[Percutaneous pedicle screw fixation combined expandable tubular retractor in the treatment of spinal metastases].

OBJECTIVE: To investigate the effectiveness of percutaneous pedicle screw fixation combined expandable tubular retractor in the treatment of patients with spinal metastases. METHODS: In the study, 12 patients of spinal metastases treated with percutaneous pedicle screw fixation combined expandable tubular retractor in our hospital were retrospectively reviewed between June 2017 and October 2019. Among the 12 patients, 9 were males and 3 were females; the median age was 62.5 years [(65.1±2.9) years]. The decompression segment of 7 patients was located at the lower thoracic spine (including 1 patient with incomplete paraplegia) and the decompression segment of 5 patients was located at the lumbar spine; Tomita score was 6.0±0.6. Perioperative data of the patients were reviewed. Visual analog scale (VAS score), Karnofsky score, and Eastern Cooperative Oncology Group (ECOG) score were compared before and after surgery. The patient's survival, adjuvant treatment, and internal fixation failure were observed in the follow-up period. RESULTS: All the 12 patients had a successful operation with percuta-neous pedicle screw fixation combined expandable tubular retractor. The average operative time, blood loss, and blood transfused of the patients were (247.0±14.6) min, (804.2±222.3) mL and (500.0±100.0) mL, respectively. The average amount of drainage was (240.8±79.3) mL. Drainage tubes were pulled out early postoperative [(3.2±0.3) d], allowing early mobilization. The patients discharged (7.8±0.8) d postoperative. All the patients were followed up for 6-30 months, and the average overall survival time was (13.6±2.4) months. During the follow-up period, 2 patients experienced screw displacement, the internal fixation was stable after conservative treatment and no revision surgery was performed. The VAS of the patients was 7.1±0.2 before surgery, which decreased to 2.3±0.1 and 2.8±0.4 at 3 and 6 months after surgery (P < 0.05). The Karnofsky score of the patients was 59.2±1.9 before surgery, which increased to 75.0±1.9 and 74.2±3.1 at 3 and 6 months after surgery (P < 0.05). The ECOG of the patients was 2.3±0.2 before surgery, which decreased to 1.7±0.1 and 1.7±0.2 at 3 and 6 months after surgery (P < 0.05). CONCLUSION: For selected patients with spinal metastases, minimally invasive surgical treatment of spinal metastases (percutaneous pedicle screw internal fixation combined with expandable tubular retractor) can effectively relieve the clinical symptoms and improve the quality of life, with satisfactory clinical outcome.

Choline supply during negative nutrient balance alters hepatic cystathionine ß-synthase, intermediates of the methionine cycle and transsulfuration pathway, and liver function in Holstein cows.

Although choline requirements for cows are unknown, enhanced postruminal supply may decrease liver triacylglycerol and increase flux through the Met cycle to improve immunometabolic status during a negative nutrient balance (NNB). Our objectives were to investigate the effects of postruminal choline supply during a feed restriction-induced NNB on (1) hepatic activity cystathionine ß-synthase and transcription of enzymes in the transsulfuration pathway and Met cycle; (2) hepatic metabolites in the Met cycle and the transsulfuration pathway, bile acids, and energy metabolism; and 3) plasma biomarkers of liver function, inflammation, and oxidative stress. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements) with abomasal infusion of water, or R plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected on d 5 after infusions ended, and blood was collected on d 1, 3, and 5. Statistical contrasts were A0 versus R0 (CONT1), R versus the average of choline doses (CONT2), and tests of linear and quadratic effects of choline dose. Activity of cystathionine ß-synthase was lower with R (CONT1) and decreased linearly with choline. Hepatic glutathione was not different with R or choline, but taurine tended to be greater with choline (CONT2). Betaine and carnitine were greater with R (CONT1) and further increased with choline (CONT2). Concentrations of NAD+ were greater with choline (CONT2). Cholic and glycol-chenodeoxycholic acids were decreased by R and choline, while taurocholic and tauro-chenodeoxycholic acids were not altered. Plasma aspartate aminotransferase and bilirubin were greater with R (CONT1) but decreased with choline (CONT2). Paraoxonase was lower with R and increased with choline (CONT2). Data suggest that enhanced supply of choline during NNB decreases entry of homocysteine to the transsulfuration pathway, potentially favoring remethylation to Met by acquiring a methyl group from betaine. As such, Met may provide methyl groups for synthesis of carnitine. Along with production data indicating that 12.5 g/d choline ion improved milk yield and liver fatty acid metabolism during NNB, the changes in blood biomarkers also suggest a beneficial effect of choline supply on liver function and oxidative stress.

