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1.
Zhonghua Gan Zang Bing Za Zhi ; 31(3): 332-336, 2023 Mar 20.
Article in Chinese | MEDLINE | ID: mdl-37137864

ABSTRACT

Sarcopenia is one of the common complications of cirrhosis. Studies have demonstrated that patients with cirrhosis combined with sarcopenia have a high mortality rate. The occurrence of sarcopenia may be associated with inflammatory states and metabolic abnormalities caused by changes in the gut microbiota environment, but such studies are currently relatively lacking. This article elaborates on the correlation between changes in the gut microbiota environment, as well as the diagnosis and treatment, in order to provide reference and assistance for the treatment of patients with cirrhosis and sarcopenia.


Subject(s)
Gastrointestinal Microbiome , Sarcopenia , Humans , Sarcopenia/complications , Liver Cirrhosis/therapy
2.
Zhonghua Yi Xue Za Zhi ; 99(25): 1981-1984, 2019 Jul 02.
Article in Chinese | MEDLINE | ID: mdl-31269605

ABSTRACT

Objective: To investigate serum status of folate, vitamin B(12), homocysteine (Hcy) and hydroxyvitamin D (25OHD) and their trends in different gender and age groups. Methods: This was a cross-sectional study. The enrolled subjects were those received medical examination in Beijing Hospital from September to November 2018 and were identified as appeared healthy persons. 1220 subjects were recruited and were divided into groups of young and middle age group (30-49 years, 50-59 years) and the elderly group (60-69 years, 70-79 years and ≥80 years). We measured folate, vitamin B(12), and 25OHD using electrochemiluminescence by chemiluminescence immunoassay. Hcy was measured by autobiochemical analyzer. Results: Total folate levels in male and female subjects were 7.16 (4.74-10.75) and 9.17 (6.49-13.55) µg/L respectively. Total vitamin B(12) levels in the male and female were 505.60 (386.80-700.90) and 582.60 (430.70-846.98) ng/L respectively. Hcy levels were 14.68 (12.25-18.58) and 11.29 (9.65-13.58) µmol/L. 25OHD levels were 21.60 (16.40-28.70) and 16.80 (12.30-24.15) µg/L respectively. Total folate and vitamin B(12) levels in female were higher than that in male subjects (Z=-7.796, -4.772, P<0.001). However, total Hcy and 25OHD levels in male were higher than that in female subjects (Z=-15.230, -8.447, P<0.001). Comparing with the substances in the above age groups, folate level in the elderly was lower than that in the younger age and middle age groups.However, vitamin B(12), 25OHD and Hcy levels were higher in the elderly groups. Furthermore, the levels of folate, vitamin B(12) and 25OHD were getting higher in the group of ≥80 years female compared with the rest of the age groups, but it turned lower in the male group of ≥80 years. Conclusions: There are some differences in the serum values of folate, vitamin B(12), Hcy and 25OHD among various age groups as well as between males and females. These should be considered in the development of national reference ranges.


Subject(s)
Vitamin B 12 Deficiency , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , Folic Acid , Healthy Volunteers , Homocysteine , Humans , Male , Middle Aged , Vitamin B 12 , Vitamins
3.
Eur Rev Med Pharmacol Sci ; 22(23): 8220-8226, 2018 12.
Article in English | MEDLINE | ID: mdl-30556861

ABSTRACT

OBJECTIVE: To explore the role of microRNA-1266 in colorectal cancer (CRC) and its underlying mechanism. PATIENTS AND METHODS: The expression level of microRNA-1266 in 48 CRC tissues and paracancerous tissues was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). The relationship between microRNA-1266 expression and basic characteristics of CRC patients was analyzed. The effect of microRNA-1266 on the viability of CRC cells was detected by cell counting kit-8 (CCK-8) assay. Subsequently, a potential target gene for microRNA-1266 was predicted through bioinformatics. Finally, the binding condition between microRNA-1266 and the target gene was verified by RNA binding protein immunoprecipitation (RIP) and luciferase reporter gene assay, respectively. RESULTS: MicroRNA-1266 was lowly expressed in 48 cases of CRC tissues than that of paracancerous tissues. Clinical data demonstrated that microRNA-1266 expression was correlated to tumor size and TNM of CRC patients. Knockdown of microRNA-1266 promoted proliferation of CRC cells. FTO was predicted to be the target gene for microRNA-1266, which was negatively regulated by microRNA-1266. CONCLUSIONS: MicroRNA-1266 is lowly expressed in CRC tissues than that of paracancerous tissues. Lowly expressed microRNA-1266 promotes the occurrence and progression of CRC by directly targeting FTO.


Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Cell Proliferation , Colorectal Neoplasms/enzymology , MicroRNAs/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Caco-2 Cells , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease Progression , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Male , MicroRNAs/genetics , Middle Aged , Signal Transduction , Tumor Burden
4.
Zhonghua Liu Xing Bing Xue Za Zhi ; 39(10): 1381-1386, 2018 Oct 10.
Article in Chinese | MEDLINE | ID: mdl-30453441

ABSTRACT

Objective: To investigate the distribution patterns of mosquitoes, midges and related arboviruses in Sichuan province. Methods: Blood-sucking insects were collected from houses and pens, using the ultraviolet lights. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes. Sequences of the virus were identified and analyzed by molecular biological software, such as BioEdit 7.0.5.3, MEGA 6.0. Results: In total, 17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017. Among them, 79.4% (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1% (1 897/17 019) as Armigeres subalbatus, 5.5% (930/17 019) were Anopheles sinensis and 4.0% (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as typeⅠ Japanese encephalitis virus. Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification. With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates. Conclusions: Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan. Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.


Subject(s)
Arboviruses , Culicidae , Encephalitis Virus, Japanese/genetics , Animals , Encephalitis Virus, Japanese/isolation & purification , Encephalitis, Japanese/diagnosis , Genes, Viral , Nucleic Acid Amplification Techniques , Phylogeny , Polymerase Chain Reaction
5.
Zhonghua Yi Xue Za Zhi ; 98(10): 733-737, 2018 Mar 13.
Article in Chinese | MEDLINE | ID: mdl-29562396

ABSTRACT

Objective: To evaluate the efficacy of ultrasound-guided spinal nerve posterior ramus pulsed radiofrequency treatment of lower back post-herpetic neuralgia. Methods: One hundred and twenty-eight cases of lower back or anterior abdominal wall acute post-herpetic neuralgia patients were enrolled. They were randomly divided into two groups. Group A: oral treatment only with gabapentin+ celecoxib+ amitriptyline. Group B: while taking these drugs, patients were treated with radiofrequency pulses using a portable ultrasound device and the paravertebral puncture technique.In both groups, sudden outbreaks of pain were treated with immediate release 10 mg morphine tablets. Before and one week, two weeks, four weeks, eight weeks after treatment, visual analogue scale (VAS) was used for pain score, Pittsburgh sleep quality index scale (PSQI) was used to evaluate sleep quality, and morphine consumption was recorded. Eight weeks after treatment analgesic efficacy was evaluated. Treatment efficiency and significant efficiency was calculated while the occurrence of complications were documented. Results: In the control group, the VAS of T1,T2,T3 and T4 were 6.7 ±1.2, 5.2 ± 1.0, 3.3 ±1.1, 3.0±0.9.However, at the same time points, the VAS in the treatment group were 3.1±1.0, 2.2±0.7, 1.4±0.5, 1.2±0.5, respectively.The radio frequency group decreased significantly compared with the control group, the difference was statistically significant (t=17.925, 19.662, 12.580, 13.987, all P<0.05). Four weeks after treatment, the TNF-α in the control group was (11.04±2.36)ng/L, and the TNF-ß in the radio frequency group was (8.07±2.13) ng/L. After the treatment, the TNF-α of the radio frequency group was lower than the control group, and the difference was statistically significant (t=-6.267, P<0.05). The IL-1ß in the control group was (3.47±1.09) ng/L after treatment.The IL-1ß in the radio frequency group was (1.96 ±0.56) ng/L, the IL-1ß in the radio frequency group was lower than the control group and the difference was statistically significant (t=-8.266, P<0.05). Pearson correlation analysis showed that before and after the treatment of TNF-α were positively correlated with VAS score (r=0.455, 0.327, all P<0.05). IL-1ß before and after treatment were positively correlated with VAS score (r=0.369, 0.357, all P<0.05). At T1,T2,T3 and T4, PSQI in the control group were 8.5±1.5, 7.3±1.4, 6.2±1.3 and 6.0±0.9 respectively.PSQI in the radio frequency group at the same time points were 6.5±1.4, 5.1±1.2, 4.0±1.1 and 3.9±0.5.Comparison between the two groups after treatment, radio frequency group was lower than the control group significantly, and the difference was statistically significant (t=7.798, 9.545, 10.335, 16.318, all P<0.05). Morphine consumption of the control group at T1,T2,T3,T4 were (22.5±2.2), (15.5±2.9), (6.8±1.5) and (4.2±0.9)mg.However, morphine consumption of the radio frequency group were (13.2±2.5), (7.2±2.7), (3.2±0.6) and (2.2±0.5)mg.Comparison between the two groups after treatment, morphine consumption of the radio frequency group decreased significantly than the control group, and the difference was statistically significant (t=22.341, 16.758, 17.827, 15.541, all P<0.05). During the operation , no error occurred with needle penetrating the abdominal cavity, chest, offal or blood vessels. Conclusion: Ultrasound-guided spinal nerve posterior ramus pulsed radio frequency treatment of lower back or anterior abdominal wall post-herpetic neuralgia proves effective and can reduce patient morphine usage and lead to fewer adverse reactions.


