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The phytochemical study on the stems and leaves of

Background: Validamycin A (Val-A) is one of the most widely used agricultural antibiotics in East Asia especially for controlling rice sheath blight disease. Fermentation contamination of the industrial Val-A producing strain is a common occurrence. Methods: Fermentation culture of S. hygroscopicus 5008 has a special smell that could be distinguished from other tainted samples. The change of the volatiles in untainted and tainted samples was characterized using headspace solid phase microextraction (HS-SPME) combined with gas chromatography- mass spectrometry (GC-MS). Results: Seventy-one volatile compounds (including alkanes, amines, alcohols, esters, aldehydes and others) were identified and there were significant differences in the composition of volatiles among different samples. Principal component analysis (PCA) based on the GC-MS data was used to identify the important volatile compounds that contributed to the differentiation of the fermentation samples under different fermentation stages, as well as among different pollution species and fermentation media. Contamination could be discovered in time irrespective of the stage of fermentation and the contaminating bacteria in broth. Conclusion: It is the first report to detect contamination by volatile compounds in the antibiotic fermentation and it was proved that HS-SPME/GC-MS is an effective contamination detection method in Val-A production.

In this paper, the common diseases about bridge deck pavement of the completed highway in Heilongjiang province have been made practical investigation and analyze why these diseases happen. It also combines the climate features in north cold areas，from the way of using High quality modified asphalt，reducing the catalytic point temperature of pitch to improve the low temperature anti-crack property, using high strength coarse and fine aggregate，adjusting gradation of mixture aggregate and the ratio of oil to stone to improve temperature and water stability and durability for Concrete pavement project of Nao Li river bridge connected Jian San river with Hu Lin highway .From trabecular bending test、Freeze-thaw splitting test、Rutting test and Immersion Marshall test，that compares different influence research on pavement performance of bridge with Bitumen concrete bridge deck which have different polyester fiber content .The experimental result indicates the proportion of polyester fiber can clearly ameliorate the temperature stability、hypothermia anti-cracking performance and water stability of Asphalt .And within a certain range of polyester fiber content，various road performances will be improved with the increasing of polyester fiber content，and on this basis，the optimum polyester fiber content is raised.

As the problem of asphalt concrete bridge deck pavement is becoming more and more serious, how to improve its road performance has become the focus of the study. As the lack of shear strength and bond strength between layers, flaking, raveling and other diseases were occurred on steel bridge deck asphalt pavement. This paper combines the common damages of bridge deck and asphalt concrete pavement in cold area of northeast region, focuses on the analysis of the cause of asphalt concrete pavement damage causes, puts forward preventive measures and recommendations, and present the bridge deck pavement structure in cold area combined with the engineering practice. The research results can effectively guide the construction of bridge deck pavement.

Artocarpus heterophyllus (jack tree) is an evergreen fruit tree belonging to the genus Artocarpus (Moraceae), which is widely distributed in subtropical and tropical regions of Asia. Its fruits (jackfruit), well-known as the world's largest tree-borne fruit, are being consumed in our daily diets as a very popular tropical fruit throughout the world and have been confirmed to hold various health benefits. In this study, five new prenylated chromones, artocarheterones A-E (1-5), as well as seven known prenylated chromones (6-12) were purified and isolated from the ripe fruits of A. heterophyllus (jackfruit). Their chemical structures were determined through comprehensive spectroscopic methods. This is the first report on prenylated chromones isolated from A. heterophyllus. The anti-HIV-1 effects of all isolated chromones were assessed in vitro. As a result, prenylated chromones (1-12) showed remarkable anti-HIV-1 effects with EC50 values ranging from 0.09 to 9.72 μM. These research results indicate that the isolation and characterization of these prenylated chromones with remarkable anti-HIV-1 activities from the ripe fruits of A. heterophyllus could be significant to the discovery and development of new anti-HIV-1 drugs.

