A prospective phase II study of combined androgen blockade in patients with androgen receptor-positive metastatic or locally advanced unresectable salivary gland carcinoma.

Background: There is no standard first-line chemotherapy for recurrent/metastatic (RM) or unresectable locally advanced (LA) salivary gland carcinoma (SGC). Patients and methods: We conducted a single institution, open-label, single arm, phase II trial of combined androgen blockade (CAB) for androgen receptor (AR)-positive SGC. Leuprorelin acetate was administered subcutaneously at a dose of 3.75 mg every 4 weeks. Bicalutamide was administered orally at a daily dose of 80 mg. Patients were treated until progressive disease or unacceptable toxicities. Results: Thirty-six eligible patients were enrolled. Thirty-three patients had RM disease and three patients had LA disease. The pathological diagnoses were salivary duct carcinoma (34 patients, 94%) and adenocarcinoma, NOS (two patients, 6%). The best overall response rate was 41.7% [n = 15, 95% confidence interval (CI), 25.5%-59.2%], the clinical benefit rate was 75.0% (n = 27, 95% CI, 57.8%-87.9%). The median progression-free survival was 8.8 months (95% CI, 6.3-12.3 months) and the median overall survival was 30.5 months (95% CI, 16.8 months to not reached). Additional analyses between treatment outcomes and clinicopathological factors or biomarkers including AR positivity, human epidermal growth factor receptor 2 status, and its complex downstream signaling pathway gene mutations showed no statistically significant differences. Elevated grade 3 liver transaminases and increased serum creatinine were reported in two patients, respectively. Discontinuation of leuprorelin acetate or bicalutamide due to adverse event occurred in one patient. Conclusion: This study suggests that CAB has equivalent efficacy and less toxicity for patients with AR-positive RM or unresectable LA SGC compared with conventional chemotherapy, which warrants further study. Clinical Trial Registration: UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape.

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.

Deletion of Ia-2 and/or Ia-2ß in mice decreases insulin secretion by reducing the number of dense core vesicles.

AIMS/HYPOTHESIS: Islet antigen 2 (IA-2) and IA-2ß are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV. METHODS: Insulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2ß [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca(2+) currents, DCV half-life, lysosome number and size and autophagy. RESULTS: Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca(2+) currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV. CONCLUSIONS/INTERPRETATION: We conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2ß, is due to a decrease in the number of DCV.

Lipopolysaccharide induces proinflammatory cytokines and chemokines in experimental otitis media through the prostaglandin D2 receptor (DP)-dependent pathway.

Otitis media is one of the most common and intractable ear diseases, and is the major cause of hearing loss, especially in children. Multiple factors affect the onset or development of otitis media. Prostaglandin D2 is the major prostanoid involved in infection and allergy. However, the role of prostaglandin D2 and prostaglandin D2 receptors on the pathogenesis of otitis media remains to be determined. Recent studies show that D prostanoid receptor (DP) and chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) are major prostaglandin D2 receptors. In this study, homozygous DP single gene-deficient (DP⁻(/)⁻) mice, CRTH2 single gene-deficient (CRTH2⁻(/)⁻) mice and DP/CRTH2 double gene-deficient (DP⁻(/)⁻ CRTH2⁻(/)⁻) mice were used to investigate the role of prostaglandin D2 and its receptors in otitis media. We demonstrate that prostaglandin D2 is induced by lipopolysaccharide (LPS), a major component of Gram-negative bacteria, and that transtympanic injection of prostaglandin D2 up-regulates macrophage inflammatory protein 2 (MIP-2), interleukin (IL)-1ß and IL-6 in the middle ear. We also show that middle ear inflammatory reactions, including infiltration of inflammatory cells and expression of MIP-2, IL-1ß and IL-6 induced by LPS, are reduced significantly in DP⁻(/)⁻ mice and DP⁻(/)⁻ CRTH2⁻(/)⁻ mice. CRTH2⁻(/)⁻ mice display inflammatory reactions similar to wild-type mice. These findings indicate that prostaglandin D2 may play significant roles in LPS-induced experimental otitis media via DP.

Geographical accessibility to dental care in the Japanese elderly.

