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1.
Radiat Prot Dosimetry ; 179(2): 101-107, 2018 Apr 01.
Article in English | MEDLINE | ID: mdl-29069480

ABSTRACT

Radon exposure in schools is different from that in dwellings because the residence pattern is very different each other. So, when effective dose is calculated in schools, different approach should be considered from in dwellings. The aim of this study was to estimate actual effective dose due to inhaled radon considering the residence time in schools. It could help avoid overestimation when effective dose is calculated in schools. The range of radon concentration in 376 schools was 18.1-2810 Bq m-3 and that of annual effective dose was estimated 0.0902-8.92 mSv y-1 considering the residence time in spring and autumn semesters.


Subject(s)
Air Pollutants, Radioactive/analysis , Air Pollution, Indoor/analysis , Radiation Monitoring/methods , Radon/analysis , Schools , Humans , Radiation Dosage , Republic of Korea
2.
Osteoarthritis Cartilage ; 24(8): 1461-9, 2016 08.
Article in English | MEDLINE | ID: mdl-27049030

ABSTRACT

OBJECTIVE: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. DESIGN: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. RESULTS: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. CONCLUSIONS: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.


Subject(s)
Cartilage, Articular , Animals , Cathepsin K , Cattle , Collagen Type II , Collagenases , Horses , Humans
3.
Vet J ; 208: 55-9, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26639830

ABSTRACT

Doxycycline is regarded as an effective treatment for periodontal inflammation. In humans, it has been shown that the long-term administration of a subantimicrobial dose of doxycycline (SDD) does not induce antimicrobial effects on the subgingival microflora and furthermore does not affect antimicrobial susceptibility. The present study was designed to evaluate the influence of oral administration of SDD on normal periodontal microflora and antimicrobial susceptibility in dogs. Experimental periodontitis was induced in 12 experimental dogs using a silk and wire-twisted ligature for 60 days. After the periodontitis induction period, the ligature was removed, and dental cleaning (subgingival and supragingival ultrasonic scaling) was performed. The dogs were randomly assigned to one of two groups: an SDD group with six dogs receiving 2 mg/kg PO once daily and a control group with six dogs receiving a placebo. At weeks 0, 4 and 8, clinical periodontal parameters were evaluated. After the clinical assessments, subgingival plaque was sampled and then cultured in an anaerobic system for one week, and the total anaerobes, Porphyromonas spp., Bacteroides spp. and Pasteurella spp. counts were investigated. Using the agar dilution method, the minimum bactericidal concentration of doxycycline was evaluated and the resistance for doxycycline was monitored during this experimental phase. The clinical periodontal status of the SDD group was significantly improved compared to the control group (P <0.05). Bacterial counts were not significantly different between the two experimental groups (P > 0.05), and antibacterial resistance was not established in the SDD group during the experimental periods (P <0.05). These results suggest that the once daily oral regimen of 2 mg/kg of doxycycline could serve as a SDD in dogs.


Subject(s)
Bacteria, Anaerobic/drug effects , Dental Plaque/veterinary , Dog Diseases/drug therapy , Doxycycline/administration & dosage , Doxycycline/pharmacology , Periodontitis/veterinary , Administration, Oral , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacology , Dental Plaque/drug therapy , Dental Plaque/microbiology , Dog Diseases/microbiology , Dogs , Drug Resistance, Bacterial , Female , Male , Periodontitis/drug therapy , Periodontitis/microbiology
4.
Radiat Prot Dosimetry ; 167(4): 620-5, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25377749

ABSTRACT

The detailed indoor radon survey was conducted during a year (from September 2012 to August 2013) quarterly in Bonghwa county, one of the provisional radon-prone areas in Korea. The surveyed area was selected on the basis of previously conducted nationwide radon survey results. In order to minimise statistical and environmental uncertainties, ∼3 % of the entire dwellings were carefully selected based on the statistical annual report of Bonghwa county. The measurement is carried out by using solid-state nuclear track detector. The range of indoor radon concentration in each dwelling was 4.36-858 Bq m(-3) and that of annual effective dose due to inhaled radon of the resident in each dwelling was 0.19-23.5 mSv y(-1). Each dwelling was determined for geology criterion using one-way Analysis of Variance for the purpose of comparing indoor radon distribution with geology. Geographical distribution of indoor radon is closely related to the geological characteristics of basement rocks. In addition, the comparison between geographical distribution of indoor radon and terrestrial gamma radiation was done.