The chemical constituents from the stems and leaves of Clausena excavata were isolated and purified by column chromatography with silica gel, ODS, Sephadex LH-20 and RP-HPLC. The chemical structures of the isolated compounds were identified on the basis of physicochemical properties, spectroscopic analysis, as well as the comparisons with the data reported in literature. Nineteen compounds were isolated from the 90% ethanol extract of the stems and leaves of C. excavata, which were identified as methyl orsellinate(1), syringaresinol(2), lenisin A(3), scopoletin(4), osthenol(5), N-benzoyltyrarnine methyl ether(6), N-p-coumaroyltyramine(7), aurantiamide acetate(8), 1H-indole-3-carboxaldehyde(9), furostifoline(10), clausenalansine E(11), 3-formylcarbazole(12), clausine L(13), clausine E(14), methyl carbazole-3-carboxylate(15), glycosinin(16), murrayafoline A(17), clausine H(18) and 2,7-dihydroxy-3-formyl-1-(3'-methyl-2'-butenyl)carbazole(19). Among these isolated compounds, compounds 1-11 were isolated from C. excavata for the first time, and compounds 1, 2 and 10 were isolated from the genus Clausena for the first time. In addition, this study evaluated the anti-rheumatoid arthritis activities of compounds 1-19 by measuring their anti-proliferative effects on synoviocytes in vitro according to MTS method. Compounds 10-19 displayed remarkable anti-rheumatoid arthritis activities, which exhibited the inhibitory effects on the proliferation of MH7 A synovial fibroblast cells with the IC_(50) values ranging from(27.63±0.18) to(235.67±2.16) μmol·L~(-1).

Eight cadinane-type sesquiterpenes, including a new cadinane-type sesquiterpene, named as mappianiodene (

Eight cadinane-type sesquiterpenes, including a new cadinane-type sesquiterpene, named as mappianiodene (1), and seven known analogues (2–8), were isolated and identified from the stems and leaves of Mappianthus iodoides. The chemical structure and absolute configurations of 1 was elucidated by extensive spectral methods and the known compounds were identified by comparing their experimental spectral data with the reported spectral data in the literature. The anti-inflammatory and anti-HIV activities of those isolated cadinane-type sesquiterpenes were tested. As a result, cadinane-type sesquiterpenes 1–8 displayed notable inhibitory effects on NO (nitric oxide) production with IC50 values equivalent to that of the hydrocortisone. Moreover, compounds 1–8 exhibited pronounced anti-HIV-1 reverse transcriptase (RT) activities with the EC50 values in range of 0.17 to 9.28 µM.

Two new prenylated chromones, artoheterophines A (1) and B (2), five known prenylated chromones (3–7), as well as five known biogenetically related prenylated flavonoids (8–12) were isolated and characterized from the stems and leaves of A. heterophyllus. Their chemical structures were unambiguously determined through comprehensive spectral data analyses. The antiproliferative and anti-inflammatory effects of all these isolated prenylated chromones and flavonoids were evaluated in vitro. As a result, compounds 1–12 showed notable inhibitory effects against various human cancer cell lines with IC50 values ranging from 0.36 ± 0.02 to 22.09 ± 0.16 μM. Meanwhile, compounds 1–12 exhibited significant inhibitory activities on nitric oxide (NO) production holding IC50 values in the range of 0.48 ± 0.05–19.87 ± 0.21 µM. These research results suggest that the isolation and characterization of these prenylated chromones (1–7) and flavonoids (8–12) holding significant antiproliferative and anti-inflammatory activities could be significant to the discovery and development of new natural anti-tumor and anti-inflammatory drugs. The findings also provides a phytochemical evidence for further development and utilization of the stems and leaves of A. heterophyllus in health and pharmaceutical products.

A phytochemical investigation on the stems and leaves of Wikstroemia chuii resulted in the isolation of three new daphnane diterpenes, wikstroechuins A–C (1–3), together with eight known analogues (4–11). The structures of new daphnane diterpenes (1–3) were determined on the basis of extensive spectroscopic methods and the known daphnane diterpenes (4–11) were identified by comparing their observable spectroscopic data with those reported spectral data in the literature. The anti-inflammatory effects as well as anti-HIV activities in vitro of all isolated daphnane diterpenes 1–11 were assessed. As a consequence, daphnane diterpenes 1–11 displayed remarkable inhibitory activities on NO (nitric oxide) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells showing IC50 values in the range of 0.12 ± 0.03 to 10.58 ± 0.16 µM. Meanwhile, daphnane diterpenes 1–11 displayed significant anti-HIV-1 reverse transcriptase (RT) effects showing EC50 values ranging from 0.09509 to 8.62356 µM. These research results indicated that the discovery of these new daphnane diterpenes with remarkable anti-inflammatory and anti-HIV activities from W. chuii, especially these new ones, could be extremely meaningful to the discovery of new anti-inflammatory agents and anti-HIV drugs as well as their potential practical values in the health and pharmaceutical products.

