MSC/ECM Cellular Complexes Induce Periodontal Tissue Regeneration.

Transplantation of mesenchymal stem cells (MSCs), which possess self-renewing properties and multipotency, into a periodontal defect is thought to be a useful option for periodontal tissue regeneration. However, developing more reliable and predictable implantation techniques is still needed. Recently, we generated clumps of an MSC/extracellular matrix (ECM) complex (C-MSC), which consisted of cells and self-produced ECM. C-MSCs can regulate their cellular functions in vitro and can be grafted into a defect site, without any artificial scaffold, to induce bone regeneration. Accordingly, this study aimed to evaluate the effect of C-MSC transplantation on periodontal tissue regeneration in beagle dogs. Seven beagle dogs were employed to generate a premolar class III furcation defect model. MSCs isolated from dog ilium were seeded at a density of 7.0 × 104 cells/well into 24-well plates and cultured in growth medium supplemented with 50 µg/mL ascorbic acid for 4 d. To obtain C-MSCs, confluent cells were scratched using a micropipette tip and were then torn off as a cellular sheet. The sheet was rolled up to make round clumps of cells. C-MSCs were maintained in growth medium or osteoinductive medium (OIM) for 5 or 10 d. The biological properties of C-MSCs were evaluated in vitro, and their periodontal tissue regenerative activity was tested by using a dog class III furcation defect model. Immunofluorescence analysis revealed that type I collagen fabricated the form of C-MSCs. OIM markedly elevated calcium deposition in C-MSCs at day 10, suggesting its osteogenic differentiation capacity. Both C-MSCs and C-MSCs cultured with OIM transplantation without an artificial scaffold into the dog furcation defect induced periodontal tissue regeneration successfully compared with no graft, whereas osteogenic-differentiated C-MSCs led to rapid alveolar bone regeneration. These findings suggested that the use of C-MSCs refined by self-produced ECM may represent a novel predictable periodontal tissue regenerative therapy.

R1 resection for aggressive or advanced colorectal liver metastases is justified in combination with effective prehepatectomy chemotherapy.

AIMS: Here we reassess anticipated inability to obtain a microscopically clear surgical margin as an absolute contraindication to surgery for colorectal liver metastases in view of improvements in treatment modalities adjunctive to surgery. METHODS: We retrospectively analysed 310 patients treated at our institution to estimate the survival benefit from R1 hepatectomy performed to remove liver metastases from colorectal cancer. RESULTS: Considering all 310 patients evaluated, the R1 resection group (positive margin; n = 55) showed a lower disease-free rate (P < 0.01) and worse overall survival (P < 0.01) than the R0 resection group (negative margin; n = 255). When patients were divided according to initial resectability, similar differences in disease-free rate and overall survival (P = 0.03) between R1 (n = 19) and R0 (n = 182) were observed in patients whose metastases were resectable. However, superior impact of R0 resection (n = 73) compared to R1 resection (n = 36) on disease-free rate (P = 0.44) and overall survival (P = 0.50) was not confirmed in patients with initially unresectable or marginally resectable metastases, especially those with a favourable response to prehepatectomy chemotherapy. CONCLUSIONS: A predicted positive surgical margin after resection no longer should be an absolute contraindication to surgery for aggressive or advanced liver metastases.

Histamine ameliorates anti-glomerular basement membrane antibody-induced glomerulonephritis in rats.

Anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis involves T-helper type 1 (Th1) responses leading to rapid crescent formation. As many inflammatory and immune responses in general are affected by histamine, we examined the effects of histaminergic ligands on immune renal injury in the rat. Female Wistar-Kyoto rats were injected intraperitoneally with an antibody against the GBMs. Histaminergic ligands were then injected twice daily for 5 days after which renal function was assessed by proteinuria. Treatment with histamine led to significant dose-dependent reductions in proteinuria compared to the control antibody-injected group and markedly decreased the number of crescentic glomeruli and macrophage infiltration of the glomeruli. Furthermore, histamine significantly decreased the plasma concentration of interleukin-12, a Th1-type cytokine compared to the antibody-injected control animals. Dimaprit, an H(2)/H(4) agonist, mimicked the effects of histamine on proteinuria and crescent formation. Clozapine, an H(4) agonist, tended to mimic the effects of histamine, whereas an H(1), mepyramine, or an H(2) antagonist, ranitidine, did not reverse the protective effect of histamine. We suggest that histamine may alleviate renal injury in anti-GBM glomerulonephritis by suppressing the immune response.

Down-regulation of GABA-transporter function by hippocampal translation products: its possible role in epilepsy.

