Phosphodiesterases 1 and 2 regulate cellular cGMP level in rabbit submandibular gland cells

In rabbit salivary glands, stimulation of muscarinic cholinergic receptors causes production of cGMP through intracellular Ca2+ and nitric oxide. In this study, we investigated a role of cyclic nucleotide phosphodiesterase (PDE) in regulating the cellular cGMP level by using cells dispersed from the submandibular gland. Methacholine, a cholinergic agonist, rapidly elevated the cGMP level. The elevation was greatly enhanced by IBMX, a non-specific inhibitor for most isoforms of the 11 PDEs. The cGMP level was also elevated by MM-IBMX and EHNA, which inhibit the activities of PDE1 and PDE2, respectively. The elevation by the simultaneous application of the two drugs corresponded to 90% of that by IBMX. Therefore, PDE1 and PDE2 are the main PDEs that act to degrade cGMP in methacholine-stimulated cells. The presence of the two PDEs was confirmed by assaying their activities of the cell lysate. In unstimulated cells, the cGMP level was elevated by MM-IBMX and little elevated by EHNA. While the PDE2 activity was thus low, it was estimated that methacholine increases its activity approximately 50-fold. The strong activation can be explained by the elevation of the cGMP level because PDE2 is a cGMP-stimulated PDE. SNAP, a nitric oxide donor, causes production of cGMP without a receptor-operated increase in intracellular Ca2+ concentration. In SNAP-stimulated cells, MM-IBMX elevated the cGMP level higher than in methacholine-stimulated cells although the PDE1 activity is dependent on Ca2+/calmodulin. Besides Ca2+, other factors may regulate the PDE1 activity in living cells.     

35 kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T

Using a tropomyosin-coupled affinity column, we have demonstrated a direct association between the chymotryptic 35 kDa fragment of h-caldesmon, which is located at the C-terminal of the parent molecule, and gizzard tropomyosin. We have subsequently determined the nucleotide sequence of cDNA clones encoding the 35 kDa fragment from the cDNA library prepared from chick embryo gizzards, and have deduced the amino acid sequence. Calculating from the predicted sequence, the 35 kDa fragment is composed of 306 amino acid residues. In agreement with the tropomyosin-binding ability, the 35 kDa fragment conserves two consensus sequences of the tropomyosin-binding domain in troponin T. These results suggest that the 35 kDa fragment of h-caldesmon, at least in part, has a common property to the striated muscle troponin T.     

Chemotherapy Induces Oral Mucositis in Mice Without Additional Noxious Stimuli

Oral mucositis (OM) is a serious side effect of cancer chemotherapy. The pathobiology of oral mucositis remains incompletely understood due to lack of appropriate models which recapitulate the human condition. Existing rodent models are intraperitoneal and require radiation, chemical or mechanical injury to the chemotherapy protocol to induce oral lesions. We aimed to develop an OM mouse model that is induced solely by chemotherapy and reproduces macroscopic, histopathologic and inflammatory characteristics of the human condition. Female C57BL/6 mice were given intravenous 5-Fluorouracil (5-FU) injections every 48 hours, for 2 weeks. A high daily dose of intraperitoneal administration was tested for comparison. Mice were monitored daily for weight loss. Epithelial histomorphometric analyses in tongue, esophageal and intestinal tissues were conducted coupled with assessment of apoptosis, cell proliferation, neutrophilic infiltration and the integrity of adherens junctions by immunohistochemistry. Neutropenia was assessed in peripheral blood and bone marrow. Tissues were analyzed for pro-inflammatory cytokines at the protein and mRNA levels. Daily intraperitoneal administration of 5-FU led to rapid weight loss and intestinal mucositis, but no oral inflammatory changes. Intravenous administration triggered atrophy of the oral and esophageal epithelium accompanied by reduction in cell proliferation and increased apoptosis. Coincidental with these changes were up-regulation of NF-κB, TNFα, IL-1β, GM-CSF, IL-6 and KC. Despite neutropenia, increased oral neutrophilic infiltration and reduced E-cadherin was observed in oroesophageal mucosae. We developed a novel experimental tool for future mechanistic studies on the pathogenesis of chemotherapy-induced OM.     

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.     

