CXCR4 antagonist AMD3100 enhances the response of MDA-MB-231 triple-negative breast cancer cells to ionizing radiation.

Radiation therapy (RT) is one of the primary modalities for triple-negative breast cancer (TNBC) treatment. However, due to the pro-metastatic potential of radiation and the intrinsic radiation resistance of some tumors, many patients experience RT failure, which leads to cancer relapse and distant metastasis. This preclinical study evaluated the efficacy of the antagonist of the SDF-1 receptor CXCR4, AMD3100, as a radiosensitizer in TNBC models. The combined effect of ionizing radiation and AMD3100 was determined in vitro by surviving fraction, cell cycle distribution, Bax and Bcl-2 expression, and apoptosis assays in a TNBC cell line (MDA-MB-231). For in vivo studies, human xenograft athymic nude mice were used. Treatment of TNBC cells with AMD3100 significantly augmented cellular radiosensitivity. Radiosensitivity was enhanced specifically through increased Bax expression, reduced Bcl-2 expression, prolonged G2-M arrest, and increased apoptosis. Combined treatment with AMD3100 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.5 to 1.8. These findings support the evaluation of antagonists of the SDF-1 receptor CXCR4, such as AMD3100, as potent radiosensitizers in TNBC.

Physiochemical properties of rice starch for production of vermicelli with premium quality.

Rice vermicelli is a main food consumed in China and Southeast Asia. Quality of rice vermicelli varies with rice cultivars. Parameters including amylose content, amylopectin distribution, thermal and pasting characteristics, gel texture and starch granules of three rice cultivars "Zhongjiazao 17", "Xiangzaoxian 24" and "Thai Jasmine Rice", were studied for their impacts on vermicelli quality. Results showed significant differences for the measurements of the quality traits and indicated that a favorable quality of vermicelli was not determined by any single factor instead of a combination of multi-parameters. A vermicelli with a favorable quality could be produced from a rice variety with a high apparent amylose content (>25%), a protein content of 11%, an intermediate gelatinization temperature and gel consistency, and a gel hardness (~3 N for a Rapid Viscosity Analyzer pasting) and moderate retrogradation capacity (a setback viscosity of 30-100 RVU).

CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission.

Despite the heterogeneity of acute myeloid leukemia (AML), overexpression of the interleukin-3 receptor-α (CD123) on both the more differentiated leukemic blast and leukemic stem cells (LSCs) provides a therapeutic target for antibody treatment. Here we present data on the potential clinical activity of the monoclonal antibody CSL362, which binds to CD123 with high affinity. We first validated the expression of CD123 by 100% (52/52) of patient samples and the correlation of NPM1 and FLT3-ITD mutations with the high frequency of CD123 in AML. In vitro studies demonstrated that CSL362 potently induced antibody-dependent cell cytotoxicity (ADCC) of AML blasts including CD34+CD38-CD123+ LSCs by natural killer cells (NKs). Importantly, compared with healthy donor (HD) NKs, NKs drawn from AML patients in remission had a comparable ADCC activity against leukemic cells; of note, during remission, immature NKs were five times higher in AML patients than that in HDs. Significantly, we report a case where leukemic cells were resistant to autologous ADCC; however, the blasts were effectively lysed by CSL362 together with donor-derived NKs after allogeneic hematopoietic stem cell transplantation. These studies highlight CSL362 as a promising therapeutic option following chemotherapy and transplant so as to improve the outcome of AML patients.

The cadmium and lead content of the grain produced by leading Chinese rice cultivars.

The cadmium (Cd) and lead (Pb) content in both white and wholemeal flour milled from 110 leading rice cultivars was assessed. The white flour Cd content ranged from <0.0025 to 0.2530mg/kg (geometric mean (GM)=0.0150mg/kg), while its Pb content ranged from <0.0250 to 0.3830mg/kg (GM=0.0210mg/kg). The indica types took up higher amounts of Cd and Pb than did the japonica types. Although the heavy metal content of wholemeal flour tended to higher than that of white flour, nevertheless 84.5% (Cd) and 95.4% (Pb) of the entries were compliant with the national maximum allowable concentration of 0.2000mg/kg of each contaminant. An analysis of the Cd content in the white flour of three indica type cultivars grown in two consecutive years at two locations indicated that Cd content may be significantly affected by the conditions prevailing in the growing season.

