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1.
Zhonghua Liu Xing Bing Xue Za Zhi ; 44(6): 891-898, 2023 Jun 10.
Article in Chinese | MEDLINE | ID: mdl-37380409

ABSTRACT

Objective: To investigate the spatial distribution characteristics and correlation between the prevalence of dental fluorosis and the chemical elemental composition of drinking water sources in coal-fired fluorosis areas. Methods: Based on the survey data on the prevalence of dental fluorosis at CDC in Guizhou Province in 2022, 274 original surface drinking water sources were collected in typical coal-fired fluorosis areas, and fluoride (F), calcium (Ca), magnesium (Mg), aluminum (Al), titanium (Ti), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), molybdenum (Mo), cadmium (Cd), barium (Ba), lead (Pb) 17 elements; apply Moran's I index, Getis-Ord Gi* hotspot analysis of the global spatial autocorrelation of chemical elements in drinking water and the degree of aggregation of each element on the local area, and correlation analysis with the prevalence of dental fluorosis in the region. Results: Except for Cu, Zn, and Cd, global spatial autocorrelation Moran's I was negative, and all other elements were positive. F, Ca, Al, Ti, As, Mo, Cd, and Cu elements showed high values of aggregation in the southeastern low-altitude area; Mg, Ba, Pb, Cr, Mn, and Fe elements were mainly aggregated in the central altitude terrain transition area, Zn and Se elements in water sources are significantly positively correlated with the prevalence of dental fluorosis (P<0.05). In contrast, F, Mg, Al, Ti, As, Mo, Cd, Ba, and Pb elements negatively correlate (P<0.05). Elements in the central region were high-high aggregation, as a hot spot aggregation area with high disease incidence, while F, Al, Mn, Mo, Cd, and Ba elements in the western region were low-low aggregation, as a cold spot aggregation area with a low incidence of fluorosis. Conclusions: The risk of population fluoride exposure in surface drinking water sources is shallow. However, the chemical element content of drinking water sources in coal-fired polluted endemic fluorosis areas has prominent spatial geographical distribution characteristics. There is a significant spatial aggregation effect with the prevalence of dental fluorosis, which may play a synergistic or antagonistic effect on the occurrence and prevalence of dental fluorosis.


Subject(s)
Arsenic , Drinking Water , Fluorosis, Dental , Selenium , Humans , Prevalence , Coal , Fluorides/adverse effects , Cadmium , Fluorosis, Dental/epidemiology , Lead
2.
Zhonghua Yi Xue Za Zhi ; 96(48): 3902-3905, 2016 Dec 27.
Article in Chinese | MEDLINE | ID: mdl-28057162

ABSTRACT

Objective: To study the prevalence of obstructive sleep apnea hypopnea syndrome (OSAHS) and its relationship with traffic accidents in the professional drivers. Methods: Questionnaires of OSAHS were sent to 950 professional drivers who had annual physical examination at the Central Hospital of Jiading District in Shanghai from October 2014 to September 2015. Those with moderate to severe snoring and/or Epworth Sleepiness Scale (ESS)≥9 performed the home sleep testing. All drivers were divided into OSAHS and non-OSAHS according to the survey and monitoring. The following parameters were compared such as driving ages, neck circumference, body mass index (BMI), average night sleep time, ESS, hypertension, diabetes, hypertrophy of tonsil and the incidence of traffic accidents. The risk factors of traffic accidents were analyzed by multivariate Logistic regression. Results: Totally 826 responses were eligible, including 578 (70.0%) with self-reported snoring. There was measurement failure involving 3 of 233 the home sleep testing due to sensor off, 823 subjects were included in the study. The prevalence of OSAHS was 13.5% (111/823). The mild, moderate and severe OSAHS were 47, 38 and 26 cases respectively. There were 712 drives without OSAHS. The neck circumference[(39.8±3.8) vs (39.0±3.0) cm]and BMI[(26.7±4.2) vs (24.4±3.8) kg/m2]were significantly higher in the drivers suffering from OSAHS (all P<0.05). The percentage of ESS≥ 9 (57.7% vs 12.6%), hypertension (27.9% vs 5.9%), diabetes (4.5% vs 1.1%), hypertrophy of tonsil (7.2% vs 2.3%) were higher in the drivers with OSAHS (all P<0.05). There were no significant difference in driving ages and average night sleep time between two groups (all P>0.05). The overall incidence of traffic accidents was 5.8% (48/823) in a year. The percentage was respectively 17.1% (19/111) in OSAHS and 4.1% (29/712) in non-OSAHS (P<0.001). Multiple logistic regression analysis showed that sleepiness (OR=30.578, 95%CI: 10.699-87.394; P<0.001), OSAHS (OR=14.062, 95%CI: 4.791-41.269; P<0.001) and vehicle years (OR=2.345, 95%CI: 1.183-4.646; P<0.05)were the risk factors, while the average night sleep time (OR=0.037, 95%CI: 0.014-0.098; P<0.001) was the protective factor. Conclusion: Professional drivers have higher prevalence of OSAHS, which contributes to the increased risk of traffic accidents.