Subject(s)
Neuralgia , Spinal Nerves , Humans , Morphine , Pain Measurement , Pulsed Radiofrequency Treatment
7.
J Virol Methods ; 234: 28-33, 2016 08.
Article in English | MEDLINE | ID: mdl-27063641

ABSTRACT

Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.


Subject(s)
Antibodies, Viral/blood , Bluetongue virus/immunology , Bluetongue/diagnosis , Protein Array Analysis/methods , Animals , Bluetongue/blood , Bluetongue/epidemiology , Bluetongue/virology , Bluetongue virus/genetics , Cattle/virology , Cattle Diseases/diagnosis , Cattle Diseases/virology , China/epidemiology , Enzyme-Linked Immunosorbent Assay/methods , Protein Array Analysis/instrumentation , Sensitivity and Specificity , Sheep/virology
8.
Sci Total Environ ; 562: 812-821, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27110992

ABSTRACT

An Aerodyne high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS) was deployed along with other observation instruments to measure the characteristics of PM1 (particulate matter with a vacuum aerodynamic diameter of ≤1µm) during the biomass burning period (October 1 to 27; BBP) and the coal combustion period (December 10 to 31; CCP) in Beijing in 2014. The average PM1 mass concentrations during the BBP and CCP were 82.3 and 37.5µgm(-3), respectively. Nitrate, ammonium and other pollutants emitted by the burning processes, especially coal combustion, increased significantly in association with increased pollution levels. Positive matrix factorization (PMF) was applied to a unified high-resolution mass spectra database of organic species with NO(+) and NO2(+) ions to discover the relationships between organic and inorganic species. One inorganic factor was identified in both periods, and another five and four distinct organic factors were identified in the BBP and CCP, respectively. Secondary organic aerosols (SOAs) accounted for 55% of the total organic aerosols (OAs) during the BBP, which is higher than the proportion during the CCP (oxygenated OA, 40%). The organic nitrate and inorganic nitrate were first successfully separated through the PMF analysis based on the HR-ToF-AMS observations in Beijing, and organic nitrate components accounted for 21% and 18% of the total nitrate mass during the BBP and CCP, respectively. Although the PM1 mass concentration during the CCP was much lower than in the BBP, the average concentration of polycyclic aromatic hydrocarbons (PAHs) during the CCP (107.3±171.6ngm(-3)) was ~5 times higher than that in the BBP (21.9±21.7ngm(-3)).