A HPLC method for the determination of three effective components in Scutellaria indica from different habitats was established. Analysis was performed on Waters Spherisorb ODS2 (4.6 mm × 250 mm, 5 μ m) with gradient mobile phase of 0.5% glacial acetic acid aqueous solution-acetonitrile. The flow rate was 1 m L/min and the detection wavelength was set at 275 nm. The results showed that the chromatographic elution curves with good resolution and reproducibility were obtained. By comparing with the reference materials, three effective components were determined, namely, scutellarin, scutellarin and apigenin. The method is sensitive, accurate and repeatable, and can be used for the determination of three components in Scutellaria indica. The results also showed that there were significant differences in the chemical composition of Scutellaria indica from different areas. The quality differences of Scutellaria indica from different areas should be considered in the selection of Scutellaria indica.

In this paper, an aptasensor with high sensitivity and rapid response was developed for the detection of staphylococcus enterotoxin B (SEB) by using thiol-modified piezoresistive cantilever. Thiol-modified aptamers, acting as the functionalized sensing elements, were immobilized on the nanogold-coated surface of the sensing cantilever as agents for detecting SEB. By using the functionalized aptasensors, different concentrations of SEB were detected with a wide detection range of 6-100 ng/mL and a quick response in milk. The experimental results indicated that the cantilever-based aptasensors had sufficient sensitivity for the detection of SEB in real food commodities and might provide an economical platform for on-site detections of different toxicants with the advantages of portability, high sensitivity, and rapid response.

In this paper, we present two metamaterial absorbers (MMA) at mid-infrared using multi-resonant structures. The dual band MMAs employ the typical metal/dielectric/metal structure with 80 nm gold ground plane at the bottom, 190 nm SiO2 dielectric spacer in the middle and periodic gold patterns on top. The top resonant structure in MMA1 consists of a gold cross resonator ringed by four gold split-ring resonators (SRR) at the ends of the cross, while in the unit cell of MMA2, gold SRRs are placed at the four quadrants of the cross resonator. MMA1 shows two absorption peaks of 90.3% and 88.4% at 4.17μm and 4.86μm respectively, and the absorption peaks of MMA2 are observed to be 72.4% at 3.90μm and 48.0% at 5.66μm.

This study assessed the importance of the Traditional Chinese Medicine (TCM) pattern on an acupoint-specific effect.This was a TCM pattern subdivision analysis of the first intervention data from a multicenter, randomized, controlled trial (ISRCTN24863192) (the main trial).The main trial recruited participants from six hospitals in three provinces in China.Five hundred and one (501) participants diagnosed with primary dysmenorrhea (PD) were enrolled in the main trial.The main trial randomly and equally divided participants into three treatment groups with bilateral electroacupuncture at three sites, respectively: Sanyinjiao (SP6), Xuanzhong (GB39), and an adjacent nonacupoint. Participants were diagnosed with TCM patterns before the treatment. The intervention was carried out when the visual analogue scale (VAS) score of participant's menstrual pain was ≥ 40 mm on the first day of menstruation and lasted for 30 minutes.The immediate improvement of pain was measured with a 100-mm VAS before the intervention, at 5 minutes, 10 minutes, and 30 minutes during the intervention, and at 30 minutes after the completion of this intervention.Three (3) TCM patterns (n=320) were eligible for analysis, including Cold and Dampness Stagnation pattern (n=184), Qi and Blood Stagnation pattern (n=84), and Qi and Blood Deficiency pattern (n=52). In Cold and Dampness Stagnation pattern, the SP6 group had a significant reduction in VAS scores compared with the GB39 group (mean difference -7.6 mm) and the nonacupoint group (mean difference -8.2 mm), respectively. There was no difference between the latter two groups. There were no group differences in VAS scores in the other two patterns.It suggested that TCM pattern might affect acupoint specific effect on the immediate pain relief obtained for participants with PD.

