The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells.

Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl(-) channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (V te) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 µM, which corresponded to the value obtained on whole-cell Cl(-) currents in Capan-1 single cells. The effects of adenosine on V te, an equivalent short-circuit current (I sc), and whole-cell Cl(-) currents were inhibited by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitor. The adenosine A2B receptor agonist, BAY 60-6583, increased I sc and whole-cell Cl(-) currents through CFTR Cl(-) channels, whereas the A2A receptor agonist, CGS 21680, had negligible effects. The A2B receptor antagonist, PSB 603, inhibited the response of I sc to adenosine. Immunohistochemical analysis showed that the A2A and A2B receptors colocalized with Ezrin in the luminal membranes of Capan-1 monolayers and in rat pancreatic ducts. Adenosine elicited the whole-cell Cl(-) currents in guinea pig duct cells. These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl(-) channels via adenosine A2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion.

Gas Exchanges and Dehydration in Different Intensities of Conditioning in Tifton 85 Bermudagrass: Nutritional Value during Hay Storage.

The present study aimed at evaluating the intensity of Tifton 85 conditioning using a mower conditioner with free-swinging flail fingers and storage times on dehydration curve, fungi presence, nutritional value and in vitro digestibility of Tifton 85 bermudagrass hay dry matter (DM). The dehydration curve was determined in the whole plant for ten times until the baling. The zero time corresponded to the plant before cutting, which occurred at 11:00 and the other collections were carried out at 8:00, 10:00, 14:00, and 16:00. The experimental design was randomised blocks with two intensities of conditioning (high and low) and ten sampling times, with five replications. The high and low intensities related to adjusting the deflector plate of the free iron fingers (8 and 18 cm). In order to determine gas exchanges during Tifton 85 bermudagrass dehydration, there were evaluations of mature leaves, which were placed in the upper middle third of each branch before the cutting, at every hour for 4 hours. A portable gas analyser was used by an infrared IRGA (6400xt). The analysed variables were photosynthesis (A), stomatal conductance (gs), internal CO2 concentration (Ci), transpiration (T), water use efficiency (WUE), and intrinsic water use efficiency (WUEi). In the second part of this study, the nutritional value of Tifton 85 hay was evaluated, so randomised blocks were designed in a split plot through time, with two treatments placed in the following plots: high and low intensity of cutting and five different time points as subplots: cutting (additional treatment), baling and after 30, 60, and 90 days of storage. Subsequently, fungi that were in green plants as well as hay were determined and samples were collected from the grass at the cutting period, during baling, and after 30, 60, and 90 days of storage. It was observed that Tifton 85 bermudagrass dehydration occurred within 49 hours, so this was considered the best time for drying hay. Gas exchanges were more intense before cutting, although after cutting they decreased until ceasing within 4 hours. The lowest values of acid detergent insoluble nitrogen were obtained with low conditioning intensity after 30 days of storage, 64.8 g/kg DM. The in vitro dry matter of Tifton 85 bermudagrass did not differ among the storage times or the conditioning intensities. There was no fungi present in the samples collected during the storage period up to 90 days after dehydration, with less than 30 colony forming units found on plate counting. The use of mower conditioners in different intensities of injury did not speed up the dehydration time of Tifton 85.

A novel predictive method for assessing the quality of isolated pancreatic islets using scanning electrochemical microscopy.

INTRODUCTION: The current methods for evaluating islet potency are not useful in clinical transplantation. Therefore, we need reliable, rapid methods enabling accurate prediction of islet quality. MATERIALS AND METHODS: We evaluated respiratory activity using scanning electrochemical microscopy (SECM), glucose-stimulated respiratory activity, glucose-stimulated insulin release, ADP/ATP assays, insulin/DNA levels, and Trypan blue exclusion tests as predictive methods for the ability of isolated rat islets to cure syngeneic diabetic rats. RESULTS: Although glucose-stimulated respiratory activity, basal respiratory activity, ADP/ATP ratio, and glucose-stimulated insulin release were significantly correlated with the outcome of transplantation into diabetic rats, there was no correlation between outcomes, insulin/DNA ratios, and Trypan blue exclusion tests. The glucose-stimulated respiratory activity in islet preparations that could cure diabetic rats was significantly greater than those unable to cure diabetes. Rat islets with >1.5-fold glucose-stimulated respiratory activity consistently cured diabetic rats, whereas those with a value <1.5 hardly cured any rats. CONCLUSION: Measurement of the glucose-stimulated respiratory activity using SECM technique is a novel method that may be useful as a rapid, potent predictor of the outcome of clinical islet transplantation.