Subject(s)
Accidents, Traffic , Automobile Driving , Sleep Apnea, Obstructive , Body Mass Index , China , Humans , Hypertension , Polysomnography , Prevalence , Risk Factors , Sleep , Sleep Stages , Snoring , Surveys and Questionnaires , Syndrome
3.
Int J Parasitol ; 44(5): 291-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24583111

ABSTRACT

Traditional healers in Sarawak, Malaysia, use plants such as Picria fel-terrae, Linariantha bicolor and Lansium domesticum to treat gastrointestinal infections. This study aimed to test whether their nematocidal activities could be confirmed in vitro using highly standardised Caenorhabditis elegans models. We applied eight different ethanol solubilised plant extracts and two commercial anthelmintic drugs to larval and adult stages of C. elegans in vitro. Seven C. elegans strains were evaluated, one wild type and six strains with GFP-tagged stress response pathways to help characterise and compare the pathways affected by plant extracts. Our in vitro screen confirmed that both of the commercial anthelmintic drugs and five of the eight traditionally used plant extracts had significant nematocidal activity against both larval and adult C. elegans. The most effective extracts were from P. fel-terrae. The plant extracts triggered different stress response pathways from the commercial anthelmintic drugs. This study showed that using traditional knowledge of plant medicinal properties in combination with a C. elegans in vitro screen provided a rapid and economical test with a high hit rate compared with the random screening of plants for nematocidal activities. The use of transgenic C. elegans strains may allow this approach to be refined further to investigate the mode of action of active extracts.


Subject(s)
Anthelmintics/pharmacology , Caenorhabditis elegans/drug effects , Drug Evaluation, Preclinical/methods , Plant Extracts/pharmacology , Plants, Medicinal/chemistry , Animals , Anthelmintics/isolation & purification , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Larva/drug effects , Plant Extracts/isolation & purification , Tracheophyta/chemistry
4.
Br J Dermatol ; 164(1): 16-25, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20846312

ABSTRACT

BACKGROUND: Endoplasmic reticulum (ER) Ca(2+) depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation. OBJECTIVES: To investigate the role of Ca(2+) depletion-induced ER stress in keratinocyte differentiation and barrier repair in vivo and in cell culture. METHODS: The SERCA2 Ca(2+) pump inhibitor thapsigargin (TG) was used to deplete ER calcium both in cultured keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, caspase 14, lipid synthesis and intracellular Ca(2+) were compared after both TG treatment and barrier abrogation. RESULTS: We showed that these components of terminal differentiation and barrier repair were signalled by physiological ER stress, via release of stratum granulosum (SG) ER Ca(2+) stores. We first found that keratinocyte and epidermal ER Ca(2+) depletion activated the ER-stress-induced transcription factor XBP1. Next, we demonstrated that external barrier perturbation resulted in both intracellular Ca(2+) emptying and XBP1 activation. Finally, we showed that TG treatment of intact skin did not perturb the permeability barrier, yet stimulated and mimicked the physiological processes of barrier recovery. CONCLUSIONS: This report is the first to quantify and localize ER Ca(2+) loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca(2+), via induction of physiological ER stress.


Subject(s)
Calcium/metabolism , Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/metabolism , Epidermis/metabolism , Keratinocytes/cytology , Transcription Factors/metabolism , Animals , Caspase 14/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Enzyme Inhibitors/pharmacology , Epidermis/drug effects , Epidermis/pathology , Humans , Immunoblotting , Keratinocytes/drug effects , Keratinocytes/pathology , Lipids/analysis , Membrane Proteins/metabolism , Mice , Polymerase Chain Reaction , Regulatory Factor X Transcription Factors , Thapsigargin/pharmacology , X-Box Binding Protein 1
5.
Bull Environ Contam Toxicol ; 80(5): 465-8, 2008 May.
Article in English | MEDLINE | ID: mdl-18465071

ABSTRACT

Total concentrations and speciation of metals had been studied in TSP of Guiyang from April 2006 and January 2007, PR China. The average concentration ranged from 14.48 ng m(-3) for Cd to 1,161.45 ng m(-3) for Zn. The concentrations of Cd, Cr, Pb and Zn were significantly higher during winter than those at other seasons. The environmentally mobile fractions of Cd and Zn were the highest in the three stages. The highest proportion of Pb was the fraction that bound to carbonate and oxide. Cr and Cu were clearly restricted to the fraction that bound to silicate and organic matter.


