Coexistence of fibrotic and chondrogenic process in the capsule of idiopathic frozen shoulders.

OBJECTIVE: To analyze changes in the capsule from idiopathic frozen shoulders and clarify their etiology. MATERIALS AND METHODS: Samples (the rotator interval capsule, middle glenohumeral ligament (MGHL), and inferior glenohumeral ligament (IGHL)) were collected from 12 idiopathic frozen shoulders with severe stiffness and 18 shoulders with rotator cuff tears as a control. The number of cells was counted and the tissue elasticity of the samples was calculated by scanning acoustic microscopy (SAM). The amount of glycosaminoglycan content was assessed by alcian blue staining. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Furthermore, the total genes of the two groups were compared by DNA microarray analysis. RESULTS: The number of cells was significantly higher and the capsular tissue was significantly stiffer in idiopathic frozen shoulders compared with shoulders with rotator cuff tears. Staining intensity of alcian blue was significantly stronger in idiopathic frozen shoulders. Gene expressions related to fibrosis, inflammation, and chondrogenesis were significantly higher in idiopathic frozen shoulders compared with shoulders with rotator cuff tears assessed by both qPCR and DNA microarray analysis. CONCLUSION: In addition to fibrosis and inflammation, which used to be considered the main pathology of frozen shoulders, chondrogenesis is likely to have a critical role in pathogenesis of idiopathic frozen shoulders.

Increase in tumour permeability following TGF-beta type I receptor-inhibitor treatment observed by dynamic contrast-enhanced MRI.

BACKGROUND: To enhance the success rate of nanocarrier-mediated chemotherapy combined with an anti-angiogenic agent, it is crucial to identify parameters for tumour vasculature that can predict a response to the treatment of the anti-angiogenic agent. METHODS: To apply transforming growth factor (TGF)-beta type I receptor (TbetaR-I) inhibitor, A-83-01, to combined therapy, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out in mice bearing colon 26 cells using gadolinium (Gd)-DTPA and for its liposomal formulation to evaluate changes in tumour microvasculature following A-83-01. Tumour vascular parameters from DCE-MRI were compared with histological assessment and apparent diffusion coefficient of water in tumour generated by diffusion-weighted MRI. RESULTS: Contrary to evaluations reported for anti-angiogenic agents, A-83-01 treatment increased the initial area under the Gd concentration-time curve (IAUGC60), volume transfer constant (K(trans)) and fractional plasma volume (v(p)) significantly within 24 h, that was positively related to alpha-smooth muscle actin-positive pericyte coverage and tumour cell proliferation, and was correlated inversely with the apparent diffusion coefficient. The vascular function of the tumour improved by A-83-01 treatment was well assessed on post-liposomal Gd-DTPA-enhanced MR images, which predicted delivery of a liposomal drug to the tumour. CONCLUSION: These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumour vasculature by A-83-01 treatment.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

Sequence and characteristics of the Bifidobacterium longum gene encoding L-lactate dehydrogenase and the primary structure of the enzyme: a new feature of the allosteric site.

The gene ldh, encoding L-lactate dehydrogenase (LDH; EC 1.1.1.27) of Bifidobacterium longum aM101-2, was cloned in Escherichia coli using an oligodeoxyribonucleotide hybridization probe. The amino acid (aa) sequence, deduced from the sequence of the cloned DNA, was consistent with the results of protein chemical analysis of B. longum LDH. The transcription start points (tsp) in B. longum were identified by S1 nuclease mapping. A sequence, GTAGCAA-(14 bp)-TTATAGA, which is located a few bp upstream from the tsp, was assigned as the promoter of this ldh gene. In the 3'-noncoding region, there were two structures that strongly resembled the Rho-independent transcriptional termination signal of E. coli. Therefore, the B. longum ldh gene might form a monocistronic unit. The deduced primary structure of B. longum LDH had 40% identity with LDHs from Thermus caldophilus, Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle. Most bacterial LDHs are allosterically regulated by fructose 1,6-bisphosphate (FBP), while the vertebrate LDHs are not. The anion-binding site of vertebrate LDHs has been thought to correspond to the FBP-binding site of bacterial LDHs. Although the B. longum LDH was regulated by FBP, the charge properties of aa residues in the putative FBP-binding site of the LDH were closer to those of the vertebrate LDHs than to those of bacterial LDHs.