OBJECTIVE: The current research aims to clarify the factors relevant to elderly people's access to dental care in Japan, particularly focusing on geographical accessibility. METHODS: The sample was taken from among the Japanese elderly, aged 65 and over, who responded to a postal survey conducted in 2003 (n = 2,192). Six types of geographical accessibility to the dental clinics were calculated using Geographic Information Systems. Logistic regression analysis was conducted using 'having a regular dentist' as a dependent variable and geographical accessibility as an explanatory variable. RESULTS: The results showed an association between having a regular dentist and geographical accessibility only for females. In the univariate model, distance to the closest dental clinics (OR = 0.62 (95% CI: 0.43-0.90)), number of dental clinics at the school district level (OR = 1.14 (95% CI: 1.03-1.26)), number of dental clinics at the municipality level (OR = 1.02 (95% CI: 1.00-1.05)), and density distribution of dental clinics (OR = 1.56 (95% CI: 1.11-2.19)) showed significant relations with having a regular dentist. After controlling for demographic, socioeconomic, and health related variables, only the density distribution of dental clinics showed significant relations at the 5% level, although distance and number of dental clinics kept a marginal significance. CONCLUSION: The current study verifies that geographical accessibility correlates with access to dental care among women, and that there were large gender differences concerning the issue of geographical access.

[Intermittent opening of a mechanical mitral valve prosthesis due to pannus formation; report of a case].

A 76-year-old woman with a history of severe mitral valve stenosis had undergone mitral valve replacement with a 27 mm St. Jude Medical (SJM) valve in 1991. Follow-up transthoracic echocardiography revealed an increase in the pressure gradient across the mitral prosthesis 16 years after the surgery. Prosthetic valve dysfunction was suspected, but transesophageal echocardiography and cineradiography failed to show mechanical valve dysfunction. Two years later, she presented with dyspnea on exertion and leg edema. Cineradiography revealed intermittent restriction of the opening of the mechanical valve leaflet approximately every 10 beats. Thus, we diagnosed intermittent prosthetic valve dysfunction and performed a reoperation. On inspection of the prosthesis, we observed semicircular pannus formation around the posterior leaflet in the ventricular side. It was considered that the pannus tissue had interfered with 1 leaflet opening of the mitral valve prosthesis, resulting in intermittent valve dysfunction. We replaced the prosthesis with a new 25 mm SJM valve. The patient was discharged after confirmation of normal prosthetic function.

Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.

Prostaglandin (PG)D2, which has long been implicated in allergic diseases, is currently considered to elicit its biological actions through the DP receptor (DP). Involvement of DP in the formation of allergic asthma was recently demonstrated with DP-deficient mice. However, proinflammatory functions of PGD2 cannot be explained by DP alone. We show here that a seven-transmembrane receptor, CRTH2, which is preferentially expressed in T helper type 2 (Th2) cells, eosinophils, and basophils in humans, serves as the novel receptor for PGD2. In response to PGD2, CRTH2 induces intracellular Ca2+mobilization and chemotaxis in Th2 cells in a Galphai-dependent manner. In addition, CRTH2, but not DP, mediates PGD2-dependent cell migration of blood eosinophils and basophils. Thus, PGD2 is likely involved in multiple aspects of allergic inflammation through its dual receptor systems, DP and CRTH2.

Eradication of the commensal intestinal microflora by oral antimicrobials interferes with the host response to lipopolysaccharide.

The host components and commensal microorganisms of the intestinal microenvironment play roles in the development and maintenance of the host defence. Recent observations have suggested that toll-like receptors (TLRs) are involved in the recognition of innate immunity against intestinal microbes. However, little is known regarding the role of TLR in the maintenance of systemic host defence by intestinal microorganisms. We studied the expression and function of TLR4 and TLR2 on alveolar and peritoneal macrophages in mice after 3 weeks of oral administration of streptomycin and cefotaxime. After active treatment, the intestinal microorganisms were nearly completely eradicated, and the surface expression of TLR4 and TLR2 on the peritoneal macrophages was prominently downregulated. When the actively treated mice were challenged with lipopolysaccharide (LPS), a TLR4 ligand, the host response was markedly impaired. Our results suggest that the oral administration of antimicrobials downregulates the expression of surface TLR on the peritoneal macrophages and modulates the host immune responses against LPS by modifying the intestinal environment.