Subject(s)
Air Pollutants/analysis , Environmental Monitoring/methods , Metals, Heavy/analysis , Air Pollutants/classification , Atmosphere , China , Industrial Waste , Metals, Heavy/classification
6.
J Cell Biochem ; 88(2): 290-5, 2003 Feb 01.
Article in English | MEDLINE | ID: mdl-12520529

ABSTRACT

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.


Subject(s)
Cell Differentiation/drug effects , Keratinocytes/drug effects , Signal Transduction/physiology , Trans-Activators/drug effects , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Animals , Calcium/metabolism , Calcium-Binding Proteins/drug effects , Calcium-Binding Proteins/genetics , Cell Differentiation/genetics , Gene Expression Regulation/drug effects , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Signal Transduction/drug effects , Trans-Activators/physiology , Type C Phospholipases/drug effects , Type C Phospholipases/metabolism
7.
J Biol Chem ; 276(44): 41079-85, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11500521

ABSTRACT

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.


Subject(s)
Calcium/physiology , Cell Differentiation/physiology , Keratinocytes/cytology , Receptors, Cell Surface/physiology , Amino Acid Sequence , Blotting, Northern , DNA, Complementary , Humans , Microscopy, Confocal , Molecular Sequence Data , Receptors, Calcium-Sensing , Receptors, Cell Surface/genetics , Transfection
8.
Am J Physiol Endocrinol Metab ; 281(1): E180-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11404236

ABSTRACT

Parathyroid cells express Ca2+ -conducting currents that are activated by raising the extracellular Ca2+ concentration ([Ca2+]o). We investigated the sensitivity of these currents to dihydropyridines, the expression of voltage-dependent Ca(2+) channel (VDCC) subunits, and the effects of dihydropyridines on the intracellular free [Ca2+] ([Ca2+]i) and secretion in these cells. Dihydropyridine channel antagonists dose dependently suppressed Ca2+ -conducting currents, and agonists partially reversed the inhibitory effects of the antagonists in these cells. From a bovine parathyroid cDNA library, we isolated cDNA fragments encoding parts of an alpha(1S)- and a beta(3)-subunit of L-type Ca(2+) channels. The alpha(1S)-subunit cDNA from the parathyroid represents an alternatively spliced variant lacking exon 29 of the corresponding gene. Northern blot analysis and immunocytochemistry confirmed the presence of transcripts and proteins for alpha(1)- and beta(3)-subunits in the parathyroid gland. The addition of dihydropyridines had no significant effects on high [Ca2+]o-induced changes in [Ca2+]i and parathyroid hormone (PTH) release. Thus our studies indicate that parathyroid cells express alternatively spliced L-type Ca2+ channel subunits, which do not modulate acute intracellular Ca2+ responses or changes in PTH release.


Subject(s)
Calcium Channels, L-Type/drug effects , Dihydropyridines/pharmacology , Ion Channels/drug effects , Parathyroid Glands/metabolism , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Amino Acid Sequence , Animals , Blotting, Northern , Calcium/pharmacology , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Cattle , Cell Separation , Cloning, Molecular , DNA/genetics , Electrophysiology , Immunohistochemistry , In Vitro Techniques , Molecular Sequence Data , Parathyroid Glands/cytology , Parathyroid Glands/drug effects , Parathyroid Hormone/biosynthesis , Parathyroid Hormone/genetics , Patch-Clamp Techniques , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction
9.
Mol Cell Endocrinol ; 177(1-2): 161-71, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11377831