Long-lasting activation of cation current by low concentration of endothelin-1 in mouse fibroblasts and smooth muscle cells of rabbit aorta.

1. Recombinant human ETA receptors were expressed in a mouse fibroblast cell line (Ltk- cell) and functional coupling of the receptors with Ca2+ permeable channels at low concentrations of endothelin-1 (ET-1) was investigated using whole-cell recordings and monitoring the changes in intracellular free Ca2+ concentrations ([Ca2+]i) with a Ca2+ indicator, fluo-3. A similar type of coupling was investigated in freshly dispersed vascular smooth muscle cells (VSMCs) of rabbit thoracic aorta by use of whole-cell recordings. 2. In Ltk- cells expressing recombinant human ETA receptors, concentrations of ET-1 (10(-8) M, 10(-9) M) evoked an initial transient peak and a subsequent sustained elevation in [Ca2+]i whereas a lower concentration of ET-1 (10(-10) M) evoked only a sustained elevation of [Ca2+]i. After removal of extracellular Ca2+, ET-1 evoked only an initial peak without a sustained elevation of [Ca2+]i. The sustained elevation induced by 10(-10) M ET-1 was blocked by 300 microM mefenamic acid (a cation channel blocker) but not by 10 microM nifedipine (a blocker of voltage-operated Ca2+ channel). 3. In whole-cell recordings with Ltk- cells, a brief (3-5 min) application of ET-1 (10(-10) M) induced a sustained inward current at a holding potential of -60 mV. The current-voltage relationship revealed that the reversal potential of the ET-1-induced current was close to 0 mV (1.9 mV) and was not altered by reducing the concentration of Cl- in the bath solution, indicating that the current is carried by cations. The current was reversibly blocked by 300 microM mefenamic acid, and it persisted after all cations in the bath solution had been replaced by Ca2+ (5 or 30 mM) and nonpermeant cation N-methyl-D glucamine,indicating that the ET-1-activated channel is permeable to Ca2+. Activation of the current was independent of membrane potential and the current was induced even after addition of a high concentration (10 mM) of a Ca2+ chelator, EGTA, to the pipette solution.4. In whole-cell recordings from rabbit aortic VSMCs, ET-l (101-10 M) induced a sustained inward current at a holding potential of -60 mV. The reversal potential was - 12 mV and was not altered when the concentration of Cl- in the pipette solution was decreased, indicating that the current is carried by cations. Again activation of the current was independent of membrane potential and was observed even after addition of a high concentration (10 mM) of a Ca2+ chelator, EGTA to the pipette solution. The current was reversibly blocked by 300 microM mefenamic acid and was permeable to Ca2+,showing marked similarities to ET-1-induced cationic current in Ltk- cells.5. These results indicate that in Ltk- cells transfected with cDNA for recombinant ETA receptors andVSMCs, ETA receptors can functionally couple with a nonselective cation channel permeable to Ca2+.Thus the present data suggest that the cation channel plays an essential role in the sustained elevation of[Ca2+]i at low concentrations of ET-l by causing Ca2+ entry through the channel.

Inhibitory effect of nitrovasodilators and cyclic GMP on ET-1-activated Ca(2+)-permeable nonselective cation channel in rat aortic smooth muscle cells.