The genetically epileptic mouse strain (El) is used as a model for human temporal lobe epilepsy. To address the question of whether altered function of the neuronal GABA transporter GAT1 is involved in the pathology of epilepsy of El mice, we expressed in Xenopus oocytes cloned GAT1 of mouse brain by injection of complementary ribonucleic acid (cRNA) and co-injected messenger ribonucleic acid (mRNA) isolated from the hippocampus of non-epileptic control mother strain (ddY) mice and from El mice. GABA transporter activity was investigated by measurements of [(3)H]-GABA uptake as well as by steady-state and transient current measurements under voltage clamp.Co-injection of hippocampal mRNA into oocytes reduced GAT1-mediated transport. This effect was more pronounced for mRNA from ddY mice than for that from El mice that never experienced seizures, El(-), and being absent for mRNA from El mice that have had high seizure experience, El(+). The pronounced inhibition of GABA transport after injection of mRNA from the ddY strain results from reduced expression of functional GAT1, but to about one third also from a reduced GABA translocation rate. The reduced translocation can be attributed to a reduced forward rate of a step associated with extracellular Na(+) binding. If the results can be applied to the mouse brain, we may hypothesise that in ddY mice some GAT down-regulating factor translated from hippocampal mRNA may be involved to keep GAT1 activity low, and hence GABA concentration in synaptic cleft high. In El(-) mice such regulatory mechanism may be reduced or counteracted by another unknown factor present in El(-) brain. The repeated seizure experience in El(+) mice enhances this compensatory effect.

The role of the hypothalamic nitric oxide in the pressor responses elicited by acute environmental stress in awake rats.

We quantitatively investigated the change in nitric oxide (NO) in the hypothalamic paraventricular nucleus (PVN) and its effect on cardiovascular regulation during shaker stress (SS) using brain microdialysis in awake rats. Male Wistar rats were fed either N(G)-nitro-L-arginine methyl ester (L-NAME, 0.7 g/L) or tap water for 2 weeks. Two days after implantation of an arterial catheter and guide shaft, a microdialysis probe was placed to perfuse the PVN with degassed Ringer solution at 2 microl/min in awake normotensive Wistar (CONTROL) and chronic L-NAME-treated hypertensive rats. After the rat was placed in a plastic cage set on a shaker, the blood pressure and heart rate was monitored and 10-min SS was loaded at a frequency of 200 cycles/min. Dialysate samples were analyzed by NO analyzer (based on the Griess reaction) every 10 min, and NOx (NO(2)(-) + NO(3)(-)) was measured. Plasma NOx was also measured before and after SS. Pressor responses elicited by SS were significantly greater in L-NAME-treated rats than in the CONTROL. Although NOx in the PVN dialysate were increased by SS in the CONTROL, these responses were attenuated in chronic L-NAME-treated rats. Resting plasma NOx were higher in the CONTROL than in L-NAME-treated rats. SS elicited no difference between two groups in plasma NOx. These results indicated that NO within the PVN, but not in systemic circulation, may play a role on the attenuation of the pressor responses elicited by SS. The dysfunction of NO release within the PVN may, in part, play a role in the exaggerated pressor responses in acute environmental stress.

A genome-wide linkage analysis of orchard grass-sensitive childhood seasonal allergic rhinitis in Japanese families.

Seasonal allergic rhinitis (SAR) is an inflammatory disease of the nose and eyes that follows sensitization to air-born pollens. We conducted a genome-wide linkage screening of 48 Japanese families (188 members) with orchard grass (OG)-sensitive SAR children (67 affected sib-pairs) in a farming community in central Japan where OG was planted for apple farming and OG pollen is a major cause of SAR. We used the GENEHUNTER program to performed nonparametric multipoint linkage analysis for OG-sensitive SAR as a qualitative trait and for log total serum IgE levels and OG-RAST IgE levels as quantitative traits. Genotyping data of 400 microsatellite markers suggested linkage of SAR to chromosomes 1p36.2, 4q13.3, and 9q34.3 (P < 0.001), linkage of serum total IgE levels to 3p24.1, 5q33.1, 12p13.1, and 12q24.2 (P < 0.001), and linkage of OG-RAST IgE levels to 4p16.1, 11q14.3, and 16p12.3 (P < 0.001). Weak evidence for linkage of SAR to 5q33.1 was also observed (P = 0.01). All these regions, with the exception of 9q34.3, have been previously reported to be linked to asthma and/or atopy. These data suggest that, although loci linked to SAR are likely to be common to asthma, a strong contribution by specific gene(s) to OG-sensitive SAR is unlikely.

[Transarterial chemoembolization (TAE) with degradable starch microspheres (DSM) in advanced hepatocellular carcinoma].

