Insulinoma induces a hyperinsulinemia-mediated decrease of GLUT2 and GLP1 receptor in normal pancreatic ß-cells.

There have been several clinical reports of transient postoperative hyperglycemia in patients with insulinoma, but the effect of insulinoma on normal ß-cells has not been investigated. We examined the glucose transporter 2 (GLUT2) and glucagon-like peptide 1 receptor (GLP1R) expression in normal pancreatic ß-cells of five patients with insulinoma and five patients with normal glucose tolerance (NGT) as controls. The positive rate of GLUT2-or GLP1R-positive islets in the nontumor area was calculated by the ratio with the analyzed islets. For functional in vitro analyses, q-PCR and Western blotting were performed after insulin loading on MIN6 cells. The expression rates of both GLUT2 and GLP1R were significantly lower in nontumor area islets of insulinoma patients than in patients with NGT (GLUT2: 31.6 ± 15.3% vs 95.9 ± 6.7%, p < 0.01, GLP1R: 66.8 ± 15.0% vs 96.7 ± 5.0%, p < 0.01). Exposure of MIN6 cells to high concentrations of insulin resulted in a significant decrease in GLUT2 protein for 12 h and GLP1R protein for 24 h (GLUT2; 1.00 ± 0.079 vs 0.81 ± 0.04. p = 0.02, GLP1R; 1.00 ± 0.10 vs 0.50 ± 0.24, p = 0.03) but not in those mRNAs. Our findings show that insulinoma is associated with the downregulation of GLUT2 and GLP1R expression in nontumor area islets. These phenomena may be caused by high levels of insulin.

Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. MATERIAL AND METHODS: Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. RESULTS: Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. CONCLUSION: Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.

Wound healing effects of gingival fibroblasts cultured in animal-free medium.

OBJECTIVE: The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal-free medium (HFDM1). METHODS: We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. RESULTS: The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5-2% was added to HFDM1. Wound healing cytokines, including transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL-6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. CONCLUSION: Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.

Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans.

INTRODUCTION: The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated. METHODS: Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. RESULTS: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems. CONCLUSION: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

Prognostic significance of germ line polymorphisms of the CYP1A1 and glutathione S-transferase genes in patients with non-small cell lung cancer.

CYP1A1 is responsible for the metabolic activation of benzo(a)pyrene in cigarette smoke, and high susceptibility to smoking-related lung cancer has been associated with the MspI polymorphism of the CYP1A1 gene. Individuals with a susceptible CYP1A1 genotype have been found to be at remarkably high risk when the genotype is combined with a deficient Mu-class glutathione S-transferase (GSTM1) genotype. In this study, we investigated the relationship between germ line polymorphisms of these genes and clinical characteristics or survival rates in 232 patients with non-small cell lung cancer (NSCLC). Statistical analysis revealed a significant association (P < 0.05) of the MspI polymorphism of the CYP1A1 gene with histological type, performance status (general conditions of patients), and the extent of the primary tumor (T-factor). On the other hand, the GSTM1 polymorphism was significantly associated with performance status, the extent of regional lymph node metastasis (N-factor), and the extent of distant metastasis (M-factor). NSCLC patients with at least one susceptible allele of the MspI polymorphism of the CYP1A1 gene [heterozygous genotype B or a rare homozygous genotype C; n = 131; median survival time (MST) = 24.2 months] were associated with a shortened survival compared with those with nonsusceptible homozygous alleles (genotype A; n = 101; MST = 65.2 months; P = 0.005 by log-rank test). Smokers with susceptible genotypes (n = 104; MST = 18.2 months) were markedly associated with a shortened survival compared with those with genotype A (n = 76; MST = 69.2 months; P = 0.024); such an association was not found among nonsmokers by genotypes. Genotype-dependent survival was also observed in patients at an advanced stage of disease (P = 0.010), but not in those at an early stage of disease (P = 0.382). Patients with the susceptible CYP1A1 genotype had remarkably shortened survivals when the genotype was combined with a deficient genotype GSTM1(-) (P = 0.017; degree of freedom = 3). Multivariate analysis by the Cox proportional hazards model also revealed that the CYP1A1 polymorphism was an independent prognostic factor in patients at a nonresectable advanced stage of NSCLC (P = 0.005; hazard ratio = 1.98; 95% confidence interval, 1.24-3.17).