1. In single vascular smooth muscle cells (VSMCs) isolated from the aortae of male Wistar rats, we examined the effects of nitric oxide (NO) donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), and 8-bromo-guanosine-3':5'-cyclic monophosphate (8-bromo-cyclic GMP) on endothelin-1 (ET-1)-activated Ca(2+)-permeable nonselective cation channel by use of whole-cell recordings of patch-clamp technique and monitoring of intracellular free Ca(2+)-concentration ([Ca2+]i) with fura-2 real-time digital microfluorometry. 2. ET-1 evoked an initial transient peak and a subsequent sustained elevation in [Ca2+]i. After removal of extracellular Ca2+. ET-1 evoked only an initial transient peak without a sustained phase. Nifedipine (1 microM), a specific blocker of the L-type voltage-operated Ca2+ channel (VOC), reduced the sustained phase to about 40% of the control level. The remaining part of the sustained phase was abolished by 30 microM SK&F 96365, a blocker of nonselective cation channels. 3. The nifedipine-resistant sustained elevation in [Ca2+]i was abolished by 100 microM SNP, 10 microM SNAP and 300 microM 8-bromo-cyclic GMP. Neither SNP, SNAP nor 8-bromo-cyclic GMP significantly affected the basal level of [Ca2+]i. 4. In a VSMC clamped at a holding potential of -60 mV with K+ in the pipette solution replaced by Cs+, application of 10(-8) M ET-1 induced an inward current with an increase in baseline fluctuation. With fluctuation analysis, unit conductance of the ET-1-induced current was calculated to be about 21 pS. The ET-1-induced current was linearly related to the membrane potentials with its reversal potential of -5.5 mV. 5. The ET-1-induced current was reversibly and completely inhibited by 30 microM SK&F 96365 or 500 microM Cd2+. The current inhibited by SK&F 96365 or Cd2+ was linearly related to membrane potential with a reversal potential of about -5 mV. 6. The ET-1-induced current was reversibly and completely inhibited by 100 microM SNP, 10 microM SNAP and 300 microM 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about -5 mV. 7. In a bath solution in which all cations were replaced by 30 mM Ca2+ and 100 mM nonpermeant cation N-methyl-D-glucamine (NMDG), ET-1 evoked a current with a reversal potential of -11 mV, from which PCa2+/Pcs1 was calculated to be 2.1. This Ca2+ current was also abolished by 100 microM SNP, 10 microM SNAP and 300 microM 8-bromo-cyclic GMP. The current inhibited by SNP, SNAP or 8-bromo-cyclic GMP showed linear voltage-dependence and reversed at about -11 mV. 8. These results taken together indicate that NO through a cyclic GMP signalling pathway inhibits ET-1-activated Ca(2+)-permeable nonselective cation channels, thereby suppressing the sustained increase in [Ca2+]i. Thus, the present study indicates that this Ca(2+)-permeable nonselective cation channel is an important target for nitrovasodilators.

The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin.

1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca2+ concentrations ([Ca2+]i) in dispersed rabbit vascular smooth muscle cells with a Ca2+ indicator fura-2. The actions of trypsin were compared with those of thrombin. 2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. 3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin. 4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca2+, indicating that most of the contraction is dependent on extracellular Ca2+. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca2+ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca2+ channels are involved in these contractions. 5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it's protease activity. 6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca2+]i, but an application of trypsin could still induce an increase in [Ca2+]i in dispersed rabbit vascular smooth muscle cells. 7. These data suggest that in addition to activation of a thrombin receptor, trypsin can contract rabbit aortae by a proteinase-activated receptor 2 or a novel mechanism.

Activation of three types of voltage-independent Ca2+ channel in A7r5 cells by endothelin-1 as revealed by a novel Ca2+ channel blocker LOE 908.

1. We have shown that in addition to voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channel (NSCC) in A7r5 cells: its lower concentrations (< or = 1 nM; lower [ET-1]) activate only an SK&F 96365-resistant channel (NSCC-1), whereas its higher concentrations (> or = 10 nM; higher [ET-1]) activate an SK&F 96365-sensitive channel (NSCC-2) as well. 2. We now characterized the effects of a blocker of Ca2+ entry channel LOE 908 on NSCCs and store-operated Ca2+ channel (SOCC) in A7r5 cells, and using two drugs, clarified the involvement of these channels in the ET-1-induced increase in the intracellular free Ca2+ concentrations ([Ca2+]i). Whole-cell recordings and [Ca2+]i monitoring with fluo-3 were used. 3. LOE 908 up to 10 microM had no effect on increases in [Ca2+]i induced by thapsigargin or ionomycin, but SK&F 96365 abolished them. 4. In the cells clamped at -60 mV, both lower and higher [ET-1] induced inward currents with linear iv relationships and the reversal potentials of -15.0 mV. Thapsigargin induced no currents. 5. In the presence of nifedipine, lower [ET-1] induced a sustained increase in [Ca2+]i, whereas higher [ET-1] induced a transient peak and a sustained increase. The sustained increases by lower and higher [ET-1] were abolished by removal of extracellular Ca2+, and they were suppressed by LOE 908 to 0 and 35%, respectively, with the LOE 908-resistant part being abolished by SK&F 96365. 6. These results show that LOE 908 is a blocker of NSCCs without effect on SOCC, and that the increase in [Ca2+]i at lower [ET-1] results from Ca2+ entry through NSCC-1 in addition to VOC, whereas the increase at higher [ET-1] involves NSCC-1, NSCC-2 and SOCC in addition to VOC.

Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells.

1. In A7r5 cells loaded with the Ca2+ indicator fura-2, we examined the effect of a Ca2+ channel blocker SK&F 96365 on increases in intracellular free Ca2+ concentrations ([Ca2+]i) and Mn2+ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed. 2. Higher concentrations (> or = 10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca2+]i: removal of extracellular Ca2+ abolished only the latter. A blocker of L-type voltage-operated Ca2+ channel (VOC) nifedipine at 1 microM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 microM). 3. Lower [ET-1] (< or = 1 nM) evoked only a sustained elevation in [Ca2+]i which depends on extracellular Ca2+. The elevation was partly sensitive to nifedipine but not SK&F 96365. 4. In the presence of 1 microM nifedipine, higher [ET-1] increased the rate of Mn2+ quenching but lower [ET-1] had little effect. 5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of -60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca2+ in the bath solution with Mn2+. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365. 6. In a bath solution containing only Ca2+ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn2+, whereas that at higher [ET-1] was partly sensitive to SK&F 96365. 7. These results indicate that in addition to VOC, ET-1 activates two types of Ca2+-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn2+.

Pharmacological characterization of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using LOE 908 and SK&F 96365.

We have recently shown that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin or ionomycin, whereas SK&F 96365 abolished them. The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or =0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. SK&F 96365 inhibited the LOE 908-resistant contractions induced by higher concentrations of ET-1 with IC50 values similar to those for contractions induced by thapsigargin or ionomycin. These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.

The incidence of reflux oesophagitis after cure of Helicobacter pylori in a Japanese population.

AIM: To investigate the incidence of reflux oesophagitis after antibacterial therapy for Helicobacter pylori infection in our patient population. METHODS: Subjects were 451 H. pylori-infected patients (primary symptom: peptic ulcer disease in 347, nonulcer dyspepsia in 100, and reflux oesophagitis in four): 11 of these patients had reflux oesophagitis on study entry. H. pylori infection was treated by a proton pump inhibitor/amoxycillin-clarithromycin regimen for either 7 or 14 days. Each patient was examined by endoscopy before treatment and more than 6 months after treatment to compare oesophageal findings. In addition, 227 patients were interviewed regarding reflux symptoms, using symptom questionnaires, before and more than 6 months after treatment. RESULTS: Among 440 patients who did not have reflux oesophagitis prior to antibacterial treatment (340 peptic ulcer patients and 100 nonulcer dyspepsia patients), 23 patients whose infection was eradicated developed reflux oesophagitis (5.4%). The 11 patients who had reflux oesophagitis prior to treatment were all successfully cured of infection. Six of these patients showed no change in their oesophagitis, while the condition improved in three and worsened in two. Symptom scores improved in 34 of the 36 patients who reported reflux symptoms. Among 19 patients who showed persistent infection, only one developed reflux oesophagitis (5.2%), while none complained of newly developed symptoms following treatment. CONCLUSIONS: Development of reflux oesophagitis after treatment of H. pylori infection was observed in a Japanese population. However, the incidence of this condition was comparable between those with persistent H. pylori infection and those in whom the infection was eradicated.

Blood and brain tissue gaseous strategy for profoundly hypothermic total circulatory arrest.

Brain tissue carbon dioxide tension, pH, and oxygen tension were measured in dogs undergoing hypothermic circulatory arrest below 20 degrees C with three types of blood gas manipulation. During core cooling, dogs were given pure oxygen (group I, n = 8), 5% carbon dioxide in oxygen (group II, n = 10), or 7% carbon dioxide in oxygen (group III, n = 4). During core cooling, brain tissue carbon dioxide tension decreased significantly in group I. During circulatory arrest, carbon dioxide tension rose by 21.5 mm Hg in group I, 35.3 mm Hg in group II, and 57.0 mm Hg in group III, nearly doubling in each group. From the last 5 minutes of core cooling to the end of rewarming, carbon dioxide tension was significantly higher in groups II and III than in group I. Brain tissue pH fell by 0.33 to 0.35 during 60 minutes of circulatory arrest and did not recover in groups II and III. Brain tissue oxygen tension decreased significantly during the latter two thirds of the circulatory arrest period in all three groups. To reduce progressive tissue hypercapnia and acidosis during and after circulatory arrest, a more hyperventilatory manipulation of blood gases than that achieved by alpha-stat strategy was thought beneficial for core-cooling perfusion.

