High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans

Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant. V. vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1. The potential of V. vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus). When cultivated at 25 degrees C in tryptone-yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease. However, at 37 degrees C with 0.9% NaCl, V. anguillarum neither grew nor produced the metalloprotease. In human serum, only V. vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease. This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans.     

Primary structures of guinea pig high- and low-molecular-weight kininogens

Guinea pig high-molecular-weight and low-molecular-weight (HMW and LMW) kininogen cDNA were amplified from liver mRNA by RT-PCR. Their nucleotide sequences were analyzed and deduced to amino acid sequences. The HMW kininogen, composed of 607 amino acid residues with a 18-residue signal sequence, possessed the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. The amino acid sequence preceding the bradykinin domain was found not to be -Leu-Met-Lys- but -Leu-Thr-Arg-. Therefore, kallidin (Lys-bradykinin) and Met-kallidin are not liberated from the guinea pig kininogens. We purified the HMW kininogen protein from plasma and prepared the kinin-free form using guinea pig plasma kallikrein. Although the amino-terminal of the HMW kininogen was modified, the 25 amino-terminal residues of the light chain of the kinin-free kininogen corresponded to the deduced sequence just after the bradykinin moiety of the HMW kininogen. With regard to the LMW kininogen, the nucleotide sequence down to T(1200) as well as the amino acid sequence till Thr(382) was identical to that of the HMW kininogen. We also examined the localization of the guinea pig kininogen gene on the prometaphase lymphocyte chromosomes by fluorescence in situ hybridization method. Two pair signals were observed on a pair of homologous chromosomes, each of which is composed of two chromatids. Based on these findings, we concluded that HMW and LMW kininogens are produced from the single kininogen gene in guinea pig as in the cases of the other mammalian species reported so far.     

Characteristics of patients with chronic hepatitis C who develop hepatocellular carcinoma after a sustained response to interferon therapy

BACKGROUND: The objective of the current study was to determine the characteristic features of sustained responders who develop hepatocellular carcinoma after treatment with interferon for chronic hepatitis C. METHODS: This study included 3626 patients with chronic hepatitis C who had received interferon monotherapy. Cox proportional hazards analysis was used to compare sustained responders who did and did not develop hepatocellular carcinoma, and nonsustained responders who developed hepatocellular carcinoma in a multicenter, retrospective cohort study. RESULTS: Among 1197 sustained responders, 27 patients developed hepatocellular carcinoma (2.3%). Compared with sustained responders who did not develop hepatocellular carcinoma, patients who developed disease more often were male (P = 0.0212), were older (P = 0.0068), and had advanced-stage histologic disease before interferon therapy (P = 0.0345). Conversely, compared with patients with hepatocellular carcinoma who were not sustained responders, patients who were sustained responders tended to be older at the time of the initiation of interferon therapy (P = 0.0552) and at the time hepatocellular carcinoma was detected (P = 0.0593), and they also were predominantly male (P = 0.0507). The histologic staging and serum aminotransferase levels at the initiation of interferon therapy, the interval to the detection of tumor, and the tumor size showed no significant differences between the two groups. CONCLUSIONS: Sustained responders in the group at high risk for developing hepatocellular carcinoma after interferon therapy were older, more often were male, and had more advanced histologic disease stage. Such patients should be followed carefully periodically for > 10 years after they complete interferon therapy.     

Distinct cellular expressions of creatine synthetic enzyme GAMT and creatine kinases uCK-Mi and CK-B suggest a novel neuron-glial relationship for brain energy homeostasis

The creatine/phosphocreatine shuttle system, as catalysed reversibly by creatine kinases, is thought to be essential for the storing and buffering of high phosphate-bound energy in tissues with high energy demand. In the present study, we aimed to clarify the cellular system of creatine biosynthesis and its energy metabolism in the mouse brain by immunohistochemistry for creatine biosynthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), ubiquitous mitochondrial creatine kinase (uCK-Mi) and brain-type cytoplasmic creatine kinase (CK-B). GAMT was expressed highly in oligodendrocytes and olfactory ensheathing glia and moderately in astrocytes, whereas GAMT was very low in neurons and microglia. By contrast, uCK-Mi was expressed selectively in neurons and localized in their mitochondria in dendrites, cell bodies, axons and terminals. The distinct and almost complementary distribution of GAMT and uCK-Mi suggests that the creatine in neuronal mitochondria is derived not only from the circulation, but also from local glial cells associated with these neuronal elements. By contrast, CK-B was selective to astrocytes among glial populations, and was exclusive to inhibitory neurons among neuronal populations. Interestingly, these cells with high CK-B immunoreactivity are known to be highly resistant to acute energy loss, such as hypoxia and hypoglycemia. Considering that phosphocreatine generates ATP much faster than the processes of glycolysis and oxidative phosphorylation, the highly regulated cellular expressions of creatine biosynthetic and metabolic enzymes suggest that the creatine/phosphocreatine shuttle system plays a role in brain energy homeostasis through a novel neuron-glial relationship.     