First Report of Aspergillus niger Causing Root Rot of Peanut in China.

Peanut (Arachis hypogaea) is one of the most important oil crops and food legumes worldwide. China sows approximately 3.5 million hectares each year and produces 40% of the world's peanuts. Fungal diseases are among the main biotic stresses affecting peanut production. Root rot is a serious disease caused by several fungi. Pythium spp., Fusarium spp., and Rhizopus spp. are some of the root rot fungi that have been reported in China. In 2012 and 2013, root rot symptoms were observed in several fields in Laixi District, Qingdao City, Shandong Province, China. The first symptoms appeared in July. Initial symptoms of the disease were brown spots on the stem base and root. Affected plants were stunted, with leaf chlorosis, reduced growth, or sudden wilting. As disease progressed, the infected tissues showed brown discoloration and rot, and abundant dark brown and black powdery spores were visible on the surfaces of affected parts. Eventually, affected plants collapsed and died. To isolate the causal organism, roots and stems were cut into sections, which were surface-disinfected with 70% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 50 s, rinsed with sterilized water three times, dried, placed on Czapek's Dox agar supplemented with chloramphenicol (100 µg/ml), and incubated at 28°C for 7 days. Fungal colonies were white initially and then covered with a dense layer of dark brown or black conidial heads. The conidial head was radiate; vesicles were nearly spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose (3.0 to 5.5 µm in diameter), dark brown to black, with rough cell walls. Total genomic DNA was extracted from mycelia using the EasyPure Genomic DNA Kit (TransGEN, Beijing, China). The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (2). The purified products were separately sequenced in both directions using the same primer pair. The sequences (GenBank Accession No. KJ848716) obtained were 99% similar to the ITS sequence of isolates of Aspergillus niger. This, together with the morphological characters (1) described above, suggested that the microorganism we had isolated was A. niger. Koch's postulates were completed in the laboratory by inoculating peanut. Thirty Huayu20 peanut seeds were placed in a 500-ml sterile pot with 300 g of autoclaved soil. Twenty days after seedling emergence, 15 peanut plants were wounded with a needle and inoculated with 5 ml of conidia suspension (106 ml-1). The same number of peanuts were similarly wounded and inoculated with 5 ml of sterile distilled water to serve as controls in the same pot. All peanuts were kept in a randomized complete block design at 30°C under a 12-h photoperiod. After 7 days, disease symptoms similar to those observed in the field appeared in all inoculated but not in non-inoculated peanuts. The tests were repeated three times in the greenhouse. Koch's postulates were satisfied after re-isolating the A. niger from inoculated peanuts using the method described above. To our knowledge, this is the first report of A. niger causing root rot in peanut in China. References: (1) M. A. Klich. Page 12 in: Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, 2002. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Optimisation of near-infrared reflectance model in measuring protein and amylose content of rice flour.

Near-infrared reflectance spectroscopy (NIRS) has been used to predict the cooking quality parameters of rice, such as the protein (PC) and amylose content (AC). Using brown and milled flours from 519 rice samples representing a wide range of grain qualities, this study was to compare the calibration models generated by different mathematical, preprocessing treatments, and combinations of different regression algorithm. A modified partial least squares model (MPLS) with the mathematic treatment "2, 8, 8, 2" (2nd order derivative computed based on 8 data points, and 8 and 2 data points in the 1st and 2nd smoothing, respectively) and inverse multiplicative scattering correction preprocessing treatment was identified as the best model for simultaneously measurement of PC and AC in brown flours. MPLS/"2, 8, 8, 2"/detrend preprocessing was identified as the best model for milled flours. The results indicated that NIRS could be useful in estimation of PC and AC of breeding lines in early generations of the breeding programs, and for the purposes of quality control in the food industry.

Aberrant expression of CXCR4 significantly contributes to metastasis and predicts poor clinical outcome in breast cancer.