In this paper, we report a method of fabricating silicon nano-wire based on thermal oxidation technique. In this method, we first fabricate a wider structure with traditional lithography, and then use a layer of silicon nitride as mask to oxidize silicon. At the same time, due to the lateral diffusion and oxidation of oxidant, silicon is consumed by oxidant and the width of the silicon structure will be reduced to nano range when we remove the silicon dioxide. The factors affecting the ratio of vertical and lateral oxidation, for example, the gentle slope caused by isotropic oxidation and the inhomogeneity of the sidewall of silicon nano-wire, are discussed at last. Our results should be useful in generating silicon–based nanospintronics devices with careful selection of the oxide parameters.

This paper proposes a modern service-oriented extensible authentication model base on company (EAMBCom), after researching some security authentication models commonly used in distributed environment. The advantages and disadvantages of strictly hierarchical authentication model (SHAM), and multiple-certificate-authority (multi-CA) model are analyzed. The EAMACom model can regard “company” as logical authentication unit, which can be extended easily and owns the ability of reducing burden of root certificate authority (RootCA). In addition, the EAMACom model can simplify the certification process on the premise of authentication security, so it actually can be used in modern service-oriented distributed file system for security.

Aim: To investigate risk factors which impact on common carotid artery intima media thickness (IMT).Methods: A total of 86 obese children and adolescents and 22 healthy children and adolescents with normal weight were enrolled. Moreover, 23 of 86 obese children and adolescents were diagnosed with metabolic syndrome (MetS). The clinical, biochemical data and the IMT of the common carotid artery were measured in all subjects.Results: Obese and obese with MetS subjects demonstrated a significantly (p < 0.01) thicker intima media (0.69mm, 0.66mm) as compared to the control group (0.38mm), but there was no significant difference of IMT between obese and MetS group. IMT was correlated to body weight, body mass index, waist circumference, waist to hip ratio, systolic blood pressure, diastolic blood pressure, fasting insulin, homoeostasis model assessment-insulin resistance, triglyceride, high-density lipoprotein- cholesterol, low-density lipoprotein-cholesterol, alanine aminotransferase, aspartate aminotransferase and fatty liver. Waist circumference, waist to hip ratio, triglyceride and homoeostasis model assessment-insulin resistance were independent determinants of mean IMT level.Conclusion: Obesity especially abdominal obesity, high TG and insulin resistance may be the main risk predictors of increased IMT.

Rapid and efficient methods for the determination of cured tuberculosis (TB) are lacking. A total of 85 differentially expressed serum proteins were identified by iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) analysis (fold change >1.50 or P P = 0.036 and P = 0.012, respectively) and significantly reduced ARHGDIB, C3 and FCN2 levels (P P = 0.035 and P = 0.018, respectively) were observed in cured TB patients compared with untreated TB patients. In addition, changes in ALB and FCN2 levels occurred after 2 months of treatment (P P = 0.030, respectively). We established a cured TB model with 87.10% sensitivity, 79.49% specificity and an area under the curve (AUC) of 0.876. The results indicated that ALB, ARHGDIB, C3, FCN2 and LPA levels might serve as potential biomarkers for cured TB. Our study provides experimental data for establishing objective indicators of cured TB and also proposes potential markers for evaluating the efficacy of anti-TB drugs.

Ficolin-2 (FCN2) is an innate immune pattern recognition molecule that can activate the complement pathway, opsonophagocytosis, and elimination of the pathogens. The present study aimed to investigate the association of the FCN2 gene single nucleotide polymorphisms (SNPs) with susceptibility to pulmonary tuberculosis (TB). A total of seven SNPs in exon 8 (+6359 C>T and +6424 G>T) and in the promoter region (-986 G>A, -602 G>A, -557 A>G, -64 A>C and -4 A>G) of the FCN2 gene were genotyped using the PCR amplification and DNA sequencing methods in the healthy controls group (n = 254) and the pulmonary TB group (n = 282). The correlation between SNPs and pulmonary TB was analyzed using the logistic regression method. The results showed that there were no significant differences in the distribution of allelic frequencies of seven SNPs between the pulmonary TB group and the healthy controls group. However, the frequency of the variant homozygous genotype (P = 0.037, -557 A>G; P = 0.038, -64 A>C; P = 0.024, +6424 G>T) in the TB group was significantly lower than the control group. After adjustment for age and gender, these variant homozygous genotypes were found to be recessive models in association with pulmonary TB. In addition, -64 A>C (P = 0.047) and +6424 G>T (P = 0.03) were found to be codominant models in association with pulmonary TB. There was strong linkage disequilibrium (r2 > 0.80, P < 0.0001) between 7 SNPs except the -602 G>A site. Therefore, -557 A>G, -64 A>C and +6424 G>T SNPs of the FCN2 gene were correlated with pulmonary TB, and may be protective factors for TB. This study provides a novel idea for the prevention and control of TB transmission from a genetics perspective.