Binding sites for the (Hg-Se) complex on selenoprotein P.

The mechanism underlying the interaction between mercury (Hg), selenium (Se) and selenoprotein P (Sel P) in the bloodstream has been explained by the formation of the [(Hg-Se)n]m-Sel P complex. In the present study, the binding sites for the (Hg-Se)n complex on Sel P were studied by competitive assay of the binding of the (Hg-Se)n complex to Sel P with polymeric and monomeric amino acids with simultaneous detection of the Hg, Se of selenite origin and Se of Sel P origin by the high performance liquid chromatography-inductively coupled argon plasma-mass spectrometry method. The specific binding of the (Hg-Se) complex but not Hg2+ or selenide to Sel P was explained by the unique binding sites consisting of the cationic and anionic ends such as imidazolyl and selenol groups on Sel P, respectively. The number, n, in the (Hg-Se)n complex was estimated to be approx. 100, while the number, m, in the [(Hg-Se)n]m-Sel P complex was estimated to be 35. The formation of the unit complex (Hg-Se)100, followed by its binding to Sel P at up to the 35 binding sites on Sel P was suggested.

Phase II study of 3-hour infusion of paclitaxel in previously untreated non-small cell lung cancer.

Paclitaxel has clinical activity in non-small cell lung cancer, with response rates of 21 and 24% in a 24-h infusion. Recent clinical studies have shown that a 3-h infusion of the drug with premedication did not result in hypersensitivity reactions, and that neutropenia was milder in the 3-h than in the 24-h schedule. In this Phase II study, we tried to evaluate the efficacy and toxicity of paclitaxel given over 3 h in patients with previously untreated, unresectable stage III or IV non-small cell lung cancer. In addition, we attempted to investigate the pharmacokinetics and pharmacodynamics of the drug. Paclitaxel was administered i.v. over 3 h at a dose of 210 mg/m2 every 3 weeks with premedication of dexamethasone, ranitidine, and diphenhydramine. Heparinized blood samples were obtained from 12 patients for pharmacokinetic studies. Twenty-three (38%) of 60 assessable patients achieved a partial response, with a median duration of 3.2 (range, 2.3-11.1) months. The median survival for all patients was 11.2 months, and the 1-year survival rate was 48%. Thirty (50%) patients developed grade 4 neutropenia. Nonhematological toxicities were mild, except for pulmonary toxicity in one (1.7%) patient who required mechanical ventilatory support for 4 days. The duration of the paclitaxel concentration above 0.1 microM correlated well with the percentage of decrease in the absolute neutrophil count. In conclusion, a 3-h infusion of paclitaxel was safe and probably not less effective than a 24-h infusion.

Breath alcohol concentrations in Japanese outpatients following paclitaxel and docetaxel infusion.

Patients receiving paclitaxel or docetaxel also receive a significant amount of ethanol, as both products contain ethanol as solvent. Patients in our clinics have occasionally exhibited signs of alcohol intoxication immediately after paclitaxel infusion. In 2002, the Japanese government lowered the minimum ethanol concentration for the definition of drunk driving, with the threshold breath alcohol concentration (BRAC) of 0.15 mg/l. The aim of this study was to measure BRAC in Japanese outpatients treated with paclitaxel or docetaxel and to assess intoxication according to this standard. Fifty-two Japanese patients were enrolled from October 2003 to February 2004. Patient characteristics were as follows: male/female, 13/39: median age, 71 (range: 34-78); breast/lung/ovarian cancer 24/16/12; and paclitaxel/docetaxel treatment: 36/16, respectively. The mean total doses of paclitaxel or docetaxel were 178 mg (range: 107-300) and 53 mg (30-100), respectively. Breath samples were measured three times immediately following the infusion of paclitaxel or docetaxel via ethyl alcohol detector and the mean value was recorded. BRAC was detected in 20 patients (56%) with paclitaxel and in none of the docetaxel patients. BRAC was measured again 30 min after the initial measurement in BRAC-detected cases with the patients' permission. In four of six BRAC-remeasured patients, BRAC became undetectable after 30 min. There was no correlation between the total doses of paclitaxel and BRAC or between the infusion rates of paclitaxel and BRAC. In conclusion, clinicians should recognize the potential for alcohol intoxication with paclitaxel administration. Patients should be instructed to avoid driving on the day of paclitaxel administration.