ABSTRACT

Calcium and 1,25 dihydroxyvitamin D (1,25(OH)(2)D) regulate the differentiation of keratinocytes. We have examined the mechanisms by which such regulation takes place, focusing primarily on the events leading to cornified envelope (CE) formation, in particular the mechanisms by which calcium and 1,25(OH)(2)D regulate the induction of involucrin, a component of the CE, and transglutaminase, the enzyme cross-linking involucrin and other substrates to form the CE. Both extracellular calcium (Ca(o)) and 1,25(OH)(2)D raise intracellular free calcium (Ca(i)) as a necessary step toward stimulating differentiation. Cells lacking the calcium sensing receptor (CaR) or phospholipase C-gamma 1 (PLC-gamma 1) fail to respond to Ca(o) or 1,25(OH)(2)D with respect to differentiation. Residing in the promoter of involucrin is a region responsive to calcium and 1,25(OH)(2)D, the calcium response element (CaRE). The CaRE contains an AP-1 site, mutations of which result in loss of responsiveness to Ca(o) and 1,25(OH)(2)D, indicating a role for protein kinases C (PKC). PKC alpha is the major PKC isozyme involved at least for calcium-induced differentiation. Thus, the regulation of keratinocyte differentiation by calcium and 1,25(OH)(2)D involves a number of signaling pathways including PLC and PKC activation, leading to the induction of proteins required for the differentiation process.


Subject(s)
Calcium/pharmacology , Keratinocytes/cytology , Vitamin D/pharmacology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Humans , Keratinocytes/enzymology , Keratinocytes/ultrastructure , Protein Precursors/drug effects , Protein Precursors/genetics , Protein Precursors/metabolism , Signal Transduction , Vitamin D/metabolism
10.
J Biol Chem ; 275(2): 1183-90, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625662

ABSTRACT

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.


Subject(s)
Alternative Splicing , Keratinocytes/cytology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiology , Skin/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Base Sequence , Calcium/metabolism , Cattle , Cell Differentiation , Cell Line , Cells, Cultured , Cloning, Molecular , Epidermal Cells , Epidermis/physiology , Humans , Keratinocytes/physiology , Mice , Mice, Knockout , Molecular Sequence Data , Receptors, Calcium-Sensing , Receptors, Cell Surface/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Skin/cytology , Transfection
11.
J Invest Dermatol ; 113(3): 340-5, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10469331

ABSTRACT

Changes in the concentration of extracellular calcium affect the balance between proliferation and differentiation in epidermal keratinocytes. Undifferentiated keratinocytes respond to the acute increase in the concentration of extracellular calcium with an increase of intracellular calcium concentration and inositol trisphosphate production, and, subsequently, the expression of differentiation related genes. Our previous studies demonstrated the presence of a calcium-sensing receptor in human keratinocytes, which is identical to the parathyroid calcium-sensing receptor. In this study we showed that the calcimimetic compound NPS R-467, a selective calcium-sensing receptor activator, augmented the calcium-elicited inositol trisphosphate response of cloned human keratinocyte calcium-sensing receptor expressed in human embryonic kidney cells 293. In order to define the role of the calcium-sensing receptor in calcium induced epidermal differentiation, we investigated the ability of NPS R-467 to raise intracellular Ca2+ and stimulate differentiation in normal human foreskin keratinocytes. In the presence of 0.03 mM Ca2+, NPS R-467 increased the intracellular calcium concentration response in a concentration-dependent fashion. Undifferentiated normal human foreskin keratinocyte cells responded to increased extracellular calcium concentration with increased intracellular calcium concentration. NPS R-467 potentiated this response by increasing the maximal response. Its stereoisomer, NPS S-467, was not active in raising intracellular calcium concentration. Increasing extracellular calcium concentration from 0.03 to 1.2 mM stimulated the promoter activity of the differentiation marker gene, involucrin. NPS R-467 potentiated the calcium-stimulated increase in involucrin promoter activity unlike NPS S-467 or vehicle. Northern analysis of the normal human foreskin keratinocyte cells treated with NPS R-467 demonstrated potentiation of the calcium-stimulated increases in involucrin and transglutaminase mRNA levels. These results support the hypothesis that the calcium-sensing receptor expressed in keratinocytes mediates at least part of the intracellular calcium response to extracellular calcium and calcium-induced differentiation.