Interaction of nuclear factors from young and old rat brain regions with regulatory sequences of the D2 dopamine receptor gene promoter.

Alterations in the number or functional state of D2 dopamine receptors have been implicated in the decreased motor abilities associated with normal aging, Parkinson's disease and other neurodegenerative diseases. Previous work has demonstrated a substantial decrease in D2 receptor-containing neurons, receptor proteins, steady-state mRNA levels, and the rate of mRNA synthesis with age in the rat striatum in particular and in mammalian brains in general. These observations suggest that one key area of regulatory control is at the level of transcriptional initiation and/or elongation. In the present study gel mobility shift experiments were used to assess the interaction of nuclear proteins from different rat brain regions with DNA containing putative DNA regulatory sites of the transcriptionally active rat D2 receptor gene promoter. Oligonucleotides containing either of the two SP1 binding sites immediately upstream of the primary transcriptional start site were bound by proteins found in nuclear extracts obtained from rat striatum, hippocampus, cortex, and cerebellum. Extracts from striatum and hippocampus formed predominantly low molecular weight complexes which do not contain SP1, as well as a small amount of high molecular weight complexes which may contain SP1 or an SP1-related protein. Cerebellar extracts formed two similar sets of complexes, but they were formed in roughly equal amounts. Extracts from cortex produced a more involved pattern of complexes, but still formed both high molecular weight complexes which contain SP1 and low molecular weight complexes which do not contain SP1. There were differences in the gel mobility as well as the relative amounts of complexes formed with the two SP1-specific oligonucleotides among different brain regions. With respect to possible age-related changes in transcription of the D2 dopamine receptor gene, there appeared to be no statistically significant difference in the DNA-protein complexes formed with striatal nuclear proteins from a population of young rats versus a population of old rats.

Crystallization of and preliminary crystallographic data for allosteric L-lactate dehydrogenase from Bifidobacterium longum.

L-Lactate dehydrogenase from Bifidobacterium longum aM101-2 was overexpressed in Escherichia coli and then purified. The enzyme was crystallized from a polyethylene glycol 6000 solution by the hanging drop vapor diffusion method. Crystals grown in the presence of NADH (type II), both NADH and oxamate (type III), and NADH, oxamate, and FBP (type IV) were analyzed. All three crystal forms belong to the orthorhombic system, space group P2(1)2(1)2. The cell dimensions of the type II crystals were a = 106.2 A, b = 131.6 A, and c = 63.8 A. Those of the type III and type IV crystals were a = 106.4 A, b = 131.4 A, and c = 63.8 A. The type III crystals diffract X-rays to beyond 2.5 A spacing. The type II and type III crystals were stable as to X-ray irradiation.

Sp family transcription factors regulate expression of rat D2 dopamine receptor gene.

The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. We previously reported that a negative modulator is located between nucleotides -116 and -76 (D2Neg-B) in this gene and that Sp1 as well as another unknown factor bind to this region (Minowa et al., J. Biol. Chem. 269, 11656, 1994). In the present investigation employing the in situ filter detection method, we identified this factor as Sp3. Anti-Sp3 antiserum used in gel-shift assays also revealed that Sp3 binds to the D2Neg-B sequence. Cotransfection of Drosophila Schneider's SL2 cells with Sp3 or Sp1 expression plasmids in the presence of CAT reporter plasmids containing D2 promoter regions demonstrated that Sp1 increased CAT activity in a dose-dependent manner, whereas Sp3, either alone or in the presence of Sp1, failed to activate or repress transcription. On the other hand, using the TATA-containing reporter plasmid BCAT-2, Sp3 coexpression significantly repressed Sp1-induced trans-activation, although Sp3 alone was ineffective. Thus, the transcriptional activity of Sp3 is dependent on the promoter context, and the negative regulation of D2 gene expression appears quite complex and may not depend simply on known DNA-protein interactions involving only Sp1 and Sp3.

