TS-071 is a novel, potent and selective renal sodium-glucose cotransporter 2 (SGLT2) inhibitor with anti-hyperglycaemic activity.

BACKGROUND AND PURPOSE: The renal sodium-glucose cotransporter 2 (SGLT2) plays an important role in the reuptake of filtered glucose in the proximal tubule and therefore may be an attractive target for the treatment of diabetes mellitus. This study characterizes the pharmacological profile of TS-071 ((1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol hydrate), a novel SGLT2 inhibitor in vitro and in vivo. EXPERIMENTAL APPROACH: Inhibition of glucose uptake by TS-071 was studied in CHO-K1 cells stably expressing either human SGLT1 or SGLT2. Single oral dosing studies were performed in rats, mice and dogs to assess the abilities of TS-071 to increase urinary glucose excretion and to lower plasma glucose levels. KEY RESULTS: TS-071 inhibited SGLT2 activity in a concentration-dependent manner and was a potent and highly selective inhibitor of SGLT2. Orally administered TS-071 increased urinary glucose excretion in Zucker fatty rats and beagle dogs at doses of 0.3 and 0.03 mg·kg(-1) respectively. TS-071 improved glucose tolerance in Zucker fatty rats without stimulating insulin secretion and reduced hyperglycaemia in streptozotocin (STZ)-induced diabetic rats and db/db mice at a dose of 0.3 mg·kg(-1). CONCLUSION AND IMPLICATIONS: These data indicate that TS-071 is a potent and selective SGLT2 inhibitor that improves glucose levels in rodent models of type 1 and 2 diabetes and may be useful for the treatment for diabetes mellitus.

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication.

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.

A patient with dementia with Lewy bodies and codon 232 mutation of PRNP.

The authors describe a patient who had a point mutation at codon 232 of the prion protein gene, resulting in the substitution of methionine for arginine (M232R). The patient developed dementia and died 6 years after its onset. Autopsy revealed dementia with Lewy bodies, not Creutzfeldt-Jakob disease. Although the M232R mutation has been reported to cause Creutzfeldt-Jakob disease, findings in our patient suggest that not all patients presenting progressive dementia with M232R mutation have Creutzfeldt-Jakob disease.

Reduced morning cortisol secretion in patients with multiple system atrophy.

To determine whether daytime or nighttime cortisol secretion is aberrant in patients with multiple system atrophy (MSA), the plasma cortisol concentrations in 20 patients with MSA were measured at 4-hr intervals for one day. Ten age-matched healthy individuals and 20 patients with cerebral infarction but without hypothalamic lesions were used as controls. The results showed that morning (8 A.M.) cortisol concentrations in patients with MSA were significantly lower than those in the control subjects. This phenomenon is highly associated with dysfunction in the autonomic nervous system in MSA.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene.

Friedreich ataxia (FRDA), the most common autosomal recessive neurodegenerative disease among Europeans and people of European descent, is characterized by an early onset (usually before the age of 25), progressive ataxia, sensory loss, absence of tendon reflexes and pyramidal weakness of the legs. We have recently identified a unique group of patients whose clinical presentations are characterized by autosomal recessive inheritance, early age of onset, FRDA-like clinical presentations and hypoalbuminemia. Linkage to the FRDA locus, however, was excluded. Given the similarities of the clinical presentations to those of the recently described ataxia with oculomotor apraxia (AOA) linked to chromosome 9p13, we confirmed that the disorder of our patients is also linked to the same locus. We narrowed the candidate region and have identified a new gene encoding a member of the histidine triad (HIT) superfamily as the 'causative' gene. We have called its product aprataxin; the gene symbol is APTX. Although many HIT proteins have been identified, aprataxin is the first to be linked to a distinct phenotype.

[Analysis of regional cerebral blood flow and distribution volume in Machado-Joseph disease by iodine-123I IMP single photon emission computed tomography].