Scanning electron microscopic studies on cultured heart cells after the induction of arrhythmias and fibrillation.

Scanning electron microscopic observations were made of mouse embryonic heart cells in culture to clarify the interrelationship between morphological damage and cardiac arrhythmias and fibrillation induced by changes of ion concentrations. They were studied with particular reference to the abnormalities in the heart cell and the intercellular connections. The cells exhibited arrhythmias with any alteration in ion concentration (MgSO4, CaCl2, KCl, NaCl) in the culture medium, and they developed fibrillation-like beating under certain conditions of lowered potassium, or sodium, and increased sodium with a reduction in calcium or potassium. The heart cells showing arrhythmias were revealed by scanning electron microscopy to have more or less fine morphological changes in their slender processes, and detached or raised cell edges. Fold-like undulations in the cell body and cell ledge were also numerous in cells exhibiting fibrillation-like beating. These cell body changes may, however, be characteristic of cells maintained in cultures with lowered potassium content, since only these cultures showed such changes. Changes of cell ledge were also demonstrable in cultures with altered MgSO4, CaCl2 content or increased KCl or NaCl content although the cells in these cultures did not develop fibrillation. It was noted that there was a tendency for the changes of cell body and cell edge to be conspicuous in cells exhibiting fibrillation. The results obtained indicated that fibrillation developed only in the presence of altered concentrations of certain ions, in association with subtle morphological changes in the cells and intercellular connections.

Why are infant gerbils more resistant than adults to cerebral infarction after carotid ligation?

Younger gerbils have been found to be more resistant than adults to cerebral infarction after carotid ligation. In this study, the perfused cerebral area after bilateral common carotid occlusion was evaluated in infant, young, and adult Mongolian gerbils by the carbon black perfusion method to assess the existence and significance of collateral blood vessels between the vertebrobasilar and carotid circulations. Nineteen gerbils were divided into three groups (i.e., infant, young and adult gerbils aged 3-4, 5-7, and 10-17 weeks, respectively). After bilateral common carotid artery occlusion, carbon black was injected directly into the left ventricle by cardiac puncture through the closed thorax. In five of eight infant gerbils, the whole brain was perfused by carbon black, while in the remaining three, only the cerebellum and brainstem were stained well, and marked bilateral cerebral pallor was observed. On the other hand, carbon black did not perfuse the brain region supplied by the carotid arteries, both in young and adult gerbils (11 animals in total). These results suggest that infant gerbils might have a more highly developed network of collateral blood vessels between the vertebrobasilar and carotid circulations, and the existence of such a significant network might be the basis for the fact that infant gerbils are resistant to cerebral infarction following carotid ligation. We propose that gerbils should be used as a stroke model only when they are 5 weeks old or older.

Effects of hematocrit variations on cerebral blood flow and oxygen transport in ischemic cerebrovascular disease.

The contribution of hematocrit (Ht) changes on cerebral blood flow (CBF) and brain oxygenation in ischemic cerebrovascular disease is still controversial. In the present study, effects of Ht variations of CBF and oxygen delivery were investigated in patients with ischemic cerebrovascular disease. CBF was measured by the Xe-133 intracarotid injection method in 27 patients, whose diagnoses included completed stroke, reversible ischemic neurological deficit, and transient ischemic attack. Ht values in the patients ranged from 31 to 53%. There was a significant inverse correlation between CBF and Ht in these Ht ranges. Oxygen delivery, i.e., the product of arterial oxygen content and CBF, increased with Ht elevation and reached the maximum level in the Ht range of 40-45% and then declined. The CBF-Ht and oxygen transport-Ht relations observed in our study were similar to those in the glass-tube model studies by other workers rather than to those in intact animal experiments. From these results, it is conceivable that in ischemic cerebrovascular disease, the vasomotor adjustment was impaired in such a manner that the relations among Ht, CBF, and oxygen delivery were different from those in healthy subjects. Further, an "optimal hematocrit" for brain oxygenation was also discussed.