Differential modulation of NR1-NR2A and NR1-NR2B subtypes of NMDA receptor by PDZ domain-containing proteins

The PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins MALS and PSD-95 localize to post-synaptic densities and bind the COOH-termini of NR2 subunits of the NMDA receptor. The effects of MALS-2 and PSD-95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS-2 and PSD-95 increased the current response of the NR1-NR2B receptor to l-glutamate. In contrast, the current response of the NR1-NR2A receptor was increased by PSD-95 but not by MALS-2. MALS-2 had no effect either on the potentiation of NR1-NR2A or NR1-NR2B channel activity by protein kinase C, or on Src-mediated potentiation of NR1-NR2A activity, whereas PSD-95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS-2 and PSD-95 revealed that the first two PDZ domains and two NH(2)-terminal cysteine residues are essential for the inhibitory effects of PSD-95 on protein kinase C-mediated potentiation of NR1-NR2A and NR1-NR2B channel activity, respectively. The second of the three PDZ domains of PSD-95 was required for its inhibition of Src-mediated potentiation of NR1-NR2A activity. These results indicate that the NR1-NR2A and NR1-NR2B receptors are modulated differentially by MALS-2 and PSD-95, and that similar regulatory effects of PSD-95 on these receptors are achieved by distinct mechanisms.     

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.     

Evaluation of Resting State Networks in Patients with Gliomas: Connectivity Changes in the Unaffected Side and Its Relation to Cognitive Function

In this study, we investigated changes in resting state networks (RSNs) in patients with gliomas located in the left hemisphere and its relation to cognitive function. We hypothesized that long distance connection, especially between hemispheres, would be affected by the presence of the tumor. We further hypothesized that these changes would correlate with, or reflect cognitive changes observed in patients with gliomas. Resting state functional MRI datasets from 12 patients and 12 healthy controls were used in the analysis. The tumor’s effect on three well-known RSNs including the default mode network (DMN), executive control network (ECN), and salience network (SN) identified using independent component analysis were investigated using dual regression analysis. Scores of neuropsychometric testing (WAIS-III and WMS-R) were also compared. Compared to the healthy control group, the patient group showed significant decrease in functional connectivity in the right angular gyrus/inferior parietal lobe of the ventral DMN and in the dorsolateral prefrontal cortex of the left ECN, whereas a significant increase in connectivity in the right ECN was observed in the right parietal lobe. Changes in connectivity in the right ECN correlated with spatial memory, while that on the left ECN correlated with attention. Connectivity changes in the ventral DMN correlated with attention, working memory, full IQ, and verbal IQ measures. Although the tumors were localized in the left side of the brain, changes in connectivity were observed in the contralateral side. Moreover, these changes correlated with some aspects of cognitive function indicating that patients with gliomas may undergo cognitive changes even in the absence of or before the onset of major symptoms. Evaluation of resting state networks could be helpful in advancing our hodological understanding of brain function in glioma cases.     

Structure of potato starch pastes in the ageing process by the measurement of their dynamic moduli

The effect of ageing on the rheological properties of potato starch pastes was investigated by measurement of dynamic viscoelasticity. The value of the storage modulus (G′) increased rapidly with time for the first few hours, but attained equilibrium after a long period of ageing. The relation between the concentration and the storage modulus showed a c^1·6 dependence. The Avrami exponent (n) obtained in this study was 0·66 and deviated significantly from the value (n = 1), corresponding to rod-like growth of crystal. The rate of storage modulus increase decreased with increasing concentrations up to 10%, and at higher concentrations little or no increase was observed. In addition, the rate of storage modulus increase increased dramatically when the NaCl concentration was raised to levels of 0·01 or above. An even greater increase in rate occurs on addition of AlCl₃.     

Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum‐induced cell death in hepatocytes

Chromobacterium violaceum is a Gram‐negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi‐1/‐1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi‐2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi‐1/‐1a‐encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi‐1/‐1a or Cpi‐2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi‐1/‐1a‐dependent manner. Characterization of Chromobacterium‐induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi‐1/‐1a effector, is implicated in translocator‐mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium‐induced cytotoxicity. These results strongly suggest that Cpi‐1/‐1a‐encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.     

Presence of an enzyme mediating transfer of phosphate from thiamine triphosphate to ADP in germinating maize

The presence of an enzyme involved in ATP synthesis by transfer of phosphate from thiamine triphosphate to ADP in maize germ axis was indicated by the assays on partially purified (27-fold) enzyme with luciferase method, spectrophotometric assay with hexokinase and glucose-6-phosphate dehydrogenase, and paper chromatography. Optimal activity was found at pH 9.0. The enzyme was heat-labile SH-enzyme, and its activity required the presence of Mg²⁺.