Hepatic 1-carbon metabolism enzyme activity, intermediate metabolites, and growth in neonatal Holstein dairy calves are altered by maternal supply of methionine during late pregnancy.

Maternal supply of methyl donors such as methionine (Met) during late pregnancy can affect offspring growth and development. The objective was to investigate the effect of postruminal Met supply during late pregnancy on 1-carbon, Met cycle, and transsulfuration pathways in the calf liver. During the last 28 d of pregnancy, cows were individually fed a control diet or the control diet plus rumen-protected dl-Met (MET; 0.09% dry matter intake). Liver samples obtained from calves (n = 14/group) at 4, 14, 28, and 50 d of age were used for metabolomics, real-time PCR, and enzyme activity analyses. Genes associated with 1-carbon metabolism, DNA methylation, and the cytidine 5'-diphosphocholine-choline pathway were analyzed via real-time PCR. Activity of betaine homocysteine methyltransferase, cystathionine ß-synthase, and 5-methyltetrahydrofolate homocysteine methyltransferase (MTR) was analyzed using 14C isotopes. Data were analyzed using a mixed model that included the fixed effects of maternal treatment, day, and their interaction, and the random effect was calf within maternal diet. Calves born to dams offered MET tended to have greater birth body weight and had overall greater body weight during the first 9 wk of life. However, no differences were detected for daily feed intake and average daily gain between groups. Concentrations of betaine and choline, reflecting Met cycle activity, at d 14 through 28 were greater in MET calves. Transsulfuration pathway intermediates also were altered in MET calves, with concentrations of cysteine sulfinic acid and hypotaurine (d 4 and 14) and taurine being greater (d 4, 14, 28, and 50). Despite the lack of differences in daily feed intake, the greater concentrations of the tricarboxylic acid cycle intermediates fumarate and glutamate along with NAD/NADH in MET calves indicated enhanced rates of energy metabolism. Although activity of betaine homocysteine methyltransferase was greater in MET calves at d 14, cystathionine ß-synthase was lower and increased at d 14 and 28, where it was greater compared with the control diet. Activity of MTR was lower at d 4 and 50 in MET calves. Among gene targets measured, MET calves had greater overall expression of MTR, phosphatidylethanolamine N-methyltransferase, and choline kinase α and ß. An interaction of maternal diet by time was detected for mRNA abundance of DNA methyltransferase 3α (involved in de novo methylation) due to greater values at d 4 and 14 in MET calves. Overall, the data indicate that enhanced postruminal supply of Met to cows during late pregnancy may program hepatic metabolism of the calf in the context of maintaining Met homeostasis, phosphatidylcholine and taurine synthesis, DNA methylation, and energy metabolism. These alterations potentially result in better efficiency of nutrient use, hence conferring the calf a physiologic advantage during a period of rapid growth and development. The precise biologic mechanisms remain to be established.

[Effect of intrapulpal pressure on the bonding strength of resin cement to dentin].

OBJECTIVE: To evaluate the effect of intrapulpal pressure simulation on the micro-tensile bond strength (µTBS) of resin cement to dentin. METHODS: Thirty extracted human third molars were selected. Occlusal enamel was removed to expose dentine surface and teeth with residual dentin thickness of 0.5-2.5 mm were selected. Dye permeation through dentin tubules with or without intrapulpal pressure (IPP) simulation, or after Single Bond Universal (SBU) application on dentin surface with IPP simulation were observed at the end of 0 min, 5 min, 30 min and 2 h. The teeth with residual dentin thickness of (1.0±0.1) mm were divided into 2 groups with IPP simulation of 15 or 0 cmH2O (1 cmH2O=0.098 kPa), which was maintained for 30 min before bonding procedure. SBU was applied on the dentin surface and light cured, then RelyX Ultimate (RLX) cement was heaped on the dentin surface (diameter=10 mm, height=4 mm) and light-cured. After the dentin-resin cement samples were stored in distilled water for 24 h at 37 °C, the samples were cut into beams with cross sectional area of 0.9 mm×0.9 mm for µTSB testing (n=100). The data were analyzed with two independent samples t-test (α=0.05). The fracture mode was observed using scanning electron microscopy (SEM). The results were analyzed with Fisher exact test (α=0.05). The rest of dentin-resin cement samples (five samples for each group) were cut perpendicular to the bonding interface and the morphology of the bonding interface was observed using SEM. RESULTS: The dye permeation through dentin tubules with IPP simulation was faster than those without IPP simulation. The µTSB of RLX to dentin with and without IPP simulation were (26.26±9.78) MPa and (28.70±9.0) MPa, respectively. The most frequent fracture mode was mixed-fracture mode. There was no significant difference between the two groups for neither bond strength nor fracture types distribution (P>0.05). Regarding the morphology of dentin-resin cement bonding interface, both groups showed 4-8 µm finger-like resin tags. CONCLUSION: With SBU pretreatment, the IPP simulation had no influence on the immediate bond strength of RLX to dentin.