Immunoreactive glucagon in the smooth muscle cells of the rat cerebral artery: an immunohistochemical analysis.

The presence of immunoreactive glucagon (IRG) in a smooth muscle of the cerebral arteries of rats was demonstrated immunohistochemically using two antisera against pancreatic glucagon, OAL -123 and Unger 's 30K . Based on the results and on our previous radioimmunoassay and gel filtration study, the smooth muscle cells of the blood vessels may be one of the extrapancreatic sources of IRG in the plasma.

Reversal of neurological deficits by levallorphan in patients with acute ischemic stroke.

This study was conducted to examine the effect of the intramuscular injection of levallorphan tartrate (1.0 mg), a mixed agonist-antagonist opiate, on the neurological signs, symptoms, and vital signs in 19 patients with acute ischemic stroke. A temporary improvement of hemiplegia or hemiparesis was observed within several minutes after levallorphan injection in 13 of the patients. There were no significant alterations in blood pressure or pulse rate after injection. The findings indicate that levallorphan may have a temporary improving effect on neurological deficits in acute ischemic stroke. In addition, observation of the response to levallorphan may serve to predict the prognosis of the final neurological outcome in this type of patient.

Importance of folded monomer and extended antiparallel dimer structures as enkephalin active conformation. Molecular dynamics simulations of [Met5]enkephalin in water.

Simulations of the molecular dynamics of the [Met5]enkephalin monomer and dimer structures in water have been carried out. The dynamic trajectories have been analyzed in terms of the distances between intra- or intermolecular polar atoms. The time-correlated conformational transitions of an extended monomer structure have been converged into a stationary state among the beta-bend folded forms. However, the dynamics simulation of an extended antiparallel dimer structure has shown no noticeable conformation change. These results imply that both the beta-bend monomer and the extended dimer structures exist together as the fundamental conformation of enkephalins.

Fine structure of peptidergic and catecholaminergic nerve fibers in the anterior cerebral artery and their interrelationship: an immunoelectron microscopic study.

This study shows (1) the ultrastructure of vasoactive intestinal polypeptide (VIP)-, substance P (SP)-, and neuropeptide Y (NPY)-containing nerve fibers in the walls of the cerebral arteries and (2) the relationship between these peptidergic (VIP, SP, and NPY) and catecholaminergic (CA) nerve terminals by immunohistochemistry combined with false transmitter (5-hydroxydopamine) histochemistry under the electron microscope. VIP-, SP-, and NPY-like immunoreactivity (VIPI, SPI, and NPYI) were found diffusely in the axoplasm and around the small clear vesicles in the nerve terminals. In a few cases, SPI was found within the large vesicles. Most of the VIPI terminals were ensheathed by the cytoplasm of the Schwann cells together with CA terminals, identified as those with a number of small granulated vesicles. In some cases, they were directly apposed to the smooth muscle cells at a distance of about 100 nm. SPI terminals were frequently solitary but about 30% were located together with CA and other (neither SPI nor CA) terminals ensheathed by Schwann cells, directly apposed to the smooth muscle cells at a distance of about 100 nm. On the other hand, NPYI terminals were also identified as CA terminals, indicating the coexistence of these two substances. These findings suggest a close interrelationship between peptidergic and CA nerve terminals in the neurogenic control of cerebral blood vessel function.

Multiple brain gas embolism after ingestion of concentrated hydrogen peroxide.

We present a 63-year-old man who developed multiple brain infarction after ingesting a 35% hydrogen peroxide solution. Neurologic examination revealed left hemiparesis, primarily affecting the lower limb, and mild weakness of the right lower limb. Gadolinium-enhanced MRI revealed patchy bilateral brain lesions. Oxygen gas embolization is the likely cause of the brain infarctions.

