Thymidine kinase-1/CD31 double immunostaining for identifying activated tumor vessels.

Although angiogenesis plays a crucial role in cancer growth and progression, no reliable method for assessing angiogenesis in tumor tissue sections currently is available. Using biomarkers with high specificity for proliferating endothelial cells could help quantify angiogenic activity. Thymidine kinase-1 (TK1) is an enzyme involved in the salvage pathway of DNA synthesis and its activity is correlated with cell proliferation. We investigated the use of double immunostaining for TK1 and CD31 for identifying activated tumor vessels. Differences in TK1/CD31 positive vessel rates (PVRs) between tumor and adjacent normal tissues were evaluated in 39 colorectal carcinoma (CRC) samples and compared with those of Ki67/CD31 double stained tissues. Mean TK1/CD31 PVR (23.6%) in CRCs was 13.9 fold greater than in adjacent normal tissues (1.7%)). By comparison, mean Ki67/CD31 PVR in CRCs was 20.0%, i.e. only 4.8 fold greater than in normal tissues (4.2%). Also, mean TK1/CD31 PVR in normal tissues was significantly less than mean Ki67/CD31 PVR. Our findings indicate that double immunostaining for TK1/CD31 can detect activated tumor vessels more accurately than staining for Ki67/CD31 and potentially could identify tumors that will respond to anti-angiogenic therapy.

A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disorder characterised by raised pulmonary artery pressures with pathological changes in small pulmonary arteries. Previous studies have shown that approximately 70% of familial PAH and also 11-40% of idiopathic PAH (IPAH) cases have mutations in the bone morphogenetic protein receptor type II (BMPR2) gene. In addition, mutations in the activin receptor-like kinase 1 (ALK1) gene have been reported in PAH patients. Since both the BMPR2 and ALK1 belonging to the transforming growth factor (TGF)-beta superfamily are known to predispose to PAH, mutations in other genes of the TGF-beta/BMP signalling pathways may also predispose to PAH. METHODS: We screened for mutations in ENDOGLIN(ENG), SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD8 genes, which are involved in the TGF-beta/BMP signallings, in 23 patients with IPAH who had no mutations in BMPR2 or ALK1. RESULTS: A nonsense mutation in SMAD8 designated c.606 C>A, p.C202X was identified in one patient. The father of this patient was also identified as having the same mutation. Functional analysis showed the truncated form of the SMAD8 C202X protein was not phosphorylated by constitutively active ALK3 and ALK1. The SMAD8 mutant was also unable to interact with SMAD4. The response to BMP was analysed using promoter-reporter activities with SMAD4 and/or ca-ALK3. The transcriptional activation of the SMAD8 mutant was inefficient compared with the SMAD8 wild type. CONCLUSION: We describe the first mutation in SMAD8 in a patient with IPAH. Our findings suggest the involvement of SMAD8 in the pathogenesis of PAH.

Cortical representation area of human dental pulp.

To elucidate the dental pulp-representing area in the human primary somatosensory cortex and the presence of A-beta fibers in dental pulp, we recorded somatosensory-evoked magnetic fields from the cortex in seven healthy persons using magnetoencephalography. Following non-painful electrical stimulation of the right maxillary first premolar dental pulp, short latency (27 ms) cortical responses on the magnetic waveforms were observed. However, no response was seen when stimulation was applied to pulpless teeth, such as devitalized teeth. The current source generating the early component of the magnetic fields was located anterior-inferiorly compared with the locations for the hand area in the primary somatosensory cortex. These results demonstrate the dental pulp representation area in the primary somatosensory cortex, and that it receives input from intradental A-beta neurons, providing a detailed organizational map of the orofacial area, by adding dental pulp to the classic "sensory homunculus".

Localization of palatal area in human somatosensory cortex.