[Analysis of influencing factors for pathogen culture result in patients with pyogenic spondylitis].

OBJECTIVE: To investigate the effect of clinical factors on the pathogen culture results in the patients with pyogenic spondylitis, and to find out clinical controllable factors which could increase the positive rate of the pathogen culture. METHODS: A retrospective study reviewed 40 patients who were diagnosed with pyogenic spondylitis in Peking University First Hospital from January 2011 to July 2017. The patients were divided into two groups depending on the culture results, culture negative or culture positive. The influence of clinical uncontrollable factors [the patient's age, gender, predisposing factors, infection site except spine, visual analogue score (VAS), course of disease, spinal segment, white blood cell (WBC), (neutrophilic granulocyte)% (NE%), the incidence of systemic inflammatory response syndrome (SIRS), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), the incidence of paravertebral abscess] and controllable factors (prior antibiotics exposure within 2 weeks, tissue homogenate, surgical approach) on pathogen culture results were analyzed. RESULTS: Of the 40 patients, 18 patients were female and 22 patients were male. Causative germ was identified in 24/40 patients (60.00%) and dominant by gram positive cocci (68.00%). For clinical uncontrollable factors, there was no significant difference between the two groups in the patient's age, gender, predisposing factors, infection site except spine, VAS, course of disease, spinal segment, WBC, NE% and the incidence of SIRS. ESR [(94.38±6.91) mm/h, P=0.023)], CRP [(64.74±13.51) mg/L, P=0.040], and the incidence of paravertebral abscess (75%, P=0.018) in culture negative group were lower in contrast to culture positive group. For clinical controllable factors, prior antibiotics exposure within 2 weeks (P=0.058, OR=4.030, 95%CI: 0.956-16.993) and tissue homogenate (P=0.014, OR=0.171, 95%CI: 0.042-0.695) were significantly associated with the pathogen culture result. Surgical approach was not significantly associated with pathogen culture result. CONCLUSION: Patients with high level of ESR, CRP, and paravertebral abscess, would have high positive rate of pathogenic culture. Prior antibiotics exposure was associated with lower positive pathogen culture rate. Culture with tissue homogenate was more likely to find the causative germ, especially for patients without paravertebral abscess who had low level of ESR, CRP and prior antibiotics exposure.

[Clinical characteristics and prognosis of cultured negative pyogenic spondylitis].

OBJECTIVE: There are limited data describing the clinical characteristics and prognosis of culture negative pyogenic spondylitis. The aim of this study was to investigate the treatment, prognosis and clinical characteristics of culture negative pyogenic spondylitis. METHODS: A retrospective study reviewed 74 patients who were diagnosed with spondylitis in Peking University First Hospital from January 2010 to December 2015. A total of 27 patients suffered from pyogenic spondylitis. According to the pathogenic culture results, the patients were divided into two groups: culture negative group and culture positive group. The clinical characteristics and treatment outcomes between the two groups were compared. RESULTS: The elder were more vulnerable to pyogenic spondylitis, and of the 27 patients, 12 patients were female and 15 male. All patients had no history of administration of antibiotics prior to obtaining culture samples. A causative germ was identified in 14/27 patients (51.9%) with Staphylococcus aureus being the most common pathogen. There was no significant difference between the two groups in the patient's age, gender, visual analogue score (VAS), predisposing factor, clinical symptom, sign and spinal segment (P>0.05). Erythrocyte sedimentation rate (ESR) (P=0.056) and C-reactive protein (CRP) (P=0.040) of culture negative group were lower in contrast to culture positive group. The incidence of vertebral abscess in culture negative group was higher than in culture positive group (P=0.046). After treatment, ESR dropped almost equally in both groups, and CRP dropped faster in the culture positive group (P=0.192). At last, there was no significant difference between the two groups in hospital stay, pain relief, open debridement operation rate, and recurrence rate of infection. CONCLUSION: ESR and CRP of the culture negative patient were lower than those of the culture positive patient, and the incidence rate of paravertebral abscess was higher than that of the culture positive patient. After administration of antibiotics, there was no significant difference between the two groups in duration of antibiotics, open debridement operation rate and recurrence rate of infection. So, culture negative may not necessarily be a negative prognostic factor for pyogenic spondylitis. However, we should watch out for the drug resistant bacteria or double infection, due to the long term use of wide-spectrum antibiotic in culture negative patients.