Triple negative breast cancer is known for its visceral metastasis. We have found that CXCR4 is overexpressed in triple negative breast cancer and is associated with visceral metastasis. We further investigated whether CXCR4 is a prognostic factor affecting survival following visceral metastasis in breast cancer patients. Our results indicate that increased CXCR4 expression among breast cancer patients with visceral metastasis was positively correlated with poor overall survival (P<0.001). Silencing of CXCR4 was associated with a decrease in the tumorigenic properties of MDA-MB-231 breast cancer cells, caused reversion of EMT and suppression of MMP-9, increased apoptosis, and caused a reduced incidence of tumor lung metastasis in mice. These results are indicative of CXCR4 having a predictive role in patients with visceral metastasis and indicate that shRNA knock down of CXCR4 might be a novel therapeutic strategy to prevent breast cancer metastasis when CXCR4 is overexpressed.

Selection pressure and founder effects constrain genetic variation in differentiated populations of soilborne bymovirus Wheat yellow mosaic virus (Potyviridae) in China.

To study the population genetic structure and forces driving the evolution of Wheat yellow mosaic virus (WYMV), the nucleotide sequences encoding the coat protein (CP) (297 sequences) or the genome-linked virion protein (VPg) (87 sequences) were determined from wheat plants growing at 11 different locations distributed in five provinces in China. There were close phylogenetic relationships between all sequences but clustering on the phylogenetic trees was congruent with their provenance, suggesting an origin-dependent population genetic structure. There were low levels of genetic diversity, ranging from 0.00035 ± 0.00019 to 0.01536 ± 0.00043 (CP), and 0.00086 ± 0.00039 to 0.00573 ± 0.00111 (VPg), indicating genetic stability or recent emergence of WYMV in China. The results may suggest that founder effects play a role in shaping the genetic structure of WYMV. Between-population diversity was consistently higher than within-population diversity, suggesting limited gene flow between subpopulations (average FST 0.6241 for the CP and 0.7981 for the VPg). Consistent amino acid substitutions correlated with the provenance of the sequences were observed at nine positions in the CP (but none in the VPg), indicating an advanced stage in population structuring. Strong negative (purifying) selection was implicated on both the CP and VPg but positive selection on a few codons in the CP, indicating an ongoing molecular adaptation.

First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia.

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of "Red with dark green edge," are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5'-TGACTAGAACAGTAGTAAAG-3' / CbVd-5-PR: 5'-AATTGAGGTCAAACCTCTTT-3') demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar "Red with dark green edge" 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:315, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

A modified method of labia minora reduction: the de-epithelialised reduction of the central and posterior labia minora.

OBJECTIVE: The aim of this study was to introduce a new method of labiaplasty. Here we describe the surgical procedure, outcomes and the advantages and disadvantages of this method. METHOD: The medical records of 167 patients aged between 20 and 43 years who underwent reduction of the labia minora from May 2006 to March 2011 were reviewed. The procedures performed in these studies used de-epithelialised reduction of the middle and posterior sections of the labia minora. RESULTS: All the surgeries were performed successfully, and 164 patients experienced an uneventful postoperative period. A minor dehiscence occurred in one patient, who recovered with no requirement for additional treatment. Another two women felt that the reduction was not fully achieved. All of the patients were satisfied with the eventual aesthetic appearance. CONCLUSIONS: The de-epithelialised reduction of the middle and posterior portion of the labia minora is a simple and safe method that is associated with satisfactory outcomes.

First Report of the Occurrence of Sweet potato leaf curl virus in Tall Morningglory (Ipomoea purpurea) in China.