Image of a line is not shrunk but neglected. Absence of crossover in unilateral spatial neglect

Patients with left unilateral spatial neglect following right hemisphere lesions usually err rightward when bisecting a horizontal line. For very short lines (e.g. 25 mm), however, leftward errors or seemingly 'right' neglect is often observed. To explain this paradox of crossover in the direction of errors, rather complicated models have been introduced as to the distribution of attention. Neglect may be hypothesized to occur in representational process of a line or estimation of the midpoint on the formed image, or both. We devised a line image task using a computer display with a touch panel and approached the representational image of a line to be bisected. Three patients with typical left neglect were presented with a line and forced to see its whole extent with cueing to the left endpoint. After disappearance of the line, they pointed to the right endpoint, the left endpoint, or the subjective midpoint according to their representational image. The line image between the reproduced right and left endpoints was appropriately formed for the 200 mm lines. However, the images for the shorter 25 and 100 mm lines were longer than the physical lengths with overextension to the left side. These results proved the context effect that short lines may be perceived longer when they are presented in combination with longer lines. One of our patients had an extensive lesion that involved the frontal, temporal, and parietal lobes, and the other two had a lesion restricted to the posterior right hemisphere. The image for a fully perceived line may be represented far enough into left space even when left neglect occurs after a lesion that involves the right parietal lobe. The patients with neglect placed the subjective midpoint rightward from the centre of the stimulus line for the 100 and 200 mm lines and leftward for the 25 mm lines. This crossover of bisection errors disappeared when the displacement of the subjective midpoint was measured from the centre of the representational line image. Left neglect may occur consistently in estimation of the subjective midpoint on the representational image, which may be explained by a simple rightward bias of attentional distribution.     

A new in vitro model for blood-cerebrospinal fluid barrier transport studies: an immortalized choroid plexus epithelial cell line derived from the tsA58 SV40 large T-antigen gene transgenic rat

The blood-cerebrospinal fluid barrier (BCSFB) plays a key role in the influx and efflux transport of drugs and endogenous substrates in the cerebrospinal fluid (CSF). To clarify the molecular mechanism of the BCSFB transport system, a new in vitro BCSFB model, i.e. an immortalized rat choroid plexus epithelial cell line (TR-CSFB), has been established from transgenic rats harboring a temperature-sensitive simian virus 40 large T-antigen gene. TR-CSFB cells grow well at 33 degrees C because of activation of the temperature-sensitive large T-antigen. These cells have a polygonal epithelial cell morphology and express typical choroid plexus epithelial cell markers, such as transthyretin (TTR) and Na+, K+ -ATPase, as well as the transporters, system A and ABCC1/mrp1. The localization of Na+, K+ -ATPase, and the transport direction of system A are polarized in TR-CSFB cells as is the case in vivo. TR-CSFB cells exhibit L-proline and L-glutamic acid uptake activities and may reflect the CSF-to-blood efflux transport functions involving these amino acids in vivo. Using TR-CSFB cells, we found for the first time that oatp3 is expressed at the BCSFB. TR-CSFB cells appear to be a useful in vitro model of the BCSFB for the study of drug transport, BCSFB transporters, and the regulation of BCSFB functions.     

New aspects of the blood-brain barrier transporters; its physiological roles in the central nervous system

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain, and restricts drug permeability into the brain. Our latest studies have revealed that the BBB transporters play important physiological roles in maintaining the brain milieu. The BBB supplies creatine to the brain for an energy-storing system, and creatine transporter localized at the brain capillary endothelial cells (BCECs) is involved in BBB creatine transport. The BBB is involved in the brain-to-blood efflux transport of the suppressive neurotransmitter, gamma-aminobutyric acid, and GAT2/BGT-1 mediates this transport process. BCECs also express serotonin and norepinephrine transporters. Organic anion transporter 3 (OAT3) and ASCT2 are localized at the abluminal membrane of the BCECs. OAT3 is involved in the brain-to-blood efflux of a dopamine metabolite, a uremic toxin and thiopurine nucleobase analogs. ASCT2 plays a role in L-isomer-selective aspartic acid efflux transport at the BBB. Dehydroepiandrosterone sulfate and small neutral amino acids undergo brain-to-blood efflux transport mediated by organic anion transporting polypeptide 2 and ATA2, respectively. The BBB transporters are regulated by various factors, ATA2 by osmolarity, taurine transporter by TNF-alpha, and L-cystine/L-glutamic acid exchange transporter by oxidative stress. Clarifying the physiological roles of BBB transport systems should give us important information allowing the development of better CNS drugs and improving our understanding of the relationship between CNS disorders and BBB function.     

Effects of oolong tea on plasma antioxidative capacity in mice loaded with restraint stress assessed using the oxygen radical absorbance capacity (ORAC) assay

In the present study, we investigated the antioxidative effect of oolong tea in vitro and in vivo using the oxygen radical absorbance capacity (ORAC) assay. An oolong tea extract, catechin and related compounds suppressed the oxidation of fluorescence induced by AAPH in a dose-dependent manner, that is, they prolonged the antioxidant time in vitro. Oral administration of the oolong tea extract to mice treated with restraint stress increased ORAC activity in plasma as compared with a stress control group. The extract also increased plasma vitamin C levels, and there was a good relationship between ORAC activity and the vitamin C level in plasma. The elevation of plasma ORAC and vitamin C level may have been related to the stress-relieving effect of oolong tea. These effects are probably due to the antioxidative properties of the tea. Thus, these findings suggested that oolong tea has beneficial effects on health related to its antioxidative action.