Subject(s)
Aniline Compounds/pharmacology , Calcium-Binding Proteins/drug effects , Calcium/metabolism , Keratinocytes/drug effects , Calcium-Binding Proteins/physiology , Cells, Cultured , Humans , Inositol Phosphates/metabolism , Keratinocytes/metabolism , Protein Precursors/genetics , RNA, Messenger/analysis , Transglutaminases/genetics
12.
J Biol Chem ; 273(36): 23344-52, 1998 Sep 04.
Article in English | MEDLINE | ID: mdl-9722568

ABSTRACT

We have recently reported the presence of the calcium sensing receptor (CaR) in keratinocytes and suggested that it signaled calcium-induced differentiation of these cells. cDNA clones encoding the full-length CaR were isolated from human keratinocytes. In addition, an alternatively spliced form that lacks exon 5, encoding a portion of the extracellular domain, also was found. The in frame deletion of 231 nucleotides of exon 5 resulted in the loss of function of the CaR as measured by calcium-stimulated production of inositol phosphates when transfected into HEK293 cells or keratinocytes. This variant produced a smaller CaR protein with an altered glycosylation pattern compared with the full-length CaR. Coexpression of the spliced variant with the full-length CaR reduced the function of the full-length CaR. The full-length CaR was expressed in undifferentiated keratinocytes consistent with their greater response to elevated extracellular calcium in terms of increased intracellular free calcium and production of inositol phosphates. The full-length CaR decreased as the keratinocytes differentiated with an increase in the ratio of the spliced variant to the full-length form. The relative proportions of these two forms of CaR may regulate the calcium responsiveness of keratinocytes during their differentiation.


Subject(s)
Alternative Splicing , Calcium/metabolism , Keratinocytes/cytology , Receptors, Cell Surface/genetics , Cell Differentiation , Glycosylation , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Models, Molecular , Protein Processing, Post-Translational , Reading Frames , Receptors, Calcium-Sensing , Receptors, Cell Surface/metabolism , Recombinant Proteins/metabolism , Transfection
13.
J Virol ; 66(2): 999-1006, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1731118

ABSTRACT

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.


Subject(s)
Mutagenesis, Site-Directed , Open Reading Frames , RNA Viruses/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/physiology , Base Composition , Base Sequence , Chromosome Deletion , Frameshift Mutation , Models, Structural , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides , Plasmids , Promoter Regions, Genetic , Restriction Mapping , Saccharomyces cerevisiae/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
14.
J Virol ; 63(9): 3983-90, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2668562

ABSTRACT

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.


Subject(s)
Genes, Viral , RNA Viruses/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Molecular Sequence Data , RNA-Dependent RNA Polymerase/analysis , Viral Fusion Proteins/biosynthesis
15.
J Biol Chem ; 264(6): 3404-8, 1989 Feb 25.
Article in English | MEDLINE | ID: mdl-2536747

ABSTRACT

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.


Subject(s)
Gene Expression Regulation/drug effects , Heat-Shock Proteins/genetics , Insulin/pharmacology , Carcinoma, Hepatocellular , Cycloheximide/pharmacology , DNA/biosynthesis , Dose-Response Relationship, Drug , Epidermal Growth Factor/pharmacology , Hepatitis B Surface Antigens/genetics , Humans , Kinetics , Liver Neoplasms , Platelet-Derived Growth Factor/pharmacology , RNA, Messenger/genetics , Receptor, Insulin/drug effects , Receptor, Insulin/metabolism , Transcription, Genetic , Tumor Cells, Cultured
16.
Can J Microbiol ; 22(4): 592-7, 1976 Apr.
Article in English | MEDLINE | ID: mdl-816442

ABSTRACT

An acetate-requiring leaky mutant was induced from Bacillus subtilis 168, and activities of its three alpha-keto acid dehydrogenases were compared with the respectives activities of the parent strain. Both pyruvate and alpha-ketoisovalerate dehydrogenase activities in the mutant were consideralby lower, being only 10-17% of those of the parent, but alpha-ketoglutarate dehydrogenase activity was unchanged. These dehydrogenases are complexed composed of three enzymes: a carboxylase, a lipoic reductase-transacylase, and a dihydrolipoyl dehydrogenase. The carboxylase activity of the affected complexes was no different. Total dihydrolipoyl dehydrogenase activity was only one-third. Thus dihydrolipoyl dehydrogenase is the defective enzyme in the two dehydrogenase complexes; the activity remaining in the mutant is accounted for by the activity of the intact alpha-ketoglutarate dehydrogenase.


Subject(s)
Bacillus subtilis/enzymology , Ketoglutarate Dehydrogenase Complex/metabolism , Ketone Oxidoreductases/metabolism , Mutation , Pyruvate Dehydrogenase Complex/metabolism , Acetates/metabolism , Cell-Free System , Dihydrolipoamide Dehydrogenase/metabolism , Pyruvate Decarboxylase/metabolism
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