A single-photon detector in the far-infrared range

The far-infrared region (wavelengths in the range 10 microm-1 mm) is one of the richest areas of spectroscopic research, encompassing the rotational spectra of molecules and vibrational spectra of solids, liquids and gases. But studies in this spectral region are hampered by the absence of sensitive detectors--despite recent efforts to improve superconducting bolometers, attainable sensitivities are currently far below the level of single-photon detection. This is in marked contrast to the visible and near-infrared regions (wavelengths shorter than about 1.5 microm), in which single-photon counting is possible using photomultiplier tubes. Here we report the detection of single far-infrared photons in the wavelength range 175-210 microm (6.0-7.1 meV), using a single-electron transistor consisting of a semiconductor quantum dot in high magnetic field. We detect, with a time resolution of a millisecond, an incident flux of 0.1 photons per second on an effective detector area of 0.1 mm2--a sensitivity that exceeds previously reported values by a factor of more than 10(4). The sensitivity is a consequence of the unconventional detection mechanism, in which one absorbed photon leads to a current of 10(6)-10(12) electrons through the quantum dot. By contrast, mechanisms of conventional detectors or photon assisted tunnelling in single-electron transistors produce only a few electrons per incident photon.

Modified phases of diamond formed under shock compression and rapid quenching.

Two modified forms of carbon were quenched by a rapid-cooling technique from graphite sheets shock-compressed to 65 gigapascals and 3700 K. One form, ;;n-diamond," which was obtained from the most rapidly cooled part, has a crystal structure close to that of cubic diamond. The other form, found in the relatively slow-cooled part, was comparable to an i-carbon prepared by an ion-beam technique. The n-diamond is interpreted as a metastable form, the same as hexagonal diamond, converted from graphite through a martensitic transition, for which either the region or the path may be different from that of hexagonal diamond. The second form was found to be produced through reconstruction.

A novel putative tyrosine kinase receptor encoded by the eph gene.

Growth factors and their receptors are involved in the regulation of cell proliferation and also play a key role in oncogenesis. In this study, a novel putative kinase receptor gene, termed eph, has been identified and characterized by molecular cloning. Its primary structure is similar to that of tyrosine kinase receptors thus far cloned and includes a cysteine-rich region in the extracellular domain. However, other features of the sequence distinguish the eph gene product from known receptors with tyrosine kinase activity. Thus the eph protein may define a new class of these molecules. The eph gene is overexpressed in several human carcinomas, suggesting that this gene may be involved in the neoplastic process of some tumors.

[Long-term evaluation of aortic root replacement using Svensson technique with multislice computed tomography].

The Svensson technique of aortic root composite valve graft replacement, which involves reimplantation of the left coronary ostium with a long interposed graft wrapping behind the composite graft, is technically simple and advantageous. However, surgeons must be concerned about complications regarding the long interposed graft in the long-term follow-up. In 32 cases with the Svensson technique, we evaluated their replaced aortic roots by multislice computed tomography (MDCT) at 4.5 +/- 3.6 years (maximum, 14.9 years) postoperatively. The mean age was 54.5 +/- 14.1 years and 22 patients were male. No patients have experienced any complications regarding the technique at the follow-up of 6.9 +/- 4.9 years (maximum, 16.4 years). No structural complications were detected by MDCT of all patients. In conclusions, the Svensson technique was demonstrated to be favorable with no technical problems in the long-term evaluations with MDCT. The Svensson technique can be considered as a justifiable technique in any aortic root replacements including complicated or redo aortic roots.

Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit.

NMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' alpha-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity.

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

CRTH2-dependent, STAT6-independent induction of cedar pollen dermatitis.

BACKGROUND: Airborne contact dermatitis to cedar pollen is a recently identified disease that generally affects individuals with cedar pollinosis of the nasal and/or ocular symptoms, as well as some patients with atopic dermatitis. OBJECTIVE: To elucidate the pathological mechanisms of cedar pollen dermatitis. METHODS: We established a mouse model of cedar pollen dermatitis by epicutaneous sensitization with Japanese cedar pollen antigen (Ag). RESULTS: Histologically, there was marked dermal cellular infiltrate, including eosinophils and mast cells, with epidermal thickening. The induction of dermatitis was accompanied by production of cedar pollen-specific IgE. In the lesional skin, IL-13, IL-18, eotaxin/chemokine (C-C motif) ligand (CCL) 11, regulated upon activation, normal T cell expressed and secreted/CCL5, macrophage-derived chemokine/CCL22 and thymus and activation-regulated chemokine/CCL17, but not IL-4 and IFN-gamma, were produced. Mast cell-deficient WBB6F1-W/W(v) mice failed to develop cedar pollen dermatitis, although regional lymph node cells proliferated in response to Cryptomeria japonica (Cry j) 1 and Cry j2 Ags in vitro. Surprisingly, the induction of dermatitis was independent of STAT6/IgE. In contrast, mice deficient in CRTH2, a receptor for prostaglandin D2 (PGD2), showed diminished inflammation. Consistent with this, ramatroban, a CRTH2 antagonist, significantly inhibited inflammatory cell infiltration. CONCLUSION: These data suggest that PGD2-CRTH2 signalling contributes to inflammation in cedar pollen dermatitis, and unlike cedar pollinosis of the nasal mucosa, STAT6 is not a therapeutic target for treatment.

Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases.

BACKGROUND: Non-receptor protein-tyrosine kinases often contain at least one Src homology 2 (SH2) domain, a protein module that binds with high affinity to tyrosine-phosphorylated peptides. Because SH2 domains would be predicted to bind with high affinity to proteins phosphorylated by the kinase, but not to the unphosphorylated substrate, their presence in tyrosine kinases has been puzzling. An important role for the SH2 domain of the Abl tyrosine kinase was suggested by work showing that Abl requires an intact SH2 domain in order to malignantly transform cells, and that replacement of the Abl SH2 domain with heterologous SH2 domains alters the spectrum of proteins phosphorylated detectably by Abl in vivo. RESULTS: We have used purified wild-type and mutant Abl kinases to examine the roles of the Abl's SH2 and catalytic domains in phosphorylation of p130CAS, a model substrate that has multiple potential phosphorylation sites. We find that an SH2 domain is required for efficient hyperphosphorylation of p130 in vitro. We use chimeric mutants with heterologous SH2 domains to demonstrate that the SH2 domain of the oncogenically transforming adaptor protein Crk, which is the SH2 domain predicted to bind with highest affinity (of those tested) to potential phosphorylation sites in p130, is best able to facilitate hyperphosphorylation. This is the case whether the catalytic domain of the kinase is derived from Abl or from its distant relative, Src. These studies also reveal a role for binding of Crk to Abl in mediating phosphorylation by the kinase. Using purified proteins, we demonstrate that association with Crk strikingly enhances the ability of Abl to hyperphosphorylate p130. There is an excellent correlation between the ability of mutant Crk proteins to promote hyperphosphorylation of p130 by Abl and their ability to transform rodent fibroblasts. CONCLUSION: Our data suggest that, ultimately, the substrate specificity of a non-receptor tyrosine kinase is dependent on the binding specificity of its associated SH2 domain. The SH2 domain binds tightly to a subset of proteins phosphorylated by the catalytic domain, leading to processive phosphorylation of those proteins. Substrate specificity can be broadened by an association between the kinase and proteins, such as Crk, that contain additional SH2 domains; this may play a role in malignant transformation by Crk.

Interaction of D-type cyclins with a novel myb-like transcription factor, DMP1.

The cyclin D-dependent kinases CDK4 and CDK6 trigger phosphorylation of the retinoblastoma protein (RB) late in G1 phase, helping to cancel its growth-suppressive function and thereby facilitating S-phase entry. Although specific inhibition of cyclin D-dependent kinase activity in vivo can prevent cells from entering S phase, it does not affect S-phase entry in cells lacking functional RB, implying that RB may be the only substrate of CDK4 and CDK6 whose phosphorylation is necessary for G1 exit. Using a yeast two-hybrid interactive screen, we have now isolated a novel cyclin D-interacting myb-like protein (designated DMP1), which binds specifically to the nonamer DNA consensus sequences CCCG(G/T)ATGT to activate transcription. A subset of these DMP1 recognition sequences containing a GGA trinucleotide core can also function as Ets-responsive elements. DMP1 mRNA and protein are ubiquitously expressed throughout the cell cycle in mouse tissues and in representative cell lines. DMP1 binds to D-type cyclins directly in vitro and when coexpressed in insect Sf9 cells. In both settings, it can be phosphorylated by cyclin D-dependent kinases, suggesting that its transcriptional activity may normally be regulated through such mechanisms. These results raise the possibility that cyclin D-dependent kinases regulate gene expression in an RB independent manner, thereby serving to link other genetic programs to the cell cycle clock.