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma.

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line.

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.

Visible-light-controlled homo- and copolymerization of styrenes by a bichromophoric Ir-Pd catalyst.

Visible-light-controlled polymerization was achieved by a bichromophoric organopalladium catalyst which possesses a naphthyl-substituted cyclometallated Ir(III) light-absorbing moiety. The complex was highly active toward styrene polymerization upon visible-light irradiation, and its photoactivity toward polymerization and dimerization was switchable. On the basis of the switching activity, controlled copolymerization of styrene and vinyl ether was achieved upon photo-irradiation to give the corresponding copolymers.

Clinical study of tympanostomy tube placement for patients with intractable Ménière's disease.

OBJECTIVE: To evaluate the effectiveness of tympanostomy tube placement in controlling symptoms of intractable Ménière's disease. METHODS: Fifteen patients with intractable Ménière's disease underwent tympanostomy tube placement in the affected ear. Post-operative changes in vertigo attacks and hearing level were recorded, and were evaluated according to American Academy of Otolaryngology-Head and Neck Surgery criteria. RESULTS: At 12 months after treatment, 3 patients (20 per cent) showed complete control of vertigo, 7 (47 per cent) showed substantial control and 2 (13 per cent) showed limited control; 3 patients (20 per cent) required other treatment. At 24 months after treatment, 7 patients (47 per cent) showed complete control of vertigo, 3 (20 per cent) showed substantial control and 1 (7 per cent) showed limited control; 1 patient required other treatment 15 months after tympanostomy tube placement. CONCLUSION: There is no definite pathophysiological explanation for the effect of tympanostomy tube placement in reducing vertigo attacks. This treatment is not effective for all patients with intractable Ménière's disease. However, tympanostomy tube placement might be an additional surgical therapeutic option to consider prior to contemplating other, more invasive treatments.

Molecular cloning of the rat thyroxine-binding globulin gene and analysis of its promoter activity.

We cloned the rat T4-binding globulin (rTBG) gene, characterized its organization, and studied its promoter activity and regulation. A genomic DNA library was constructed and screened using an rTBG complementary DNA (cDNA) as a probe. An 8.6-kilobase pair (kbp) clone was partially sequenced and compared with the sequence of the previously cloned cDNA. It helped complete the cDNA sequence and identify the first noncoding exon (exon 0). The transcription start site was identified using an RNase protection assay. The rTBG genomic clone contained 1.2 kbp 5'-flanking and 1.7 kbp 3'-flanking regions. The sizes of exons and introns of the rTBG gene are similar to those of the human TBG gene, belonging to the serine protease inhibitor family. The 5'-flanking region contains a TATA box, a CAAT box, and a consensus sequence for the hepatocyte nuclear factor 1-binding site. We tested the promoter activity of the 1.2-kbp 5'-flanking region using a luciferase reporter plasmid. When transfected into a hepatocyte-derived cell line (HepG2), the plasmid construct containing the fragment -1227 to +11 (transcription start site, +1) showed a 9-fold increase in luciferase activity compared with that of a promoterless luciferase vector. No promoter activity was detected in a nonhepatocyte-derived cell line (COS1). Serial 5'-deletion revealed that the construct containing the fragment -180 to +11 had 40% of the maximal promoter-induced luciferase activity. And that containing the fragment -53 to +11 showed no significant increase of luciferase activity. These results suggest that the region -180 to -53, containing hepatocyte nuclear factor 1-binding site, is essential to the liver-specific expression. We previously reported down-regulation of rTBG messenger RNA by T3 in vivo. The present study failed to show T3's effect on the promoter activity of the 1.2-kbp 5'-flanking region of the rTBG gene.

A novel mutation causing complete thyroxine-binding globulin deficiency (TBG-CD-Negev) among the Bedouins in southern Israel.

T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon 1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD.

Gene screening of thyroxine-binding globulin (TBG) deficiencies in the Japanese: only two mutations account for TBG deficiencies in the Japanese.