Subject(s)
Air Pollutants/analysis , Biomass , Coal , Environmental Monitoring , Particulate Matter/analysis , Beijing , Mass Spectrometry , Nitrates/analysis , Nitrogen Oxides/analysis , Polycyclic Aromatic Hydrocarbons/analysis
9.
Int J Cosmet Sci ; 38(2): 155-63, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26291687

ABSTRACT

BACKGROUND: The typical changes to hair associated with ageing are greying, thinning, dryness and brittleness. Research on the influence of ageing on hair properties will enable a detailed understanding of the natural ageing process. METHODS: The studies were carried out using an SEM (scanning electron microscope), a TriboIndenter and an artificial finger. Three characteristic features of tactile perception that could reflect the perceptual dimensions of the fineness, roughness and slipperiness of hair were extracted. The influences of ageing on the diameter, surface topography, nanomechanical properties and tactile perception of hair were determined. RESULTS: In the three age group hair samples, the children's group hair samples have the smallest diameter. The hair cuticles in the children and young adult groups were relatively complete and less damaged than in the elderly group. The hardness and elastic modulus of the young adult group's hair samples were higher than those in the elderly and children's groups. For all groups, loss modulus E" was smaller than storage modulus E'. Vertical deviations (R) and coefficient of friction (µ) increased, and spectral centroid (SC) decreased, with the increase in age. Ageing decreased the tactile perception of hair. CONCLUSION: Ageing influences the diameter, surface topography, hardness, loss modulus, storage modulus and tactile perception of human hair.


Subject(s)
Aging/physiology , Hair/physiology , Touch/physiology , Adolescent , Adult , Biomechanical Phenomena , Child , Child, Preschool , Humans , Infant , Middle Aged , Nanotechnology , Young Adult
10.
Asian J Androl ; 3(4): 271-5, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11753471

ABSTRACT

AIM: To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection. METHODS: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collected. Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry study under light and electron microscopes. RESULTS: In testes resected mice the size and the weight of thymus were markedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the cell density was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count, the percentage of acid alpha-naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette formation of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelial cells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheral blood were increased. CONCLUSION: After bilateral testicular resection, the thymus of aged male mice showed morphological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggested that testicular resection may improve immune function.


Subject(s)
T-Lymphocytes/immunology , Testis/immunology , Thymus Gland/anatomy & histology , Thymus Gland/immunology , Aging/immunology , Aging/pathology , Animals , Dendritic Cells/cytology , Epithelial Cells/cytology , Lymphocyte Count , Macrophages/cytology , Male , Mice , Mice, Inbred ICR , Naphthol AS D Esterase/metabolism , Plasma Cells/cytology , Regeneration , T-Lymphocytes/enzymology , Testis/cytology , Testis/enzymology , Testis/surgery , Thymus Gland/cytology , Thymus Gland/physiology
12.
Brain Res Dev Brain Res ; 126(2): 147-55, 2001 Feb 28.
Article in English | MEDLINE | ID: mdl-11248348

ABSTRACT

Incomplete neural tube fusion (iNTF), induced by alcohol, in midline floor and roof plates was found in our recent study. In this study, serotonin (5-HT) neurons, known to be born entirely in the midline raphe at brainstem, were examined during their development with fetal alcohol exposure. Weight-matched C57BL mice pregnant dams were divided into three groups on E8: one received ethanol via a chocolate Sustacal liquid diet providing 20% ethanol-derived calories as the sole source of nutrients (ALC); the second received an isocaloric Sustacal liquid diet and was pair-fed to individual dams in the ethanol-fed group (PF); the third was fed ad lib rat chow (Chow). Fetal brains were obtained on E15 and were processed for immunostaining of 5-HT and its trophic factor, S100 beta. The ascending 5-HT neurons, in normal development, appear bilaterally near midline on E12, and by E15, as seen in chow and PF groups, migrate from the midline germinal zone laterally and dorsally to their final position with rich fibers. In contrast, in the E15 ALC group, many 5-HT-im neurons were found remaining in the midline germinal region or had migrated, but with under-differentiated, sparse fibers. There were 20--30% fewer 5-HT-im neurons in ALC as compared to PF and Chow. In addition, the number of S100 beta cells was less in ALC as compared with PF and Chow groups. No difference was found between PF and Chow in number of 5-HT-im or S100 beta-im cells. The 5-HT neurons found compromised in migration and differentiation may, in part, stem from failure of access to floor plate or midline tissue induction and the insufficient support by S100 beta. As 5-HT neurons have been implicated for signaling brain maturation, fewer 5-HT neurons may have lasting effects on the development of brain or, if persistent in the adult, profoundly affect adult brain function.