To determine the 'hard palate representing' area in the primary somatosensory cortex, we recorded somatosensory-evoked magnetic fields from the cortex in ten healthy volunteers, using magnetoencephalography. Following electrical stimulation of 3 sites on the hard palate (the first and third transverse palatine ridges, and the greater palatine foramen), magnetic responses showed peak latencies of 15, 65, and 125 ms. Equivalent current dipoles for early magnetic responses were found along the posterior wall of the inferior part of the central sulcus. These dipoles were localized anterior-inferiorly, compared with locations for the hand area in the cortex. However, there were no significant differences in three-dimensional locations among the 3 selected regions for hard palate stimulation. These results demonstrated the precise location of palatal representation in the primary somatosensory cortex, the actual area being small.

Cortical neuromagnetic fields preceding voluntary jaw movements.

Slow cortical potentials (readiness potentials, RPs) reflecting the central programming of voluntary jaw movements were reported to appear preceding the movements. However, the current source producing the RP has not yet been localized. This study aimed to determine the cortical regions involved in the central programming of bilaterally symmetrical voluntary jaw movements, by locating the current source of the neuromagnetic counterpart of the RP (readiness field, RF). The RFs were found in the fronto-lateral region bilaterally, starting around 860 and 600 ms prior to the onset of masseter and digastric electromyograms (EMGs), respectively, and gradually increasing in magnitude to the peak within 100 ms before the EMG onset. Thus, the RFs appeared long before the reported onset of the excitability increase of pyramidal tract neurons. The current sources producing the RFs were located in the precentral gyrus bilaterally, with no bilateral differences in strength. We conclude that the primary motor cortex is involved bilaterally in central programming as well as in execution of bilaterally symmetrical voluntary jaw movements.

Divergence of mobile genetic elements involved in the distribution of xenobiotic-catabolic capacity.

Bacteria adapt rapidly to environmental stimuli, such as exposure to xenobiotics. Mobile genetic elements (MGEs) play a major role in such bacterial adaptation, via the dispersal of catabolic capacity; and, in fact, genes that encode the degradation enzymes for xenobiotics are often located on MGEs. The list of reported catabolic MGEs keeps growing as researchers continue to isolate and characterize xenobiotic degraders and the corresponding catabolic genes. Major catabolic MGEs include (conjugative) plasmids, transposons, and conjugative transposons. Catabolic transposons can be divided into class I elements (composite transposons) and class II elements (Tn 3 family transposons). This review includes a comprehensive list of naturally occurring discrete catabolic MGEs, together with a brief description for each. While MGEs are often rather large, genome-wide or large-scale sequence analyses have provided useful information on the whole genetic structure of MGEs, with clues to their function (transfer, maintenance, catabolism, etc.) and behavior in a complex natural environment. This review also gives an insight into MGE functions, based on the complete sequencing of several catabolic plasmids and two Pseudomonas genomes.

Lack of association between hepatocyte nuclear factor-1beta gene and common forms of type 2 diabetes in the Japanese population.

Mutations in the hepatocyte nuclear factor-1beta (HNF-1beta) gene have been shown to be a cause of maturity-onset diabetes of the young (MODY). We studied the contribution of the HNF-1beta gene to susceptibility to common forms of Type 2 diabetes in the genetically homogeneous Japanese population, by investigating the allelic association of Type 2 diabetes with two markers in the HNF-1beta region. The frequency of a nonsense mutation, R177X, which was previously reported in a Japanese family, was also studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using a mismatch primer. A total of 200 subjects were studied. There was no significant difference in allele frequencies of either of the two polymorphisms studied between patients with Type 2 diabetes and control subjects, or between subgroups of patients subdivided by the presence of mild or severe diabetic nephropathy. None of the subjects studied had R177X mutation, giving a frequency of less than 1.1% in common forms of Type 2 diabetes in Japan. These results suggest that mutations in the HNF-1beta gene derived from a limited number of founders are not a major cause of common forms of Type 2 diabetes, even in the genetically homogeneous Japanese population.

Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes.