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components.

UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.

Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis.

Cloning of a phosphatidylinositol 4-kinase gene based on fiber strength transcriptome QTL mapping in the cotton species Gossypium barbadense.

Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.

New evidence on the earliest human presence at high northern latitudes in northeast Asia.

The timing of early human dispersal to Asia is a central issue in the study of human evolution. Excavations in predominantly lacustrine sediments at Majuangou, Nihewan basin, north China, uncovered four layers of indisputable hominin stone tools. Here we report magnetostratigraphic results that constrain the age of the four artefact layers to an interval of nearly 340,000 yr between the Olduvai subchron and the Cobb Mountain event. The lowest layer, about 1.66 million years old (Myr), provides the oldest record of stone-tool processing of animal tissues in east Asia. The highest layer, at about 1.32 Myr, correlates with the stone tool layer at Xiaochangliang, previously considered the oldest archaeological site in this region. The findings at Majuangou indicate that the oldest known human presence in northeast Asia at 40 degrees N is only slightly younger than that in western Asia. This result implies that a long yet rapid migration from Africa, possibly initiated during a phase of warm climate, enabled early human populations to inhabit northern latitudes of east Asia over a prolonged period.

Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer.

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

Effects of endothelin-11-31 on cell viability and [Ca2+]i in cultured neonatal rat cardiomyocytes

We previously found that endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).

Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

[Surface roughness and gloss of novel flowable composites after polishing and simulated brushing wear].

Objective: To investigate surface properties of novel flowable composites after polishing and simulated brushing wear, compared to their pasty counterpart. Methods: Composites employed in this study were: three flowable composites (A1: Clearfil Majesty ES Flow; B1: Beautifil Flow Plus F00; C1: Filtek Bulk Fill) and three paste composites (A2: Clearfil Majesty; B2: Beautifil; C2: Filtek Z350. Eleven disk-shaped specimens were made for each material. The specimens were cured, then subjected to sandpaper finishing for 20 s, one-step polishing for 30 s, finally subjected to simulated brushing for 10 000 cycles. Surface roughness and glossiness were measured before finishing, after finishing, after polishing, after 5 000 brushing cycles and after 10 000 brushing cycles, respectively. Data obtained were analyzed using two-way ANOVA method. Scanning electron microscope was employed to examine the microscopic appearance of each material. Results: Surface roughness (0.11~0.22 µm) and glossiness (74.25~86.48 GU) of each material were similar after one-step polishing. After brushing simulation, roughness increased significantly and glossiness decreased significantly for each material (P<0.05). Group A1 presented the best gloss ([50.68±1.58] GU) after final wear (P<0.05). Flowable composites of group A1 and B1 tested in the present setup showed better surface properties compared to their pasty counterpart (group A2 and B2). Conclusions: Within the limit of this study, flowable composites tested in the present research can obtain similar surface polish or even better than the paste composite counterpart.

Interleukin-10 -1082 promoter polymorphism is associated with schizophrenia in a Han Chinese sib-pair study.

The interleukin-10 (IL-10) gene has been identified as a susceptibility gene for schizophrenia in Caucasians. A previous case-control study conducted by our group revealed a weak association between polymorphism, -592C/A, of the IL-10 gene promoter and schizophrenia. Our present study was aimed at confirming the association of the IL-10 promoter with schizophrenia using 197 Han Chinese sib-pair families. A family-based association test (FBAT) and haplotype analysis was undertaken using the FBAT v1.5.5. The global TDT was significant for a different polymorphism, -1082G/A (chi2=13.16, P=0.000285) and that the allele -1082G was preferentially transmitted to schizophrenia-affected children. Furthermore, haplotype TDT analysis showed that haplotype "GCC" was significantly associated with the disease (chi2=8.1, P=0.00443). Our results also indicate that the IL-10 gene may play a significant role in the etiology of schizophrenia among Han Chinese.

Saccharomyces cerevisiae expression of exogenous vacuolar ATPase subunits B.

