turnover in hepatic encephalopathy

The increase of the brain levels of 5-hydroxyindoleacetic acid (5-HIAA) in hepatic encephalopathy (HE) suggests an increased turnover of serotonin (5-HT). To study the role of tryptophan on the increased brain 5-HT metabolism in HE, we attempted to monitor brain levels of tryptophan in rats with thioacetamide-induced acute liver failure by intravenous infusion of branched-chain amino acids (BCAA). The effect of this treatment on 5-HT synthesis and metabolism was investigated in five brain areas. BCAA-infusions (1 and 2 gm/kg/24 h) increased the ratio BCAA/aromatic amino acids in plasma two- and fourfold, respectively, and lowered both plasma and brain levels of tryptophan. At the higher BCAA-dose all parameters suggesting an altered brain 5-HT metabolism (increased brain levels of 5-HT and 5-HIAA, increased 5-HIAA/5-HT ratio) were almost completely normalized. These results provide further evidence for the role of tryptophan in the elevation of brain 5-HT metabolism and for a potential role of BCAA in the treatment of HE.     

Preventive administration of ornithine alpha-ketoglutarate improves intestinal mucosal repair after transient ischemia in rats

Introduction; Healthcare professionals need to continually update their knowledge to provide care based on scientific evidence. In some cases it can be difficult to gain access to the different sources of medical information. In an attempt to overcome these problems, a toll-free telephone medical information service (Doctorline) was established. OBJECTIVE: To describe the development, aims, organization, and activities of this private service. METHODS: Doctorline is an independent, unbiased, toll-free medical information service that provides information on clinical, pharmacologic, and toxicologic issues; bibliographic searches; full-text articles; public and private clinics; details of forthcoming congresses; and legislative documentation. The service is available Monday through Friday, 1000 to 2000. Staff members are physicians trained in communication techniques, literature evaluation methodologies, and computerized database use. The main on-line facilities are MEDLINE, Micromedex-CCIS, and the Italian Formulary on CD-ROM. Books, bulletins, national and international drug formularies, and property files (i.e., directory of Italian public and private clinics) are also available. RESULTS: In 5 years, Doctorline has received 65 258 calls. Nearly 34% of the calls were made by general practitioners, followed by cardiologists (22%), orthopedists (15%), pharmacists (14%), gastroenterologists (13%), and urologists (10%). From 1991 to 1996, nearly 20% of the calls concerned pharmacologic issues, 43% nonpharmacologic issues, while the rest of the calls were for nonclinical requests. Approximately 21% of all questions received an answer during the same phone call (on-line answers); for the other answers (off-line answers) the mean +/- SD waiting time was 7.8 +/- 10.4 days. Although the nature of the questions has been recorded since 1991, data about the exact number of physicians who used the service are available only from 1994. Data from 1994 indicate that of the 52,181 physicians who could access the service, only 8817 (16.9%) called at least once, with a mean number of calls per physician of 3.9 (range 3.0-5.6). CONCLUSIONS: The future of Doctorline will depend on the quality and validity of the information provided (i.e., based exclusively on scientific evidence, independent of the source of funds), the promotion of the aims, organization, and clinical utility of the service (especially among physicians who made little or no use of the service), and differentiation of the service activities in relation to the physician's specific needs.     

Clinical efficacy of lactulose in cirrhotic patients with and without subclinical hepatic encephalopathy

Seventy-five cirrhotic patients with hyperammonemia in the past or at the time of the study were randomly divided into two groups (treated with lactulose or nontreated) in 14 hospitals in Japan. Thirty-six cirrhotic patients were diagnosed as having subclinical hepatic encephalopathy (SHE), and 39 were diagnosed as non-SHE. SHE was diagnosed when the results of all three of the quantitative psychometric tests used (number connection test, and symbol digit and block design tests of the Wechsler adult intelligence scale [revised]) were abnormal as compared with age-matched normal values. The mean number of abnormal psychometric test results and the prevalence of SHE were used for a quantitative evaluation of the efficacy of the lactulose treatment. Twenty-two of the SHE patients were treated with lactulose (45 mL/d) for 8 weeks, and the other 14 SHE patients did not receive lactulose. In the SHE patients administered lactulose, the results of the quantitative psychometric evaluation were significantly improved at 4 and 8 weeks after the beginning of the lactulose administration. The SHE had disappeared in 10 (50%) of the 20 treated patients at week 8, but it persisted in 11 (85%) of the untreated 13 patients. We concluded that lactulose treatment in cirrhotic patients with SHE is effective with respect to psychometric tests.     

Carrier-mediated hepatic uptake of quinolone antibiotics in the rat

The systemic clearance of many quinolone antibiotics is mainly via metabolism and urinary excretion; by contrast, biliary excretion is a major route of elimination for a new quinolone grepafloxacin (GPFX). Accordingly, we studied the hepatic uptake of GPFX because it is the first step in the drug's hepatobiliary transport. The hepatic uptake of GPFX in vivo after i.v. administration was found to approach the hepatic blood flow, suggesting the existence of an effective hepatic uptake mechanism. To clarify this transport mechanism, GPFX uptake by isolated rat hepatocytes was examined and found to consist of a saturable component (Km 173 microM, Vmax 6.96 nmol/min/mg) and a nonspecific diffusion component. The inhibition of GPFX uptake by ATP-depletors and a lack of effect after replacing Na+ with choline demonstrated that the uptake was an Na+-independent carrier-mediated active process. This uptake was inhibited by other quinolones and for lomefloxacin this was competitive in nature. Mutual inhibition studies were undertaken to investigate whether the transporter for GPFX might be the same as other transporters so far identified. GPFX inhibited the uptake of taurocholic acid, pravastatin (organic anion), cimetidine (organic cation) and ouabain (neutral steroid). However, GPFX uptake was not inhibited by these compounds. Confirmation that GPFX uptake is blood flow limited was obtained by extrapolation of the in vitro data based on mathematical modeling. In conclusion, the effective hepatic uptake of quinolone antibiotics are via carrier-mediated active transport, which is distinct from that involved in the transport of bile acids, organic anions, organic cations or neutral steroids.