Thiazolidinediones, insulin-sensitizing agents, have been reported to increase glucose uptake along with the expression of glucose transporters in adipocytes and cardiomyocytes. Recently, we have further suggested that the translocation of GLUT4 is stimulated by thiazolidinediones in L6 myocytes. However, the direct effects of thiazolidinediones on translocation of glucose transporters have not yet been determined. In this study, using hemagglutinin epitope-tagged GLUT4 (GLUT4-HA), we provide direct evidence of the effect of troglitazone on the translocation of GLUT4 in rat epididymal adipocytes. Primary cultures of rat adipocytes were transiently transfected with GLUT4-HA and overexpressed eightfold compared with endogenous GLUT4 in transfected cells. A total of 24 h of treatment with troglitazone (10(-4) mol/l) increased the cell surface level of GLUT4-HA by 1.5 +/- 0.03-fold (P < 0.01) without changing the total amount of GLUT4-HA, whereas it increased the protein level of endogenous GLUT4 (1.4-fold) without changing that of GLUT1. Thus, the direct effect on the translocation can be detected apart from the increase in endogenous GLUT4 content using GLUT4-HA. Troglitazone not only increased the translocation of GLUT4-HA on the cell surface in the basal state but also caused a leftward shift in the dose-response relations between GLUT4-HA translocation and insulin concentration in the medium (ED(50): from approximately 0.1 to 0.03 nmol/l). These effects may partly contribute to the antidiabetic activity of troglitazone in patients with obesity and type 2 diabetes.

Genetic polymorphisms and functional characterization of the 5'-flanking region of the human CYP2C9 gene: in vitro and in vivo studies.

OBJECTIVE: Genetic polymorphisms were identified in the 5'-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin. METHODS: Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5'-flanking region-luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities. RESULTS: Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of T-->C transition at position -1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1-6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present. CONCLUSION: In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.

Sequence analysis of candidate genes for common susceptibility to type 1 and type 2 diabetes in mice.

Although type 1 and type 2 diabetes are regarded as clinically distinct diseases, several lines of evidence have suggested common genetic factors between the two types of diabetes. The non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, and the Nagoya-Shibata-Yasuda (NSY) mouse, a model of type 2 diabetes, are derived from the same outbred colony, Jcl:HCR, suggesting a shared susceptibility between the two types of diabetes in mice. Genetic as well as functional studies have supported the possibility that Tcf2, which encodes the transcription factor, hepatocyte nuclear factor 1beta (HNF-1beta), is a candidate gene for the common susceptibility between NSY and NOD mice. Txn, encoding thioredoxin which is a redox (reduction/oxidation)-active protein, is also a positional and functional candidate for a common susceptibility gene. To investigate whether either of these two genes is a common susceptibility gene, the coding nucleotide sequences of these two genes were compared among the NSY, NOD and control C3H strains. The coding sequence of Tcf2 of the NOD mouse was identical to that of the C3H mouse, but was different from that of the NSY mouse. The coding sequence of Txn was identical in the three strains. These data suggest that neither of the two genes is a common susceptiblity gene between type 1 and type 2 diabetes in mice.

Transgenic overexpression of leptin rescues insulin resistance and diabetes in a mouse model of lipoatrophic diabetes.

Lipoatrophic diabetes is caused by a deficiency of adipose tissue and is characterized by severe insulin resistance, hypoleptinemia, and hyperphagia. The A-ZIP/F-1 mouse (A-ZIPTg/+) is a model of severe lipoatrophic diabetes and is insulin resistant, hypoleptinemic, hyperphagic, and shows severe hepatic steatosis. We have also produced transgenic "skinny" mice that have hepatic overexpression of leptin (LepTg/+) and no adipocyte triglyceride stores, and are hypophagic and show increased insulin sensitivity. To explore the pathophysiological and therapeutic roles of leptin in lipoatrophic diabetes, we crossed LepTg/+ and A-ZIPTg/+ mice, producing doubly transgenic mice (LepTg/+:A-ZIPTg/+) virtually lacking adipose tissue but having greatly elevated leptin levels. The LepTg/+:A-ZIPTg/+ mice were hypophagic and showed improved hepatic steatosis. Glucose and insulin tolerance tests revealed increased insulin sensitivity, comparable to LepTg/+ mice. These effects were stable over at least 6 months of age. Pair-feeding the A-ZIPTg/+ mice to the amount of food consumed by LepTg/+:A-ZIPTg/+ mice did not improve their insulin resistance, diabetes, or hepatic steatosis, demonstrating that the beneficial effects of leptin were not due to the decreased food intake. Continuous leptin administration that elevates plasma leptin concentrations to those of LepTg/+:A-ZIPTg/+ mice also effectively improved hepatic steatosis and the disorder of glucose and lipid metabolism in A-ZIP/F-1 mice. These data demonstrate that leptin can improve the insulin resistance and diabetes of a mouse model of severe lipoatrophic diabetes, suggesting that leptin may be therapeutically useful in the long-term treatment of lipoatrophic diabetes.