This study was undertaken to evaluate the clinical utility of chemoembolization using degradable starch microspheres (DSM), which resolve in a short period in patients with advanced hepatocellular carcinoma (HCC). Twenty-one patients underwent DSM chemoembolization 24 times. After a mixture of iodized oil and epirubicin was injected into the hepatic arteries, the patients were embolized with DSM alone 16 times. In the other 8 times, embolization was done in one hepatic lobe with DSM and in the other hepatic lobe with gelatin sponge (GS). There was no major complication related to chemoembolization. Tumor response (complete, partial, and minor responses) was found in 46% of patients after TAE. Tumor recurrence was found in 64% of responders after a mean period of 2.0 months. The response rate was significantly higher when chemoembolization was performed using both DSM and GS than when it was done with DSM alone (63% vs 37%, p < 0.04). Although the response rate after DSM-TAE is low, its anticancer effect is reinforced when used as an adjuvant therapy of GS-TAE.

The relationship between a leaf-rolling moth (Dactylioglypha tonica) and fungi covering the cocoon.

To discover the relationship between a leaf-rolling moth and the fungi densely covering its cocoons, the rolled nest leaves were collected in two districts in Japan and antibacterial properties of the fungi were examined. Cocoons and fungi isolated from the nest were classified into 5 categories by the growth stages of the insects, and 7 categories based on taxonomic properties and pigment productivity, respectively. The dominant genus was Penicillium in each location. However, the composition of the fungal categories was different and seemed to depend on their circumstances. From all cocoons with larvae, the strains that belonged to the same fungal category and produced the same antibiotic (deoxyherqueinone) were isolated. From these results, the species-specific relationship between the insect and fungi or fungal products was considered to be not extremely tight, and it was suggested the period of the larval spinning of the cocoon is a key stage of this unique relationship.

T-cell-immunity-based inhibitory effects of orally administered herbal medicine juzen-taiho-to on the growth of primarily developed melanocytic tumors in RET-transgenic mice.

We examined the effect of oral administration of juzen-taiho-to, one of the most popular herbal medicines in Japan, on primary melanocytic tumor growth in RET-transgenic mice. There was virtually no difference between the lengths of tumor-free stages in the juzen-taiho-to-treated mice and the untreated littermate control mice. The rate of tumor growth in the juzen-taiho-to-treated mice, however, was greatly suppressed during the entire period after the initial tumor development. Correspondingly, the life span of juzen-taiho-to-treated transgenic mice was longer (over 6 mo in mean value) than that of control mice. We partially elucidated the mechanism of the antitumor effect of juzen-taiho-to. The addition of juzen-taiho-to at any of a wide range (50-1600 microg per ml) of concentrations to in vitro cultures of Mel-Ret cells, a malignant melanoma cell line derived from a RET-transgenic mouse, caused neither cell death nor cell cycle arrest directly. The addition of 50-400 microg per ml of juzen-taiho-to to cultures of murine spleen cells, however, promoted their DNA synthesis. More importantly, peritoneal exudate cells from the juzen-taiho-to-treated transgenic mice, in which the ratio and number of T cells were increased, displayed an antitumor immunity against Mel-Ret cells in vitro. Interestingly, the peritoneal-exudate-cell-associated antitumor immunity was further augmented by the addition of 200-400 microg per ml of juzen-taiho-to in vitro. This immunity, which was primarily conveyed by Thy-1+ T cells, was antigen (RET/melanoma) specific and cytotoxic. Amongst various chemical ingredients of juzen-taiho-to examined in this study, glycirrhizin displayed an action, partially replacing that of juzen-taiho-to, in promoting anti-Mel-Ret immunity when supplementarily added in vitro. These results suggest that juzen-taiho-to suppresses once-developed primary melanocytic tumors through potentiation of T-cell-mediated antitumor cytotoxic immunity in vivo.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

A malonylated anthocyanin and flavonols in blue Meconopsis flowers.

The structures of the major anthocyanin and two flavonols from the blue flowers of Meconopsis were identified by NMR spectroscopy as being cyanidin 3-O-[(6-O-malonyl-2-O-B-D-xylopyranosyl)-beta-D-glucopyranoside]-7-O-beta-D-glucopyranoside, kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-glucopyranoside and kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-galactopyranoside respectively.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6.

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

The functional unit of Na,K-ATPase is a monomeric alphabeta protomer.