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia. Its clinical features vary greatly in different generations of the same family. Regional cerebral blood flow (rCBF) and distribution volume (Vd) in the pons, cerebellum, and cerebral cortex were measured in 12 patients with MJD by autoradiography (ARG) and the table look-up (TLU) method of iodine-123 IMP (123I-IMP) single photon emission computed tomography (SPECT). Representative cases were as follows: A 46-year-old woman first experienced gait ataxia at age 38. Computed tomography (CT) and magnetic resonance imaging (MRI) showed no atrophy in the pons or cerebellum, but rCBF measured by the 123I-IMP SPECT ARG method detected hypoperfusion in the pons, and cerebellar vermis and hemisphere. A 76-year-old woman first experienced gait ataxia at age 69. CT and MRI findings showed severe atrophy in the pons, and cerebellar vermis and hemisphere. Moreover, rCBF was decreased in the pons, whereas it was not decreased in the cerebellar vermis and hemisphere. In the pons of patients with MJD, rCBF was markedly decreased regardless of disease severity. Because this SPECT finding for the pons looked like a 'dot', we have called it the 'pontine dot sign'. In the MJD group, rCBF was significantly decreased in the pons (Student's t test, p < 0.01) and cerebellar vermis (p < 0.05). The Vd was also significantly decreased in the pons (p < 0.005) in comparison with that for normal subjects. Pearson's correlation analysis yielded a significant relationship between the rCBF in the pons and age at onset (r = 0.578, p < 0.05). There was a strong correlation between the Vd for the pons and age at onset (r = 0.59, p < 0.05). Pearson's correlation analysis also showed a significant relationship between the Vd in the cerebellar hemispheres and International Cooperative Ataxia Rating Scale (r = 0.644, p < 0.05). The pontine rCBFs in patients with early onset MJD, whose pons was not atrophic, decreased more than did those in patients with late onset MJD, whose pons was severely atrophic. This suggests that the SPECT findings are indicative of underlying neurodegenerative processes that began before the onset of clinical symptoms. Different processes seem to function in atrophy and the rCBF decrease in the pons of patients with MJD. These findings will be proved by the increase of the number of cases of MJD. Until now, there has been no report on rCBF and Vd obtained by 123I-IMP SPECT for patients with MJD identified by gene analysis. Our study shows that SPECT measurement of rCBF and Vd is useful for understanding the pathophysiology of MJD.

Regulation of the development of allodynia by intrathecally administered P2 purinoceptor agonists and antagonists in mice.

Effects of agonists and antagonists of P2X-purinoceptors on the regulation of the development of allodynia were examined in mice; the drugs were administered intrathecally to the spinal cord. Suramin (5, 10 microg) and pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS), antagonists of P2X receptors, inhibited prostaglandin (PG) E(2)-induced allodynia. PPADS did not block glutamate-induced allodynia. alpha,beta-Methylene ATP (alpha, beta-meATP), an agonist of P2X receptor, elicited allodynia. alpha, beta-me ATP-induced allodynia was blocked by co-administration of alpha,beta-meATP with PPADS, MK 801 or N(omega)-nitro-L-arginine methyl ester (L-NAME). Suramin at higher doses (20, 40 microg) induced allodynia, which was inhibited by MK 801 or L-NAME. These results suggest that ATP P2X receptors in the spinal cord are involved in the regulation of tactile allodynia. Glutamate receptor and nitric oxide systems play an important role in the development of allodynia produced by alpha,beta-meATP and suramin.

Involvement of the zinc-binding capacity of Sendai virus V protein in viral pathogenesis.

The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C(341)S and V-C(365)R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V(DeltaC), which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

Clinical experiences of stenting in patients with esophago-bronchial fistula: report of four cases.

The esophagorespiratory fistula is difficult to treat, and the patients' quality of life is generally poor due to suffering from dysphagia and dyspnea. We performed stent therapy in four cases of the esophagorespiratory fistula associated with esophageal cancer. Three of four patients showed improved symptoms, enabling oral liquid or food intake, although one died of dyspnea despite the therapy. The findings suggest that stent therapy is an effective method to close the esophagorespiratory fistula and to improve the patients' quality of life, although it is temporary and not a radical treatment.

Nocturnal decrease in vasopressin secretion into plasma in patients with multiple system atrophy.

To determine whether the nocturnal decrease in arginine vasopressin (AVP) secretion into the plasma, found in a patient with multiple system atrophy (MSA) reported previously, is a usual finding in MSA, the plasma AVP concentrations in 13 patients with MSA were measured every 4 hours during a 24 hour period. The plasma AVP concentrations in these patients showed significant daily variations and were the lowest during the night. This finding indicates that patients with MSA often exhibit nocturnal decrease in AVP secretion into the plasma. The results suggest the possibility that the system responsible for the daily variations in AVP secretion is involved in MSA.

"Tail sign" associated with microembolic signals.

BACKGROUND AND PURPOSE: Transcranial Doppler ultrasound (TCD) can detect circulating microembolic signals (MES). We focused our attention on tail signs (TS), a signal associated with MES that appeared as a small reversal signal after MES on the high time resolution spectral display. We examined MES and artifacts in an animal study to determine whether TS were specific changes associated with MES and investigated the characteristics of TS in both animal and clinical studies. METHODS: In an animal study, adult pigs with venoarterial extracorporeal membrane oxygenation and minimal anticoagulation therapy were used as a model for cerebral embolism. After performing TCD monitoring with a multigated approach, we did an offline analysis to investigate several parameters concerning MES and TS. We also examined TS in patients in a clinical study. RESULTS: From a total of 362 MES investigated in the animal study, 72.9% were followed by TS. We could not find any TS associated with artifacts. The time delay between TS and MES was negatively correlated with the velocity of MES. MES almost always appeared first in the proximal channel, whereas TS conversely appeared first in the distal channel. In the clinical study, we were also able to observe TS associated with MES. CONCLUSIONS: TS may represent emboli passing down a branch vessel or twisted downstream vessel. TS are specific for MES and can be used as another criterion for MES identification.

TAL1 and LIM-only proteins synergistically induce retinaldehyde dehydrogenase 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA3.