A cross-validation of the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQ-C30) for Japanese with lung cancer.

The EORTC QLQ-C30 was developed in English-speaking cultures. To determine if this instrument could cross a broad cultural divide and be used in Japan, the cross-cultural validity of its Japanese version was estimated. In evaluating psychometric testing, internal consistency by Cronbach's alpha, item-discrimination by multitrait scaling analysis, and validity analysis with ECOG performance score (PS) and Karnofsky Performance Status Scale (KPS) were performed. The QLQ-C30 (version 1.0) was given to 105 patients with lung cancer. Although the response rate was low in patients with PS 4, the questionnaire was well accepted by patients with PS 0-3. The Japanese QLQ-C30 has a weak scale of role functioning in terms of item discriminative validity. It also has a weak scale of cognitive functioning in items of discriminative validity and internal consistency. However, known-groups comparison showed the expected clinical validity with PS for all the scales except for financial impact, and longitudinally clinical validity with KPS was shown in scales of cognitive functioning, fatigue, and nausea and vomiting. Multitrait scaling analysis showed that the predicted scales constituting quality of life (QOL) in the English-speaking culture were extracted from the Japanese QLQ-C30, and found to be valid in Japan, indicating its possible usefulness as an instrument that is universally applicable across cultures.

Centrally-induced vasodepressor responses to diltiazem, a calcium channel blocker, in rats.

The effects of a calcium channel blocker, diltiazem, on central cardiovascular regulation were investigated by injecting it intracerebroventricularly (i.c.v.) in urethane-anaesthetized rats. The blood pressure decreased immediately after the injection returning to baseline level 20-30 min later. Both heart rate and abdominal sympathetic nerve activity decreased correspondingly. Diltiazem injected intravenously (i.v.) decreased both blood pressure and heart rate without affecting sympathetic nerve firing. Although the central pressor responses to carbachol and prostaglandin E2 were not affected by i.c.v. pretreatment with diltiazem, diltiazem attenuated the pressor responses to angiotensin II. Furthermore, electrical lesioning of the anteroventral third ventricle (AV3V) area significantly attenuated the depressor responses to diltiazem injected i.c.v. These results suggest that diltiazem injected i.c.v. affects the central nervous system to decrease sympathetic outflow, and thereby to attenuate the central vasopressor effects of angiotensin II in the brain AV3V area.

Overall distribution of vasoactive intestinal polypeptide-containing nerves on the wall of cerebral arteries: an immunohistochemical study using whole-mounts.

The overall distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity on the wall of the cerebral arteries, including its 3-dimensional profile, was investigated by means of the indirect immunofluorescence method using flat-mounts. VIP-immunoreactive fibers run spirally on the wall of the cerebral arteries. On the wall of the large arteries, such as the vertebral artery, basilar artery, internal carotid artery, within and/or without the circle of Willis, posterior and anterior communicating arteries, proximal parts of anterior, mid and posterior cerebral arteries, these fibers are richly distributed and show a dense grid-like appearance. The highest density was identified on the wall of the anterior cerebral artery, internal carotid artery and anterior communicating artery, while the lowest density was on the posterior communicating artery. On the other hand, on the walls of the branches of these arteries or along distal parts of the anterior, mid and posterior cerebral arteries, the number of VIP-immunoreactive fibers decreased markedly.

Demonstrating patency of STA-MCA anastomosis with Tc-99m albumin microspheres.

A patient with occlusion of the left internal carotid artery underwent an anastomosis between branches of the left superficial temporal and middle cerebral arteries (STA-MCA). A left carotid angiogram 9 days later showed extreme, tapered narrowing of the involved STA branch, with no filling of the MCA from the bypass. A year later, symptoms had improved slightly, and a minimally invasive study was made with Tc-99m human albumin microspheres. This showed patency of the bypass, with intracranial perfusion in the MCA field, findings later confirmed by a repeat left carotid angiogram. It appears that Tc-99m HAM scintigraphy will be useful in assessing the effectiveness of this and other bypass operations.