Subject(s)
Air Pollutants, Radioactive/analysis , Air Pollution, Indoor/analysis , Radiation Monitoring/methods , Radon/analysis , Geology , Housing , Humans , Republic of Korea
5.
Vet J ; 196(1): 103-8, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23000006

ABSTRACT

The effect of local anaesthetic on the extraocular muscles was investigated in dogs by injecting lidocaine into the space between Tenon's capsule and the sclera. A cross-over design was used with both eyes from five Beagle dogs randomly injected, under general anaesthesia, with 1 mL of 2% lidocaine (1 mL-lidocaine group), 2 mL of 2% lidocaine (2 mL-lidocaine group) or 2 mL of normal saline (control group). Each eye was assigned to all treatments with a minimum 14 day interval between injections. Changes in eye position, pupil diameter, and intraocular pressure (IOP) were evaluated during the procedure. All eyes in the 2 mL-lidocaine group exhibited akinesia and mydriasis (pupil diameter >10mm) with an onset time of 6.5 ± 4.9 and 4.2 ± 4.3 min, respectively. In the 1 mL-lidocaine group, akinesia was induced in nine eyes and mydriasis occurred in seven eyes at 10.7 ± 5.8 and 5.4 ± 2.4 min after the injection, respectively. No changes in eye position or pupil diameter were observed in the control group. Akinesia was maintained for 44.3 ± 26.7 min in the 1 mL-lidocaine group and for 88.5 ± 17.2 min in the 2 mL-lidocaine group. Duration of mydriasis was 51.7 ± 28.9 min in the 1 mL-lidocaine group and 82.9 ± 15.6 min in the 2 mL-lidocaine group. Marked chemosis and sub-conjunctival haemorrhage occurred in 16/30 and 15/30 eyes, respectively. No significant change in IOP was observed between the mean pre- and post-injection values in all groups. These results suggest that a sub-Tenon's injection of 2 mL of 2% lidocaine provided effective extraocular muscle akinesia and mydriasis in dogs.


Subject(s)
Anesthetics, Local/adverse effects , Eye/drug effects , Injections, Intraocular/methods , Lidocaine/adverse effects , Ophthalmologic Surgical Procedures/methods , Animals , Cross-Over Studies , Dogs , Female , Injections, Intraocular/veterinary , Intraocular Pressure/drug effects , Male , Ocular Physiological Phenomena/drug effects , Ophthalmologic Surgical Procedures/veterinary , Tenon Capsule/drug effects
6.
Science ; 321(5887): 411-3, 2008 Jul 18.
Article in English | MEDLINE | ID: mdl-18635805

ABSTRACT

Cyclic di-guanosine monophosphate (di-GMP) is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms by which cyclic di-GMP regulates gene expression have remained a mystery. We found that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in messenger RNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pilus formation, and flagellum biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.


Subject(s)
Aptamers, Nucleotide/metabolism , Bacteria/genetics , Cyclic GMP/analogs & derivatives , Gene Expression Regulation, Bacterial , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , Second Messenger Systems , Bacillus cereus/genetics , Bacillus cereus/metabolism , Bacteria/metabolism , Bacteriophages/genetics , Base Sequence , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Cyclic GMP/metabolism , Genes, Bacterial , Ligands , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Messenger/chemistry , Regulon , Vibrio cholerae/genetics , Vibrio cholerae/metabolism
8.
Matrix Biol ; 26(2): 140-3, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17123807

ABSTRACT

PRELP is a member of the small leucine-rich repeat proteoglycan family that is abundantly expressed in many cartilages compared to other connective tissues. To study the consequence of PRELP overexpression in tissues where it is normally expressed at low abundance, transgenic mice were generated in which the human PRELP transgene was placed under control of the CMV promoter. A connective tissue phenotype was observed in the skin, where the organization of collagen fibrils in the dermis was perturbed and the thickness of the hypodermal fat layer was diminished.


Subject(s)
Collagen/metabolism , Dermis/cytology , Extracellular Matrix Proteins/metabolism , Gene Expression , Glycoproteins/metabolism , Skin/metabolism , Adipose Tissue/metabolism , Animals , Collagen/physiology , Collagen/ultrastructure , DNA Primers , Dermis/metabolism , Humans , Mice , Mice, Transgenic , Microscopy, Electron , Plasmids/genetics , Skin/ultrastructure
9.
Osteoarthritis Cartilage ; 14(11): 1147-54, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16806997