Previously, we have shown that TAL1 and the LIM-only protein gene (LMO) are regularly coactivated in T-cell acute lymphoblastic leukemia (T-ALL). This observation is likely to relate to the findings that TAL1 and LMO are highly synergistic in T-cell tumorigenesis in double-transgenic mice. To understand the molecular mechanisms of functional synergy between TAL1 and LMO in tumorigenesis and transcriptional regulation, we tried to identify downstream target genes regulated by TAL1 and LMO by a subtractive PCR method. One of the isolated genes, that for retinaldehyde dehydrogenase 2 (RALDH2), was regularly expressed in most of the T-ALL cell lines that coexpressed TAL1 and LMO. Exogenously transfected TAL1 and LMO, but not either alone, induced RALDH2 expression in a T-ALL cell line, HPB-ALL, not expressing endogeneous TAL1 or LMO. The RALDH2 transcripts in T-ALL were, however, mostly initiated within the second intron. Promoter analysis revealed that a GATA site in a cryptic promoter in the second intron was essential and sufficient for the TAL1- and LMO-dependent transcriptional activation, and GATA3 binds to this site. In addition, forced expression of GATA3 potentiated the induction of RALDH2 by TAL1 and LMO, and these three factors formed a complex in vivo. Furthermore, a TAL1 mutant not binding to DNA also activated the transcription of RALDH2 in the presence of LMO and GATA3. Collectively, we have identified the RALDH2 gene as a first example of direct transcriptional target genes regulated by TAL1 and LMO in T-ALL. In this case, TAL1 and LMO act as cofactors for GATA3 to activate the transcription of RALDH2.

[Radiotherapy for locally advanced esophageal cancer].

The conventional treatment for patients with locally advanced esophageal cancer has been surgery or radiotherapy. The indication of the treatment depends on the location of the primary tumor, tumor stage, resectability of the lesion and operability of the patient. Radiotherapy is used in a case refusing surgery, medical contraindications to surgery, extensive locoregional disease or poor general condition. The survival rate by surgery alone or radiotherapy alone is poor. Thus, single modality therapy is not a standard treatment, but combined multimodal therapy including chemotherapy is a standard approach for locally advanced esophageal cancer. Further studies of optimization of multimodal therapies which are surgery and/or radiotherapy combined with chemotherapy are needed to improve the results in the treatment of esophageal cancer.

[Cerebral embolism due to left atrial ball thrombus without mitral stenosis--usefulness of the transesophageal echocardiography for the diagnosis].

We reported two cases of cerebral embolism associated with atrial fibrillation. Left atrial ball thrombus without mitral stenosis was diagnosed by the transesophageal echocardiography, followed by successful removal. Left atrial ball thrombus has a risk for lethal complications, and a high incidence of systemic embolism even during anticoagulation therapy. An immediate surgical treatment is needed. The transthoracic echocardiography is not useful in diagnosis for left atrial ball thrombus. It is important to examine left atrial ball thrombus by the transesophageal echocardiography in patients with cerebral embolism associated with atrial fibrillation.

Central noradrenergic mediation of nitrous oxide-induced analgesia in rats.

PURPOSE: Although several studies have demonstrated that both supra opiate receptors and spinal alpha 2 adrenoceptors play a mediating role in nitrous oxide(N2O) analgesia, controversy still exists. The present study was undertaken to evaluate further the involvement of noradrenergic (NA) neuronal activity in N2O analgesia by investigating tail-flick latency and supra- and spinal NA levels in rats. METHODS: In an analgesia study, effect of N2O 75% and its modification were evaluated using the tail-flick test in male Wistar rats. Results were expressed as % maximum possible effect (MPE). Modification of N2O analgesia was examined in rats pretreated with either the alpha 2 receptor agonist, clonidine(CLO: 150 micrograms.kg-1, i.p.), alpha 2 receptor antagonist, idazoxone(IDZ: 100 micrograms.kg-1, i.v.) by lesioning the locus coeruleus (LC) seven days before exposure to N2O, or naloxone (5 mg.kg-1, i.v.). Also, in a NAergic neuronal transmission study, the changes in NA content at LC and spinal cord were determined using HPLC-ECD. RESULTS: Nitrous oxide produced analgesia, % MPE increased to a maximum of 78% at 30 min, thereafter declining to 38% at 120 min. Clonidine potentiated the analgesic effect of N2O at 120 min (80%). The analgesic effect of N2O was attenuated by IDZ or by LC lesioning. However, naloxone, in a dose sufficient to block morphine-induced analgesia, had no effect. With N2O exposure, NA content was decreased by 52% in the LC and by 20% at spinal cord. With morphine, NA content did not differ from the control group. CONCLUSION: The data suggest that N2O-induced analgesia is principally mediated by activation of the descending inhibitory NAergic system and/or increased NA release at spinal cord which may lead to presynaptic inhibition of primary afferent neurotransmitter release and hyperpolarize the dorsal horn neurons by alpha 2 receptors.