Background Traditional Chinese Medicine (TCM) has been applied in treating tuberculosis (TB) based on the TCM syndromes with the effects of inhibiting Mycobacterium, strengthening the body immune system, and reducing the pulmonary toxicity. We used bioinformatic methods to study the clinical and pathological characteristics of pulmonary TB patients with TCM syndromes. Isobaric tags for relative and absolute quantification - coupled two dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) methods were applied to screen differentially expressed serum proteins. Methods Pulmonary TB cases were divided into four distinctive TCM syndromes: pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, deficiency of Qi and Yin (DQY) syndrome, and deficiency of Yin and Yang (DYY) syndrome. The serum samples from 214 pulmonary TB patients were collected, and the clinical and pathological data was analyzed by using iTRAQ-2DLC-MS/MS. Finally, the differentially expressed proteins were screened and tested by ELISA. Only 5 patients with DYY syndrome were recruited in 3 years, which were not enough for further research. Results The DQY cases had higher erythrocyte sedimentation rate (ESR) compared to the PYD and HFYD cases (P = 0.0178). 94.44 % (12 PYD, 18 HFYD, and 4 DQY before anti-TB treatment) of 36 treated TB cases were transformed to PYD accompanied with the reduction of ESR and absorption of pulmonary lesions. A total of 39 differentially expressed proteins (ratios of >1.3 or

In this paper, we successfully applied the PECVD method to deposit nanocrystalline silicon films (nc — Si: H) and determined the microstructure with HREM and STM technology. The nc — Si: H films consist of a large number of micrograins and interfaces. For this man-made film material with special structure, we studied the piezo- resistance effect and hydrogen content, which varied with the microstructure of the films. We suggest that the unique structure of nc — Si:H films is the main cause of larger piezo- resistance effect and higher hydrogen content in the films.

We have investigated the role of intracellular cytoplasmic sequences in the assembly of the mouse muscle nicotinic acetylcholine receptor (AChR) transiently expressed in COS cells. A chimeric protein in which the region from M1 to M4 of the alpha subunit was replaced by the corresponding region in the beta subunit was unable to support AChR assembly when substituted for the alpha subunit; a chimeric alpha subunit containing only the long cytoplasmic loop from the beta subunit was likewise inactive. Systematic mutation of short segments of the loop identified a sequence of 17 amino acids near the C-terminal end of the loop for which the beta sequence could not be substituted. Each of the inactive chimeric and mutated alpha subunits bound alpha-bungarotoxin when expressed alone and formed a heterodimer when expressed with the delta subunit. An alpha subunit truncated after M1 formed both an alpha delta heterodimer and an alpha delta beta heterotrimer, demonstrating that the cytoplasmic loop is dispensable for the early steps of assembly. A sequence in the long cytoplasmic loop of the alpha subunit thus appears to play a role in a late step of AChR assembly.

In this study we initiated a proteomic investigation of the maize thylakoid membrane by using a shotgun proteomic approach based on LC-MS(E). A total of 34 maize thylakoid membrane proteins were identified, the majority of which are primarily involved in photosynthesis, including the light-reaction and carbon assimilation. It is noteworthy that all of the core subunits of the Photosystem II were identified in our search. Proteins involved in other processes, such as iron storage, were also detected in our study. The quantity of each identified protein was also determined. Of interest, we discovered that the amount of the three ATP synthase subunits were not equivalent, suggesting that these proteins perform other functions in addition to ATP synthesis. To our knowledge this is the first extensive proteomic investigation of the maize thylakoid membrane, and will likely enable further study of maize photosynthesis and chloroplast development.