Troglitazone induces GLUT4 translocation in L6 myotubes.

A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10(-5) mol/l) caused a substantial increase in the 2-deoxy-[3H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 micromol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 x 10(-6) mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-lambda, PKC-beta2, and PKC-zeta; or 5'AMP-activated protein kinase activity. a-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle.

Polymorphism in gene for islet autoantigen, IA-2, and type 1 diabetes in Japanese subjects.

Autoantibodies against IA-2 have been detected in up to 86% of newly diagnosed patients with type 1 diabetes and appear to identify a subgroup of prediabetic subjects who rapidly progress to type 1 diabetes. We examined the association of IA-2 gene polymorphism with type 1 diabetes in Japanese subjects. A total of 276 Japanese subjects were studied for disease association and, in addition, another 53 patients were studied for association with the autoantibody status to IA-2. A microsatellite marker D2S1753E, located in the intron of the IA-2 gene, was used as a genetic marker in this study. In Japanese, two alleles (161mu and 165mu) were more frequent, and the 163mu allele was less frequent than in Caucasians (p = 0.0001). There was no significant difference in frequencies of alleles between diabetic patients and control subjects. The frequency of IA-2 gene polymorphism was not significantly different between patients stratified by age-at-onset, or between patients with and without susceptible HLA, DRB1*0405, DRB1*0802 and DRB1*0901. There was no significant difference in allele frequency of the IA-2 gene polymorphism between patients with and without autoantibody to IA-2. In conclusion, IA-2 gene polymorphism is not associated with either susceptibility to, or heterogeneity in type 1 diabetes in Japanese subjects.

[Pathology and significance of leptin resistance in obesity].

Leptin, the protein product of the ob gene, is predominantly secreted from white adipose tissue, and acts on the brain to regulate food intake, energy expenditure, and neuroendocrine function. Obese rodent and humans are mostly associated with high circulating leptin levels. These findings have led to the conclusion that obese individuals are relatively insensitive to endogenous leptin termed 'leptin resistance'. The potential sites for leptin resistance include the blood-brain-barrier transport system and the leptin signaling mechanism in leptin-responsive neurons in the hypothalamus. In this review, we describe leptin, leptin receptor, and potential hypothesis of leptin resistance.

Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway.

Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS) receptor originally isolated from the stomach, occurs in the hypothalamic arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY), suggesting the involvement of NPY in some of ghrelin actions. This study was designed to elucidate the role of ghrelin in the regulation of food intake. A single intracerebroventricular (ICV) injection of ghrelin (5-5,000 ng/rat) caused a significant and dose-related increase in cumulative food intake in rats. Ghrelin (500 ng/rat) was also effective in growth hormone-deficient spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats that received a single ICV injection of ghrelin (500 ng/rat) (approximately 160% of that in vehicle-treated groups, P < 0.05). The ghrelin's orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1 receptor antagonist (10-30 microg/rat). The leptin-induced inhibition of food intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner (5-500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35% (P < 0.05), which was abolished by ICV co-injection of ghrelin (500 ng/rat). This study provides evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of hypothalamic NPY/Y1 receptor pathway.

Prognostic results of cisplatin IP and carboplatin IV with G-CSF in patients with ovarian cancer.

We performed a dose-escalation study of carboplatin combined with a fixed dose of intraperitoneal cisplatin and G-CSF in patients with epithelial ovarian cancer, and analyzed the progression-free and overall survival. Six of the patients who entered the study with stage IC and II disease are still alive with no evidence of disease. The five-year survival rate was 61% for the 18 patients with stage III and IV disease; progression-free survival over 5 years was 32%. Our results show this to be an effective treatment regimen for epithelial ovarian cancer. Prognosis is good with this combined carboplatin/cisplatin/G-CSF therapy, especially for those patients with microscopic or no residual disease.