Subject(s)
Cell Movement/drug effects , Ethanol/administration & dosage , Neurons/drug effects , Neurons/metabolism , Serotonin/metabolism , Administration, Oral , Animals , Brain/cytology , Brain/embryology , Brain/metabolism , Cell Count , Cell Differentiation/drug effects , Ethanol/blood , Female , Food, Formulated , Mice , Mice, Inbred C57BL , Nerve Growth Factors , Neurons/cytology , Pregnancy , Prenatal Exposure Delayed Effects , Raphe Nuclei/cytology , Raphe Nuclei/drug effects , Raphe Nuclei/embryology , S100 Calcium Binding Protein beta Subunit , S100 Proteins/metabolism
13.
World J Gastroenterol ; 7(5): 690-4, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11819855

ABSTRACT

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P<0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P < 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.


Subject(s)
Cancer Vaccines , Dendritic Cells/cytology , Liver Neoplasms, Experimental/prevention & control , Animals , Antigens, CD/analysis , B7-1 Antigen/analysis , B7-2 Antigen , Cell Fusion , Dendritic Cells/chemistry , Integrin alphaXbeta2/analysis , Intercellular Adhesion Molecule-1/analysis , Liver Neoplasms, Experimental/pathology , Male , Membrane Glycoproteins/analysis , Mice , Mice, Inbred BALB C , Spleen/cytology
14.
World J Gastroenterol ; 7(6): 788-91, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11854902

ABSTRACT

AIM: To explore the etiologic role of HPV infection in esophageal carcinoma, and the association of HPV-16 E6 with the nuclear matrix of carcinoma cells. METHODS: Two esophageal carcinoma cell lines,EC/CUHK1 and EC/CUHK2, were tested for HPV-16 E6 subgenetic fragment by polymerase chain reaction amplification of virus DNA associated nuclear matrix. RT-PCR and immunocytochemistry were also used to visualize the expression of E6 subgene in the cells. RESULTS: The HPV-16 E6 subgenetic fragment was found to be present in nuclear matrix-associated DNA, E6 oncoprotein localized in the nucleus where it is tightly associated with nuclear matrix after sequential extraction in EC/CUHK2 cells. It was not detected, however, in EC/CUHK1 cells. CONCLUSION: The interaction between HPV-16 E6 and nuclear matrix may contribute to the virus induced carcinogenesis in esophageal carcinoma.


Subject(s)
Carcinoma/metabolism , Esophageal Neoplasms/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins, Viral/metabolism , Repressor Proteins , Antigens, Nuclear , Humans , Tumor Cells, Cultured
15.
Proc Natl Acad Sci U S A ; 97(17): 9665-70, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10920201

ABSTRACT

We present here a method for in vivo transposon mutagenesis of a methanogenic archaeon, Methanosarcina acetivorans C2A, which because of its independence from host-specific factors may have broad application among many microorganisms. Because there are no known Methanosarcina transposons we modified the mariner transposable element Himar1, originally found in the insect Hematobia irritans, to allow its use in this organism. This element was chosen because, like other mariner elements, its transposition is independent of host factors, requiring only its cognate transposase. Modified mini-Himar1 elements were constructed that carry selectable markers that are functional in Methanosarcina species and that express the Himar1 transposase from known Methanosarcina promoters. These mini-mariner elements transpose at high frequency in M. acetivorans to random sites in the genome. The presence of an Escherichia coli selectable marker and plasmid origin of replication within the mini-mariner elements allows facile cloning of these transposon insertions to identify the mutated gene. In preliminary experiments, we have isolated numerous mini-mariner-induced M. acetivorans mutants, including ones with insertions that confer resistance to toxic analogs and in genes that encode proteins involved in heat shock, nitrogen fixation, and cell-wall structures.