Natural occurrence of Sweet potato leaf curl virus (SPLCV) has been reported in Ipomoea batatas (sweet potato, Convolvulaceae) or I. indica (Convolvulaceae) in several countries including the United States, Sicily, and China (1-3). In September of 2007, while collecting samples showing begomovirus-like symptoms in the Chinese province of Fujian, we observed tall morningglory (I. purpurea (L.) Roth, also known as Pharbitis purpurea (L.) Voigt), plants with slightly yellow mosaic and crinkled leaves. Total DNA was extracted from leaves of these plants and tested by rolling circle amplification (4). Amplification products were digested by the restriction enzyme BamHI for 30 min. Restriction products (2.8 kb) were then cloned into pMD18T vector (Takara Biotechnology, China) and sequenced. Comparison of complete DNA sequences by Clustal V analysis revealed that these samples were infected by the same virus, and an isolate denoted F-p1 was selected for further sequence analysis. F-p1 was 2,828 nucleotides, with the typical genomic organization of begomoviral DNA-A (GenBank Accession No. FJ515896). F-p1 was compared with the DNA sequences available in the NCBI database using BLAST. The whole DNA sequence showed the highest nucleotide sequence identity (92.1%) with an isolate of SPLCV (GenBank Accession No. FJ176701) from Jiangsu Province of China. The result confirmed that the samples from the symptomatic tall morningglory were infected by SPLCV. To our knowledge, this is the first report of the natural occurrence of SPLCV in I. purpurea, a common weed species in China. References: (1). P. Lotrakul et al. Plant Dis. 82:1253, 1998. (2). R. W. Briddon et al. Plant Pathol. 55:286, 2006. (3) Y. S. Luan et al. Virus Genes 35:379, 2007. (4) D. Haible et al. J. Virol. Methods 135:9, 2006.

First Report of Ageratum yellow vein virus Causing Tobacco Leaf Curl Disease in Fujian Province, China.

A leaf curling disease was observed on 7% of tobacco plants during December 2005 in research plots in the Cangshan District of Fuzhou, Fujian, China. Tobacco plants were infested with Bemisia tabaci, suggesting begomovirus etiology. To identify possible begomoviruses, total DNA was extracted from four symptomatic leaf samples (F1, F2, F3, and F4). The degenerate primers PA and PB were used to amplify part of the intergenic region and AV2 gene of DNA-A-like molecules (3). A 500-bp DNA fragment was amplified by PCR from all four samples. The PCR products were cloned and sequenced (GenBank Accession Nos. EF531601-EF531603 and EF527823). Alignment of the 500-bp sequences for the four isolates indicated that they shared 98.5 to 99.6% nt identity, suggesting that the plants were all infected by the same virus. Overlapping primers TV-Full-F (5'-GGATCCTCTTTTGAACGAGTTTCC-3') and TV-Full-R (5'-GGATCCCACATGTTTAAAATAATAC-3') were then designed to amplify the full-length DNA-A from sample F2. The sequence was 2,754 nucleotides long (GenBank Accession No. EF527823). A comparison with other begomoviruses indicated the F2 DNA-A had the highest nucleotide sequence identity (95.7%) with Ageratum yellow vein virus (AYVV; GenBank Accession No. X74516) from Singapore. To further test whether DNAß was associated with the four viral isolates, a universal DNAß primer pair (beta 01 and beta 02) was used (4). An amplicon of approximately 1.3 kb was obtained from all samples. The DNAß molecule from F2 was then cloned and sequenced. F2 DNAß was 1,345 nucleotides long (GenBank Accession No. EF527824), sharing the highest nucleotide sequence identity with the DNAß of Tomato leaf curl virus (97.2%) from Taiwan (GenBank Accession No. AJ542495) and AYVV (88.8%) from Singapore (GenBank Accession No. AJ252072). The disease agent was transmitted to Nicotiana tabacum, N. glutinosa, Ageratum conyzoides, Oxalis corymbosa, and Phyllanthus urinaria plants by whiteflies (B. tabaci) when field infected virus isolate F2 was used as inoculum. In N. tabacum and N. glutinosa plants, yellow vein symptoms were initially observed in young leaves. However, these symptoms disappeared later during infection and vein swelling and downward leaf curling symptoms in N. tabacum and vein swelling and upward leaf curling in N. glutinosa were observed. In A. conyzoides, O. corymbosa, and P. urinaria plants, typical yellow vein symptoms were observed. The presence of the virus and DNAß in symptomatic plants was verified by PCR with primer pairs TV-Full-F/TV-Full-R and beta 01/beta 02, respectively. The above sequence and whitefly transmission results confirmed that the tobacco samples were infected by AYVV. In China, Tobacco leaf curl Yunnan virus, Tobacco curly shoot virus, and Tomato yellow leaf curl China virus were reported to be associated with tobacco leaf curl disease (1,3). To our knowledge, this is the first report of AYVV infecting tobacco in China. A. conyzoides is a widely distributed weed in south China and AYVV was reported in A. conyzoides in Hainan Island, China (2). Therefore, this virus may pose a serious threat to tobacco production in south China. References: (1) Z. Li et al. Phytopathology 95:902, 2005. (2) Q. Xiong et al. Phytopathology 97:405, 2007. (3) X. Zhou et al. Arch. Virol. 146:1599, 2001. (4) X. Zhou et al. J. Gen. Virol. 84:237, 2003.