Aim: To investigate risk factors which impact on common carotid artery intima media thickness (IMT). Methods: A total of 86 obese children and adolescents and 22 healthy children and adolescents with normal weight were enrolled. Moreover, 23 of 86 obese children and adolescents were diagnosed with metabolic syndrome (MetS). The clinical, biochemical data and the IMT of the common carotid artery were measured in all subjects. Results: Obese and obese with MetS subjects demonstrated a significantly (p < 0.01) thicker intima media (0.69mm, 0.66mm) as compared to the control group (0.38mm), but there was no significant difference of IMT between obese and MetS group. IMT was correlated to body weight, body mass index, waist circumference, waist to hip ratio, systolic blood pressure, diastolic blood pressure, fasting insulin, homoeostasis model assessment-insulin resistance, triglyceride, high-density lipoprotein- cholesterol, low-density lipoprotein-cholesterol, alanine aminotransferase, aspartate aminotransferase and fatty liver. Waist circumference, waist to hip ratio, triglyceride and homoeostasis model assessment-insulin resistance were independent determinants of mean IMT level. Conclusion: Obesity especially abdominal obesity, high TG and insulin resistance may be the main risk predictors of increased IMT.

Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit.

To investigate the relations of platelet phospholipid fatty acids to thrombotic risk factors in the middle-aged and geriatric patients with hyperlipidemia in the metropolitan area of Hangzhou, Zhejiang province.A questionnaire survey was conducted among 81 patients with hyperlipidemia, 50 males and 31 females, aged (57 +/- 8), and 65 healthy controls, 43 males and 22 females, aged (58 +/- 9) to collect the data about height, weight, and diet. Peripheral venous blood samples were collected to examine the total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), homocysteine (Hcy), 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), and thromboxane B(2) (TXB(2)) were examined by standard methods. Serum thrombotic risk factors including homocysteine and Thromboxane B(2) were determined by standard methods. Platelet phospholipid fatty acids were examined by gas chromatography. The correlation between the serum thrombotic risk factors (Hcy, TXB(2), and 6-keto-PG F1a) was analyzed by multivariate linear regression.There were no significant differences in platelet phospholipid fatty acids between the patients with hyperlipidemia and the healthy controls. Serum Hcy was significantly negatively correlated with docosahexaenoic acid (DHA) and the ratio of n-3 PUFA (polyunsaturated fatty acids)/n-6 PUFA (r = -0.277 and -0.231, both P0.01). The level of serum TXB(2) was significantly positively correlated with arachidonic acid (r = 0.176, P0.05), and significantly negatively correlated with DHA (r = -0.209, P0.01), eicosapentaenoic acid (EPA) (r = -0.194, P0.05), and n-3 PUFA/n-6 PUFA (r = -0.238, P0.01).Increasing the ratio of n-3 PUFA/n-6 PUFA in platelet phospholipid may potentially decrease the thrombotic risks such as Hcy and TXB(2) and provide a reference for diet selection.

The 43 kDa protein, a cytoplasmic peripheral membrane protein, is closely associated with the acetylcholine receptor (AChR) at the neuromuscular junction, where it is thought to anchor the receptor in the postsynaptic membrane. We have used the 43 kDa protein-induced clustering of AChRs that occurs when both proteins are transiently expressed in COS cells to investigate which parts of the AChR might interact with the 43 kDa protein. By constructing chimeric subunits, we showed that the cytoplasmic domains of neither the epsilon nor delta subunits are required for 43 kDa protein-induced clustering. Systematic mutational analysis of the long cytoplasmic loops of the alpha and beta subunits showed that most of the loops can be altered without affecting the ability of the AChR to be clustered; in each case, however, one or more sequences could not be tested, because mutation in these regions prevented AChR assembly. Our results suggest either that these regions are involved in clustering or that the 43 kDa protein can interact with multiple, alternative sites on the cytoplasmic surface of the AChR. Our experiments also show that the postulated sites of tyrosine phosphorylation in the beta subunit and of serine phosphorylation in the alpha subunit can be mutated without affecting 43 kDa protein- induced AChR clustering.

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.