Subject(s)
DNA Transposable Elements/genetics , DNA-Binding Proteins/genetics , Genes, Archaeal/genetics , Genes, Insect/genetics , Methanosarcina/genetics , Mutagenesis, Insertional/genetics , Alkanesulfonic Acids/pharmacology , Cell Wall/metabolism , Cloning, Molecular , DNA Mutational Analysis , DNA, Recombinant/genetics , Drug Resistance, Microbial/genetics , Fluoroacetates/pharmacology , Genetic Markers/genetics , Heat-Shock Proteins/genetics , Methanosarcina/drug effects , Nitrogen Fixation/genetics , Plasmids/genetics , Promoter Regions, Genetic/genetics , Transformation, Genetic , Transposases/genetics , Transposases/metabolism
16.
J Bacteriol ; 182(9): 2611-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10762266

ABSTRACT

Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA(r)) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene (ileS) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PA(r) clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA(r) were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA(r) clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 microgram/ml of medium (for the wild type, the MIC is 12 microgram/ml). The highest MICs (560 microgram/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA(r) based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.


Subject(s)
Anti-Bacterial Agents/pharmacology , Isoleucine-tRNA Ligase/genetics , Methanosarcina barkeri/drug effects , Methanosarcina barkeri/enzymology , Mupirocin/pharmacology , Base Sequence , Biomarkers , Chromosomes, Archaeal , Cloning, Molecular , DNA, Archaeal , Drug Resistance, Microbial , Escherichia coli/genetics , Genes, Archaeal , Genetic Vectors/genetics , Isoleucine-tRNA Ligase/chemistry , Isoleucine-tRNA Ligase/classification , Methanosarcina barkeri/genetics , Molecular Sequence Data , Mutagenesis , Protein Conformation , Recombination, Genetic , Sequence Analysis, DNA
17.
Brain Res Dev Brain Res ; 119(1): 33-45, 2000 Jan 03.
Article in English | MEDLINE | ID: mdl-10648870

ABSTRACT

Serotonin transporter (5-HTT), a transmembrane protein, has been shown in adult brain to be distributed not only on synaptic terminals but to a great extent on axons as well. Here we report the ontogeny of 5-HTT and its relationship with serotonin (5-HT) neurons using established 5-HTT and 5-HT antibodies. Both 5-HTT- and 5-HT-immunostaining (-im) appear in 5-HT neurons at embryonic day 12 (E12) in rostral raphe nuclei (RRN). Soon after appearing, 5-HTT-im is highly expressed on axons, similar to adult expression. However, in contrast to adult, 5-HTT-im also outlines the soma-dendrites. Rich 5-HTT-im appears along the entire length of projecting axons, extending to the growth tip. In the next 2 days, intensive 5-HTT-im axons from RRN travel a course preferentially in the floor plate and later, the medial forebrain bundle trajectory. A group of new 5-HT-im neurons and 5-HTT-im axons appear at E13 in caudal raphe nuclei. At E16-18, taking the exact trajectory course of 5-HT axons, 5-HTT-im axons reach ganglionic eminence, olfactory bulb, and cortex and disperse into many brain regions in E18-20. No 5-HTT-im cell bodies were seen in nigral, locus ceruleus, or hypothalamus. However, the transient expression of 5-HTT on non-serotonergic system was seen in cortical and striatal neuroepithelia at E12 and sensory thalamic pathways at P0-P10. Prominent 5-HTT-im fibers in thalamocortical bundles project from sensory thalamic nuclei through reticular nucleus, internal capsule bundle and form barrels in somatosensory cortices. No 5-HTT-im was seen in glia-like cells using currently available antibody. These observations indicate that 5-HTT is: (a) associated preferentially with 5-HT neurons in brainstem, (b) temporally co-expressed with 5-HT in 5-HT neurons, (c) expressed on axons prior to synaptical sites at target neurons, which strongly indicates a volumic (extrasynaptic) transmission, (d) expressed in non-5-HT neurons within a specific window, which may affect the development of the systems "borrowing" the 5-HT. The early appearance of 5-HTT may also endow functionality as well as vulnerabilities of 5-HT, sensory thalamic, and cortical neurons to 5-HTT targeting drugs during pregnancy and after birth.