Efficacy comparison of intravenous artelinate and artesunate in Plasmodium berghei-infected Sprague-Dawley rats.

This paper reports the comparative antimalarial efficacy of intravenous artelinate and artesunate in rats. Prior to efficacy experiments, a Plasmodium berghei-Sprague-Dawley rat model of malaria was developed, in which the clearance effects of intravenous drugs could be readily compared. In efficacy experiments, groups of P. berghei-infected rats were given 3 daily intravenous treatments of artelinate or artesunate at molar equivalent dose rates (total of 0-191.2 micromoles/kg). Artelinate was superior to artesunate in terms of clearance (100% clearance dose of 95.6 micromoles/kg (40 mg/kg) versus 191.2 micromoles/ kg for AS (73.4 mg/kg)) and parasite clearance time (1.7 +/- 0.5 days for AL versus 2.7 +/- 0.5 days for AS at a dose rate of 191.2 micromoles/kg, P < 0.01). No frank clinical toxicity was observed, though both artesunate and artelinate induced dose-related vascular necrosis at the site of injection. The necrosis was less severe and reversible when the drugs were administered via femoral, rather than tail/foot veins. The data suggest that the P. berghei-7-week-old Sprague-Dawley rat model of malaria is reproducible and useful for assessing the efficacy of antimalarials and that artelinate is at least as potent, and safe, as artesunate, the leading clinical treatment for severe malaria.

The properties of the Kir6.1-6.2 tandem channel co-expressed with SUR2A.

Functional ATP-sensitive K (KATP) channels have an octameric subunit structure with four pore-forming subunits (Kir6.x) and four sulfonylurea receptors (SURx). In the present study, the properties of the heteromeric KATP channel whose pore subunits are composed of Kir6.1 and Kir6.2 were examined using a heterologous expression system. In COS7 cells co-transfected with Kir6.1, Kir6.2 and SUR2A at a ratio of 1:1:2, KATP channels showed various unitary conductances between those of Kir6.1/SUR2A (33.6+/-4.2 pS) and Kir6.2/ SUR2A (67.1+/-1.6 pS). Kir6.1-6.2 tandem protein, constructed by fusing the C-terminus of Kir6.1 to the N-terminus of Kir6.2 with a ten glutamine linker sequence, also formed a channel with an intermediate conductance (58.9+/-1.5 pS). Kir6.2 and Kir6.1-6.2 showed similar sensitivity to ATP4-: half-maximal inhibition (IC50) was obtained at 14.1+/-12.8 microM and 17.6+/-9.6 microM, respectively. In the presence of Mg2+, Kir6. 1-6.2 was significantly less sensitive than Kir6.2 to MgATP (IC50=95.5+/-49.6 microM versus 18.9+/-5.0 microM). These results suggest that Kir6.1 and Kir6.2 are endowed with the potential to form a heteromeric KATP channel, which has a low sensitivity to MgATP.

Agonist-independent modulation of L-type Ca currents by basal Gs protein activities in single guinea pig ventricular myocytes.