T4-binding globulin (TBG) is the principal transport protein for thyroid hormone in the circulation. Twelve mutations in the human TBG gene have been reported, and the inheritance of those variant TBGs was shown to be X-chromosome linked. We previously reported a nucleotide deletion at the codon 352 (TBG-CDJ) and a nucleotide substitution at the codon 362 (TBG-PDJ) of the TBG gene in a Japanese male manifesting TBG complete deficiency and partial TBG deficiency, respectively. In this communication we investigate the prevalence of both mutations among 50 unrelated Japanese subjects manifesting complete or partial TBG deficiency from various areas of the Japanese Archipelago. Mutant alleles were identified by amplification of their genomic DNAs by PCR with allele-specific primers. In addition, the presence of a polymorphic mutation in codon 283 (TBG-Poly) in these variants was investigated. All male subjects manifesting complete TBG deficiency (n = 30) and all female subjects manifesting partial TBG deficiency (n = 4) were demonstrated to be hemizygotes and heterozygotes for the mutation of TBG-CDJ, respectively. All male subjects manifesting partial TBG deficiency (n = 16) were shown to have the mutation of TBG-PDJ as hemizygotes. TBG-Poly was consistently absent from these variants. We conclude that only TBG-CDJ and TBG-PDJ may account for complete and partial TBG deficiencies in the Japanese.

Dopamine transporter gene polymorphism and psychiatric symptoms seen in schizophrenic patients at their first episode.

To investigate the possible role of the dopamine transporter (DAT) gene in determining the phenotype in human subjects, allele frequencies for the 40-bp variable number of tandem repeats (VNTR) polymorphism at this site were compared between 117 Japanese normal controls and 118 schizophrenic patients, including six subgroups: early-onset, those with a family history, and those suffering from one of the following psychiatric symptoms at their first episode: delusion and hallucination; disorganization; bizarre behavior; and negative symptoms. No significant differences were observed between the group as a whole or any subgroup of schizophrenic patients and controls. The results indicate that VNTR polymorphism in the DAT gene is unlikely to be a major contributor to any of the psychiatric parameters examined in the present population of schizophrenic subjects.

Dopamine D3 receptor gene polymorphism and the psychiatric symptoms seen in first-break schizophrenic patients.

To investigate the possible effect of polymorphism at the BalI site of the dopamine D3 receptor gene (DRD3) on the phenotype in human subjects, allele and genotype frequencies for this polymorphic site were examined in 113 schizophrenic patients, including six subgroups, and 48 normal controls. The schizophrenic subgroups included patients with early onset, those with a family history, and those who suffered from one of the following psychiatric symptoms at their first episode: (1) delusion and hallucination; (2) disorganization; (3) bizarre behavior; and (4) negative symptoms. No significant differences were observed in genotype, allele and homozygosity frequencies between the whole group or any subgroup of schizophrenic patients and the controls. The present results indicate that polymorphism at the BalI site of the DRD3 is unlikely to be a major contributor to any of the psychiatric parameters examined in the present population of schizophrenic subjects.

Age-associated decrease in response of rat aquaporin-2 gene expression to dehydration.

It is well known that urine-concentrating ability decreases with aging and that this decreasing ability results from a reduced sensitivity of the renal collecting duct to arginine vasopressin (AVP). AVP regulates the water channel (aquaporin-2:AQP2) through V2 receptors and increases the water permeability of the collecting duct. To elucidate the mechanism of change with aging in urine-concentrating ability, we investigated the change of V2 receptor and AQP2 mRNA expression in young (8-week-old) and older (7-month-old) rats after dehydration for 2 days. After dehydration, plasma AVP levels in older rats were higher than young rats, and urinary osmolality in older rats was lower than young rats. By Northern blot analysis, there was no significant difference between young and older rats in both V2 receptor and AQP2 mRNA expression before dehydration. After dehydration, V2 receptor mRNA expression in young and older rats decreased in the same degree, suggesting the downregulation of V2 receptors may occur in the mRNA level. Northern blot analysis and in situ hybridization histochemistry showed that AQP2 mRNA expression increased and the increased expression in older rats was less than in young rats. The present study suggests the reduced response of AQP2 mRNA expression to dehydration, observed in 7-month-old rats, might be partially responsible for the decreasing urine-concentrating ability with aging.

Effects of self-etching primers on dentin.

The effects of self-etching dentin primers on the hardness of dentin was evaluated by determination of the Vickers Hardness of both untreated dentin and dentin treated with different primers. Several experimental primers were prepared by dilution of dicarbonates or their esterified salts with different ratios of HEMA to obtain ratios of 5% and 35% by weight. Methacryloxyethyl succinate (MES) and Methacryloxyethyl phthalate (MEP) experimental primers caused less softening of dentin than did maleic acid diluted with HEMA or a commercial dentin primer (Scotchprep, 3M). A high correlation was observed between the pH of a primer and the hardness of dentin treated with the primer. A correlation was also observed between the hardness of treated dentin and the wall-to-wall polymerization contraction of a light-curing microfilled resin (Silux, 3M).

[Blood biochemical properties in male workers analysed according to body type].