The precise function of subunit B of the vacuolar H(+)-ATPase class is unknown, but it is essential for proton pumping. We have previously reported the DNA sequence and predicted protein sequence of the vacuolar ATPase subunit B for Candida tropicalis (Gu, H.H., Gallagher, M.J., Rupkey, S. and Dean, G.E. (1990) Nucleic Acids Res. 18, 7446). When the Candida gene was expressed in a Saccharomyce cerevisiae delta vat2 mutant from which the homologous gene had been deleted, viability and vacuolar acidification was restored to apparently wild-type levels. The predicted identity between these two proteins is 90%. We have searched for vacuolar ATPase subunits B from other species that might show a difference in function, when expressed in yeast, relative to the endogenous gene. We have cloned an apparently full-length 1.8-kb bovine subunit B cDNA from adrenal medulla that is about 1 kb shorter than the previously reported bovine brain cDNA (Puopolo, K., Kumamoto, C., Adachi, I., Magner, R. and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706; Nelson, R.D., Guo, X.L., Masood, K., Brown, D., Kalkbrenner, M. and Gluck, S. (1992) Proc. Natl. Acad. Sci. USA 89, 3541-3545), but nearly identical throughout the coding nucleotide and protein sequences; it is only 74% identical to the Saccharomyces subunit B protein sequence. Upon expression of this cDNA in two different delta vat2 deletion strains, the bovine cDNA restored function only partially, as judged by both viability at high pH and vacuolar acidification. Current work is aimed at determining which regions of the bovine protein require alteration in order to fully restore the delta vat2 strain to wild-type acidification, with the eventual goal of identifying interactive residues between subunit B and other proteins required for pump function.

Identification and characterization of six new alternatively spliced variants of the human mu opioid receptor gene, Oprm.

The mu opioid receptor plays an important role in mediating the actions of morphine and morphine-like drugs. Receptor binding and a wide range of pharmacological studies have proposed several mu receptor subtypes, but only one mu opioid receptor (Oprm) gene has been isolated. Like the mouse and rat, the human Oprm gene undergoes alternative splicing. In the present studies, we have identified and characterized six new splice variants from the human Oprm gene using a reverse transcription-polymerase chain reaction strategy, yielding a total of 10 human splice variants of the mu opioid receptor MOR-1. All the variants identified contained exons 1, 2 and 3, but differed from MOR-1 itself and each other by splicing downstream from exon 3, resulting in different amino acid sequences. Northern blot analysis demonstrated expression of the variant mRNAs. Receptor binding assays established that these variants belonged to the mu opioid receptor family with limited differences in mu opioid ligand affinities and selectivity. However, adenylyl cyclase and [35S]GTPgammaS binding assays revealed major differences in both potency and efficacy among these variants. The dissociation between binding affinity, potency and efficacy for the opioids among these variants may provide insights into the wide range of opioid responses among these agents observed clinically and opens new avenues in designing selective drugs based upon their efficacy and potency rather simple binding affinity.

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats.

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.

Developmental regulation of a turkey intestinal peptide transporter (PepT1).

A cDNA encoding a turkey intestinal peptide transporter, tPepT1, was isolated from a turkey small intestinal cDNA library. The tPepT1 cDNA encodes a 714-amino acid protein with 12 predicted transmembrane domains. The amino acid sequence of tPepT1 is 94.3% identical to chicken PepT1 and approximately 60% identical to PepT1 from rat, sheep, rabbit, and human. Using a 2-electrode voltage-clamp technique in Xenopus oocytes expressing tPepT1, Gly-Sar transport was pH dependent but was independent of Na+ and K+. For the dipeptides Gly-Sar and Met-Met, the evoked inward currents indicated that the transporter was saturable and had high affinity (0.69 +/- 0.14 mM and 0.23 +/- 0.04 mM, respectively) for these substrates. However, transport of the tetrapeptide, Met-Gly-Met-Met, exhibited apparent substrate inhibition at high substrate concentrations. To study developmental regulation of PepT1 mRNA in turkey embryos, embryos (6 males and 6 females) were sampled daily from 5 d before hatch to the day of hatch (d 0). The abundance of PepT1 mRNA in the small intestine was quantified densitometrically from Northern blots after hybridization with full-length tPepT1 cDNA as probe. A 3.2-fold increase in PepT1 mRNA was observed in intestinal tissue from 5 d before hatch to d 0. This increase in PepT1 mRNA abundance indicates that the PepT1 gene is developmentally regulated and that there may be an important role for PepT1 in the neonatal poult.

Structure and characterization of the gene encoding a mouse kappa3-related opioid receptor.

A genomic clone comprising the entire cDNA sequence encoding a mouse kappa3-related opioid receptor (KOR-3) was isolated by screening a mouse genomic library with a radiolabeled mouse KOR-3 cDNA probe. Sequence analysis of the genomic clone indicates that the KOR-3 gene contains five exons separated by four introns. The transcription start point (tsp) of KOR-3 was mapped by primer extension analysis of RNAs synthesized either in vivo or in vitro. A TATA-box and several potential regulatory elements, including five GRE sites, four NF-E1 binding sites and one MRE site, are present in the 2 kb of 5'-flanking region. A putative poly(A) signal (AATAAA) is found in the 3'-flanking region.