The modulation of L-type Ca2+ currents (I(Ca,L)) by the basal activities of G proteins was studied in adult guinea pig ventricular myocytes by whole-cell patch-clamp techniques. With intrapipette guanosine triphosphate (GTP) (100 microM), a specific inhibition of G1 proteins by pertussis toxin (PTX) produced an increase in the basal density of I(Ca,L) (from 11.0+/-0.8, n = 13, to 25.0+/-2.0 pA/pF, n = 11, at OmV test potential). In addition, PTX shifted the forskolin (Fsk) concentration-I(Ca,L) response relation significantly leftward (EC50, = 63.7+/-12.5 vs 625+/-75 nM). With intrapipette guanosine diphosphate (GDP)betaS (1 mM), the Fsk-I(Ca,L) relation was also shifted leftward (EC50 = 197+/-18.3 vs 781+/-82.5 nM). However, chronic GDPbetaS dialysis accelerated the rundown of I(Ca,L) significantly, suggesting a potential contribution of Gs proteins in maintaining basal I(Ca,L). In contrast, intra-pipette GTPgammaS (100 microM) produced a transient rise in I(Ca,L) from 11.0+/-3.0 to 22.8+/-7.0 pA/pF (in 3.4 min after whole-cell formation at 0 mV, n = 9), presumably through the activation of Gs proteins. It was followed by a gradual decline in I(Ca,L) (to 15.5+/-3.5 pA/pF), which was still enhanced by Fsk (EC50 = 1450+/-98 nM), indicating that the current decay was not solely due to rundown but to activation of Gi proteins. Gs, in addition to Gi proteins, show sufficient basal activity to modulate I(Ca,L) in an agonist-independent manner.

[Establishment of a TLC identification method for xuanshen(radix scrophulariae)].

OBJECTIVE: To establish a TLC identification method for Xuanshen(Radix Scrophulariae). METHOD: Using TLC with harpagoside and harpagide as reference substances. RESULT: A TLC identification method of Xuanshen has been established. The specificity of the method has been proved by a comparative detection of Xuanshen from different habitats and several crude drugs which are easily confused with Xuanshen. CONCLUSION: The method is simple, accurate and reliable.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism.

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M(1) muscarinic receptors, we activated the ATP-sensitive potassium (K(ATP)) channel with a K(+) channel opener and recorded the whole-cell K(ATP) current. The K(ATP) current was reversibly inhibited by the stimulation of the M(1) receptor, which is linked to phospholipase C (PLC) by the G(q) protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca(2+) with 10 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M(1)-receptor stimulation failed to inhibit the K(ATP) current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP(2)) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP(2) was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5'-[beta,gamma-imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the K(ATP) channel was significantly higher when the M(1) receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the K(ATP) channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP(2) levels.

Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node.

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.

Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.

1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (KATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2DeltaC26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 microM Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1 % (mean +/- s.e.m.) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2 % (n = 13) of the Ca2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2 % (10 microM) and 4.3 +/- 2.5 % (100 microM) of the Ca2+-induced run-down. 4. MgUDP (10 mM) reversed the Ca2+-induced run-down of Kir6.2/SUR2A channels by 60.4 +/- 7.6 % (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2DeltaC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca2+; Kir6.2DeltaC26 channels were inactivated to 44.8 +/- 4.4 % (n = 14) by 100 microM Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2DeltaC26 by 89.8 +/- 7. 7 % (n = 9), and 100 microM wortmannin inhibited this reaction (1.8 +/- 2 %, n = 7). 6. Application of 10 microM phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4 %, n = 10). Even after the MgATP-dependent recovery was blocked by 100 microM wortmannin, PI-4,5-P2 reactivated the channels (102.3 +/- 8.6 %, n = 5). Similar results were obtained with Kir6.2DeltaC26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P2 or phosphatidylinositol-3,4, 5-trisphosphate may upregulate Kir6.2 channels.

[Musculocutaneous flap containing upper part of bilateral trapezius muscles in the treatment of cicatricial contracture deformity of neck].

OBJECTIVE: To evaluate the efficacy of musculocutaneous flap containing the upper part of bilateral trapezius muscles in the treatment of cicatricial contracture deformity of neck. METHODS: From January 1990 to January 1997, twelve cases were treated by musculocutaneous flap containing upper part of bilateral trapezius muscles from 14 cm x 16 cm to 28 cm x 11 cm in size, and 9 of the 12 cases were followed up for 2-3 years. RESULTS: Primary healing of the wounds was observed in all of the cases. The function and the external appearance were satisfactory, and no graft contracture was found in the 9 cases followed up. CONCLUSION: The musculocutaneous flap grafting was an ideal method in the repair of soft tissue defect of neck, with the advantage of invariable blood supply and simple operative procedure.