The ouabain-resistant and ouabain-sensitive alpha-subunit cRNAs in various molar ratios were injected into Xenopus oocytes together with cRNA for the beta-subunit. The ouabain-resistant ATPase activity, as well as ouabain-resistant Rb+ uptake, of the injected oocytes increased linearly with increasing the amount of cRNA for the ouabain-resistant alpha-subunit. When a functionless mutant was used instead of the ouabain-sensitive alpha-subunit, similar results were obtained in ATPase activity and Rb+ uptake. These results indicate that a monomeric alphabeta protomer is a functional unit of membrane-bound Na,K-ATPase, even if the enzyme exists structurally as a diprotomer or higher oligomers in membranes.

Nitric oxide is an excitatory modulator in the rostral ventrolateral medulla in rats.

Nitric oxide is a messenger molecule having various functions in the brain. Previous studies have reported conflicting results for the roles of nitric oxide in the rostral ventrolateral medulla, a major center that regulates sympathetic and cardiovascular activities. We hypothesized that in this region, nitric oxide may have a biphasic effect on cardiovascular activity. Microinjection of a low dose (1 nmol) of a nitric oxide donor sodium nitroprusside or a cyclic GMP agonist 8-bromocyclic GMP into this area increased arterial pressure, whereas injection of a nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester or a soluble guanylate cyclase inhibitor methylene blue decreased arterial pressure. Microinjection of a high dose (100 nmol) of sodium nitroprusside decreased arterial pressure and inhibited spontaneous respiration with concomitant production of peroxynitrite, a strong cytotoxic oxidant. Increases in arterial pressure caused by microinjection of L-glutamate were inhibited after preinjection of Nomega-nitro-L-arginine methyl ester or methylene blue. Increases in arterial pressure caused by microinjection of sodium nitroprusside (1 nmol) were inhibited after preinjection of a glutamate receptor antagonist kynurenate. These results suggest that low doses of nitric oxide may increase arterial pressure, whereas high doses of nitric oxide may decrease arterial pressure through cytotoxic effects in the rostral ventrolateral medulla. They also indicate that nitric oxide may stimulate neurons both through activation of the nitric oxide cyclic GMP pathway and through modulation of glutamate receptor stimulation, and therefore, increase arterial pressure in rats.

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.

Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl.

The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

[Thoracoscopic pleural biopsy under local anesthesia using a 2 mm laparoscope].

Thoracoscopy is indicated in patients with undiagnosed effusion after conventional methods. It has been usually performed under general anesthesia or using a thoracoscope with a thoracoscope with a diameter over 5 mm. However, it is an invasive diagnostic technique. We evaluated the feasibility of thoracoscopic pleural biopsy under local anesthesia using a 2 mm laparoscope. Six patients with a pleural effusion of unknown etiology after conventional methods, underwent thoracoscopy under local anesthesia. A 2 mm laparoscope and biopsy forceps (2 mm Minisite, United States Surgical Corp., USA) was used in all patients. Pleural fluid was removed, and the thoracic cavity was inspected. Thoracoscopic intercostal blocks were performed with 1% lidocaine, and then a biopsy was performed. The biopsy specimen was sent for histopathology. Three patients were shown to have carcinomatous pleurisy, two of them with localized lesions less than 10 mm. In the remaining three patients, non-specific diagnoses were made, but long-term follow-up revealed no malignant pleural disease. Although the pictures obtained using a 2 mm laparoscope were inferior in quality, they were adequate for the detection of malignant lesions in the pleural cavity. There were no procedure-related complications. These findings suggest that thoracoscopy using a 2 mm laparoscope is (1) a useful diagnostic tool in cases of pleural malignancy; (2) a minimally invasive method with the advantage of being easily performed under local anesthesia. Thus, thoracoscopic pleural biopsy using a 2 mm laparoscope appears to be useful for undiagnosed pleural effusion.

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors.

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

TLR6: A novel member of an expanding toll-like receptor family.

Drosophila Toll protein is shown to activate the innate immune system in adult and regulate the dorsoventral patterning in the developing embryo. Recently, five human homologs of Drosophila Toll, designated as Toll-like receptors (TLRs), have been identified and shown to play a role in the innate immune response. We report here the molecular cloning and characterization of a new member of Toll-like receptor family, Toll-like receptor 6 (TLR6). Human and murine TLR6 are type-I transmembrane receptors that contain both an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic Toll/IL-1 receptor (IL-1R)-like region. The amino acid sequence of human TLR6 (hTLR6) is most similar to that of hTLR1 with 69% identity. RT-PCR analysis revealed that murine TLR6 is expressed predominantly in spleen, thymus, ovary and lung. Like other TLR family members, constitutively active TLR6 activates both NF-kappaB and c-Jun N-terminal kinase (JNK). The TLR6 gene, as well as the TLR1 gene, mapped to the proximal region of murine chromosome 5 within 1.7cM of each other. These results suggest that TLR6 is a novel member of an expanding TLR family.