Subject(s)
Brain/embryology , Brain/metabolism , Carrier Proteins/biosynthesis , Membrane Glycoproteins/biosynthesis , Membrane Transport Proteins , Nerve Tissue Proteins , RNA, Messenger/biosynthesis , Animals , Axons/metabolism , Brain/cytology , Brain Mapping , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Dendrites/metabolism , Gestational Age , Immunohistochemistry , Nerve Fibers/metabolism , Neuroglia/cytology , Neuroglia/metabolism , Neurons/metabolism , Organ Specificity , Raphe Nuclei/cytology , Raphe Nuclei/embryology , Raphe Nuclei/metabolism , Rats , Rats, Sprague-Dawley , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins , Thalamus/cytology , Thalamus/embryology , Thalamus/metabolism
18.
Alcohol Clin Exp Res ; 23(6): 1023-30, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10397286

ABSTRACT

BACKGROUND: Neuropeptide Y (NPY) is a neuropeptide that has been demonstrated to produce anxiolytic effects when administered centrally. To examine the hypothesis that NPY might play a role in alcohol-seeking behavior, this study took advantage of the genetic differences of the alcohol-preferring (P) rats and alcohol-nonpreferring (NP) rats, as well as the high alcohol-drinking (HAD) rats and low alcohol-drinking (LAD) rats, in voluntary alcohol consumption to examine if NPY neurons in the brains differ between these selected lines. METHODS: The NPY immunoreactivity (NPY-I) was measured using an established radioimmunohistochemical assay in discrete brain structures including the paraventricular hypothalamic nucleus (PVN), arcuate nucleus (ARC), and central nucleus of the amygdala (CeA). RESULTS: The quantitative data indicated that there was more NPY-I in the PVN and ARC of P rats than NP rats, whereas there was less NPY-I in the PVN and ARC of HAD rats than LAD rats. However, the NPY-I in the CeA was less in both the P and HAD rats than in the NP and LAD rats, respectively. Therefore, the data indicate that there are innate differences in the NPY-I in the brain between selectively bred rats with high and low alcohol preference. CONCLUSION: Because both P rats and HAD rats have high alcohol preference, the disparate finding between these two lines of rats suggests that the hypothalamic NPY neurons are probably not associated with alcohol preference. In contrast, consistent findings in the CeA of both P rats and HAD rats suggest that NPY in the CeA of P and HAD rats may contribute to the regulation of alcohol consumption. This is substantiated by a recent report showing that NPY-knockout mice drink significantly more ethanol, and transgenic mice that overexpress the NPY gene drink less alcohol, than wild-type mice. Together, the findings support the notion that NPY agonists that would enhance NPY function in the amygdala might be useful for the treatment of anxiety and alcoholism.


Subject(s)
Alcohol Drinking/genetics , Amygdala/chemistry , Hypothalamus/chemistry , Neuropeptide Y/analysis , Animals , Arcuate Nucleus of Hypothalamus/chemistry , Biomarkers/analysis , Gyrus Cinguli/chemistry , Male , Mice , Paraventricular Hypothalamic Nucleus/chemistry , Prefrontal Cortex/chemistry , Rats , Species Specificity
19.
Biomed Environ Sci ; 11(1): 31-7, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9559100

ABSTRACT

A national system of infectious disease surveillance was established in 1959 in China. Now it consists of three subunits, namely, national disease reporting system (NDRS), nationwide disease surveillance points (DSPs), and surveillance network for specific infectious diseases. There are 35 notifiable infectious diseases, which are divided into Classes A, B, and C. The functions of the surveillance include explaining the natural history of infectious diseases, describing the distribution of case occurrence, triggering disease-control effort, monitoring epidemic of infectious diseases during natural disasters, predicting and controlling epidemics and providing the base of policy adjustment.


Subject(s)
Communicable Diseases/epidemiology , Population Surveillance , China/epidemiology , Communicable Diseases/classification , Cross-Sectional Studies , Disasters , Disease Notification , History, 20th Century , Humans , Risk Assessment
20.
Appl Environ Microbiol ; 64(2): 768-70, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9464421

ABSTRACT

To simplify the incubation of Methanosarcina spp. on solid agar medium, a two-port, manual, rectangular air lock was modified to serve as an anaerobic incubator. In one operation, it is possible to incubate 153 petri plates, the equivalent of 11 standard anaerobic jars, with plating efficiencies identical to those of traditional protocols.


Subject(s)
Methanol/metabolism , Methanosarcina/growth & development , Anaerobiosis
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