Structure-activity relationship of orally potent tripeptide-based HIV protease inhibitors containing hydroxymethylcarbonyl isostere.

We designed and synthesized a new class of peptidomimetic human immunodeficiency virus protease inhibitors containing a unique unnatural amino acid, allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid], with a hydroxymethylcarbonyl isostere as the active moiety. From a structure-activity relationship study of HIV-1 protease inhibition, enzyme selectivity for other aspartyl proteases, the antiviral activity and pharmacokinetics in rats, 24c (KNI-227) and 24d (KNI-272, our first clinical candidate) were found to be selective and orally potent HIV protease inhibitors. Moreover, an improvement of the pharmacokinetic features of KNI-272 provided two long-lasting and highly bioavailable compounds (24g: JE-2178, 24h: JE-2179).

Age-related association of MHC class I chain-related gene A (MICA) with type 1 (insulin-dependent) diabetes mellitus.

To assess the contribution of the HLA class I region to susceptibility to and heterogeneity of type 1 diabetes, we investigated the association of polymorphism of MHC class I chain-related gene A (MICA) with age-at-onset as well as susceptibility to type 1 diabetes. One hundred one Japanese patients and 110 healthy control subjects were studied. The frequency of A4 allele was significantly higher and that of A6 allele was significantly lower in patients than in control subjects. The frequency of A5.1 allele was highest in early-onset patients (23.0%), intermediate in intermediate-onset patients (9.2%) and lowest in late-onset patients (7.7%) (trend chi-squared test, p = 0.0098). A5. 1 allele was strongly associated with HLA-B7 and Cw7, suggesting that MICA*A5.1-B7-Cw7 haplotype contains a gene responsible for age-at-onset. A4 allele was associated with a susceptible haplotype, DR4-DQB1*0401, and A6 allele was associated with a protective haplotype, DR2-DQB1*0601, suggesting that the association of MICA with type 1 diabetes susceptibility may be due to linkage disequilibrium with class II haplotypes. These data suggest that MICA gene is associated with age-at-onset and that a gene (or genes) responsible for age-at-onset of type 1 diabetes is located in the HLA class I region, probably near the region of MICA-B-C.

Comparative evaluation of conjugate vaccines in the Haemophilus influenzae infection model.

We used a murine model of Haemophilus influenzae type b (Hib) infection to analyze the immunologic response to two commercially available PRP conjugate vaccines (HbOC, PRP-T). The mortality rate in mice infected with a large dose of the bacteria after vaccination with HbOC or PRP-T at two and three doses was significantly lower than in non-vaccinated mice and mice vaccinated by one dose. Furthermore, for infections caused by a small bacterial dose, the mortality rate in mice vaccinated with one, two, or three doses was significantly lower than in non-vaccinated mice. The induction level of anti-PRP antibodies, especially IgG, in serum of mice vaccinated by two or three doses was higher than in those vaccinated with a single dose. Our results indicate that the dose of vaccine influences its efficacy in protecting against Hib infection. Our results also showed a lack of difference between two different PRP conjugate vaccines.

[Insulin resistance, role of leptin and leptin receptor].

Leptin, the obese gene product, is an adipocyte-derived satiety factor which is involved in the regulation of food intake and energy expenditure. Obesity often accompanies insulin resistance and high levels of leptin. In in vitro studies, leptin has been reported to increase fatty acid oxidation and decrease fatty acid synthesis in adipocytes and hepatocytes. The direct effects of leptin on glucose metabolism and insulin signaling have not been clarified yet. In in vivo studies, however, leptin has been reported to improve insulin sensitivity and glucose metabolism in normal and obese rodents acting mainly through hypothalamus. Moreover leptin has been reported to have antidiabetic effects in insulin-deficient diabetes rats and lipoatrophic diabetes mice. It is suggested that leptin modulates insulin sensitivity and glucose disposal and that leptin may have a pathophysiological and therapeutic implications in diabetes.