The characteristics of body type, serum lipids and blood pressure in male blue workers were evaluated. The subjects were 1,705 healthy men aged 40.3 +/- 4.33 years, and were classified into 4 groups according to body mass index: BMI; lean (group L: BMI < 20), normal (group N: 20 < or = BMI < 24), mildly obese (group MO: BMI 24 to 26.5) and obese group (group O: BMI > 26.5). The physical characteristics, blood pressure and 14 blood constituent items were measured. Groups L, N, MO and O accounted for 8.5, 53.4, 28.6, and 9.4% of all subjects, that is the obese body type (MO and O groups) occupied 38%. Rest systolic and diastolic blood pressure were positively related to BMI. Most blood constituent items biochemically analyzed increased in proportion to BMI values. The Trigriceride level was 88.5, 112.2, 152.8 and 171.9 mg/dl in groups L, N, MO and O, respectively. The Trigriceride level in groups MO and O were about the upper limit of the normal range. The total cholesterol level was in the normal range in 4 groups, but was higher in the MO and O groups than in groups L and N. The HDL-cholesterol level was low in inverse proportion to BMI, that is 58.2, 51.3, 47.6, and 42.6 mg/dl in groups L, N, MO, and O, respectively. Especially the values in group O were similar to the values in the aged and adult disease patients. A greater percentage of Japanese male workers were obese men and they risked developing atherosclerosis and adult diseases. the present study suggests that health education regarding exercise prescription and nutrition intake in daily life should be provided.

[Ifosfamide in the treatment of small cell carcinoma of the lung].

A total of 36 cases with small cell carcinoma of the lung were treated with Ifosfamide. Fourteen cases (38.9%) out of 36 cases showed good or marked clinical response, and 120 mg/kg of Ifosfamide was necessary as a minimum dose to obtain effective response. As side effects of Ifosfamide, gastrointestinal disturbance (66.7%), depilation (66.7%), leukopenia (38.9%) and hematuria (36.1%) were observed. Mean survival time (M.S.T.) was prolonged by using Ifosfamide, comparing with the groups treated by with other anticancer drugs. Ifosfamide, therefore, should be used for the treatment of small cell carcinoma of the lung.

[Autopsy findings of pontine lesions in the elderly].

The characteristics of multiple spongy necrosis of the basis pontis (MSN), central pontine myelinolysis (CPM), and cerebrovascular disease (CVD) of the pons developing in the elderly have not been fully clarified. We therefore studied 305 patients (aged 60-107, mean: 83.9) autopsied in Nagoya City Kohseiin Geriatric Hospital. MSN was found in four patients (1.3%). The major histologic finding was multiple necrosis of the basis pontis, characterized by loss of myelin and axons, no reactive astrocytes or inflammatory cells were found. Small foci of spongy necrosis appeared to unite with each other to form larger lesions. The central basis pontis was weakly stained by Klüver-Barrera stain in 15 patients, which suggested the presence of CPM. However, the diagnosis was only confirmed in one patient, (by the myelinolysis, loss of oligodendroglia, infiltration of macrophages, and reactive astrocytosis). With regard to CVD, none of the 305 patients had macroscopic pontine hemorrhage, but histologically small and old hemorrhagic lesions were found in 15. These lesions were associated with hypertensive or arteriosclerotic changes in the pontine vessels. Pontine infarction was evident in 77 patients (25.2%). In most lesions (74%) the area of infarction was smaller than 1 mm2. In 27 of the 74 patients with lacunar or microscopic infarcts, the pontine infarcts were found in areas where the blood was supplied through the short circumferential branch.

[Role of institutions for the elderly in senile dementia].

The purpose of this study was to assess the role of institutions for the elderly, and special nursing homes in particular, with regard to senile dementia. The subjects consisted of 81 cases who were institutionalized in our special nursing home from early November, 1992 to late October, 1993. Of these, 37 cases (45.7%) showed clinical dementia at the time of institutionalization. The severity and respective percentages of dementia were as follows: slight degree 32.4%, moderate degree 27.0% and severe degree 40.6%. The severity of dementia demonstrated significant correlation with age. Hasegawa's Dementia Scale, activity of daily living and physical conditions. The admission rate (40.5%) of the demented group for physical diseases was significantly higher than that (22.7%) of the non-demented group. The majority of families of both groups first sought advice at institutions other than our special nursing home and the welfare office in the city, prior to being institutionalized. The referral rate from such institutions was far higher than that for direct entry from their homes. We consider that special nursing homes, in addition to carrying for daily living requirements, should pay great attention to physical diseases in patients suffering from senile dementia, and cooperate with medical institutes, particularly referring hospitals, and other welfare and health facilities, in this regard.