Optimal blood flow for cooled brain at 20 degrees C.

BACKGROUND: Optimal conditions for deep hypothermic perfusion and protective brain blood flow remain unclear. METHODS: Dogs (n = 52) underwent 120 minutes of cardiopulmonary bypass at 20 degrees C with perfusion flow rates of 2.5, 5, 10, 20, 40, and 100 mL x kg(-1) x min(-1). We examined the effect of the various flow rates and different perfusion pressures on brain blood flow, metabolism, and intracellular pH. RESULTS: The brain was ischemic and acidotic when the perfusion flow rate was less than 5 mL kg(-1) x min(-1) and pressure was less than 10 mm Hg. When perfusion pressure was higher than 10 mm Hg, cerebral cortex blood flow was more than 9 mL x 100 g(-1) x min(-1) and intracellular pH, higher than 6.95. The cerebral metabolic rate for oxygen decreased at a flow rate of 2.5 mL x kg(-1) min(-1). The cerebral metabolic ratio of glucose to oxygen and the cerebral vascular resistance were lowest when perfusion pressure was 10 to 30 mm Hg. Full-flow (100 mL x kg(-1) x min(-1)) perfusion caused paradoxical brain acidosis; a flow of 40 mL x kg(-1) x min(-1) provided the best results. CONCLUSIONS: Both extremely low-flow perfusion and excessive perfusion cause brain acidosis. Low-flow perfusion at a pressure of 20 mm Hg provides cerebral vasorelaxation and aerobic metabolism during operations at 20 degrees C.

Evaluation of multidisciplinary treatment of bladder cancer, especially in chemoimmunotherapy (ADM and OK-432) as a consolidation therapy.

The relapse rate of bladder cancer (transitional cell Ca) is said to be about 45%-80% even after tumor resection. Multidisciplinary treatment was designed and studied to prevent such recurrence. This treatment was designed to have three steps: induction, consolidation, and maintenance therapy. Following surgical tumor removal, OK-432 and Adriamycin (ADM) were administered as consolidation therapy, followed by administration of PSK and carboquone (CQ) in small amounts as maintenance therapy continuously for about 3 years, and the course was observed. In both consolidation and maintenance groups various non-specific immunoparameters were superior in groups receiving combined immunotherapeutic agents. Thus, the use of immunotherapeutic agents in combination with chemotherapeutic agents was considered to be effective. The 3-year recurrence rate was only 8% in the multidisciplinary treatment group, while that in the non-multidisciplinary treatment group was 61%. This approach, especially with chemoimmunotherapy (ADM and OK-432) as a consolidation therapeutic mode, is therefore considered to be useful for the prevention of recurrence.

Role of nonselective cation channels as Ca2+ entry pathway in endothelin-1-induced contraction and their suppression by nitric oxide.

The present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin- in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imida zole . HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 microM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.

Comparison of the efficacy of 400mg and 800mg of clarithromycin used with lansoprazole and amoxicillin in eradication regimens for Helicobacter pylori infection in a Japanese population.

We conducted a randomized prospective comparative study to determine whether a 1-week lansoprazole-amoxicillin-clarithromycin (LAC) regimen with 800 mg of clarithromycin a day was more effective such a regimen with 400 mg daily in the Japanese population. One hundred and seventy-five Helicobacter pylori-positive patients were randomly assigned to receive one of two different 7-day regimens, one with clarithromycin 400 mg (LAC 400 regimen) and the other with clarithromycin 800 mg (LAC 800 regimen). The cure rates for both regimens were similar, although adverse effects were significantly more frequent in the LAC 800 regimen, suggesting that 400 mg of clarithromycin may be sufficient in our patient population.

Microalgal cultivation in a solution recovered from the low-temperature catalytic gasification of the microalga.

Microalgal cultivation in a solution recovered from the low-temperature catalytic gasification of the microalga itself was studied. The growth of Chlorella vulgaris in 75-300-fold diluted recovered solution containing phosphate, magnesium ions and micro-elements was comparable to that in the standard culture medium. It was suggested that C. vulgaris could use ammonium in the recovered solution as its nitrogen source and at the same time could provide a source of biomass which was recycled via gasification.