ABSTRACT

OBJECTIVE: Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase-2 (ADAMTS-5) have been named for their ability to degrade the proteoglycan aggrecan. While this may be the preferred substrate for these enzymes, they are also able to degrade other proteins. The aim of this work was to determine whether the aggrecanases could degrade biglycan and decorin. METHODS: Biglycan, decorin and aggrecan were purified from human and bovine cartilage and subjected to degradation by recombinant aggrecanase-1 or aggrecanase-2. In vitro degradation was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting, and the cleavage site in biglycan was determined by N-terminal amino acid sequencing. SDS/PAGE and immunoblotting were also used to assess in situ degradation in both normal and arthritic human articular cartilage. RESULTS: Both aggrecanase-1 and aggrecanase-2 are able to cleave bovine and human biglycan at a site within their central leucine-rich repeat regions. Cleavage occurs at an asparagine-cysteine bond within the fifth leucine-rich repeat. In contrast, the closely related proteoglycan decorin is not a substrate for the aggrecanases. Analysis of human articular cartilage from osteoarthritic (OA) and rheumatoid arthritic (RA) joints showed that a biglycan degradation product of equivalent size is present in the extracellular matrix. No equivalent degradation product was, however, detectable in normal adult human articular cartilage. CONCLUSION: Biglycan, which is structurally unrelated to aggrecan, can act as a substrate for aggrecanase-1 and aggrecanase-2, and these proteinases may account for at least part of the biglycan degradation that is present in arthritic cartilage.


Subject(s)
Endopeptidases/metabolism , Extracellular Matrix Proteins/metabolism , Proteoglycans/metabolism , ADAM Proteins/metabolism , ADAMTS4 Protein , ADAMTS5 Protein , Adolescent , Adult , Aged , Aggrecans/metabolism , Animals , Arthritis, Rheumatoid/metabolism , Biglycan , Cartilage/metabolism , Cartilage, Articular/metabolism , Cattle , Child , Child, Preschool , Decorin , Electrophoresis, Polyacrylamide Gel/methods , Humans , Infant , Middle Aged , Osteoarthritis/metabolism , Procollagen N-Endopeptidase/metabolism , Protein Denaturation
10.
Am J Physiol Lung Cell Mol Physiol ; 281(5): L1164-72, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11597908

ABSTRACT

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl(-) transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl(-) transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl(-) gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl(-) currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl(-) solution, indicating restoration of Cl(-) secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl(-) secretion.


Subject(s)
Chlorides/metabolism , Cholestanols/pharmacology , Cystic Fibrosis/metabolism , Respiratory Mucosa/metabolism , Amiloride/pharmacology , Animals , Cells, Cultured , Cholestanols/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Diuretics/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Ion Transport , Ionophores/pharmacology , Membrane Potentials , Mice , Mice, Transgenic , Molecular Structure , Nasal Mucosa/metabolism , Patch-Clamp Techniques , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects
11.
Nutr Cancer ; 39(1): 35-41, 2001.
Article in English | MEDLINE | ID: mdl-11588900

ABSTRACT

Vitamin D has recently emerged as a potentially protective agent against colorectal neoplasia. We assessed the associations between dietary vitamin D, plasma 25-hydroxyvitamin D [25(OH)D], dietary calcium, and colorectal adenomas in a large screening sigmoidoscopy-based case-control study in Southern California. Because conversion of serum 25(OH)D to serum 1,25-vitamin D is highly regulated by serum calcium, we also assessed modification of the 25(OH)D-adenoma association by calcium intake. Cases were 473 subjects with a primary adenoma, and controls were 507 subjects who had no adenomas at sigmoidoscopy and no history of adenomas. Compared with those in the lowest quartile of intake, those in the highest quartile of dietary vitamin D had an adjusted odds ratio (OR) of 0.83 [95% confidence interval (CI) = 0.49-1.41] and those in the highest quartile of dietary calcium had an OR of 0.82 (95% CI = 0.49-1.25). There was a suggestion that plasma 25(OH)D may be protective in this population (OR for highest vs. lowest quartile = 0.74, 95% CI = 0.51-1.09). A significant protective effect of 25(OH)D was clearly evident only in those with calcium intakes below (OR = 0.40 for highest vs. lowest quartile, 95% CI = 0.22-0.71, p for trend = 0.005) and above (OR = 1.17, 95% CI = 0.69-1.99, p for trend = 0.94) the median calcium intake.


Subject(s)
Adenoma/etiology , Calcium, Dietary/administration & dosage , Colorectal Neoplasms/etiology , Vitamin D/analogs & derivatives , Vitamin D/administration & dosage , Vitamin D/blood , Adenoma/epidemiology , Aged , Case-Control Studies , Colorectal Neoplasms/epidemiology , Female , Humans , Male , Middle Aged , Odds Ratio , Prevalence , Risk , Risk Factors
12.
Cancer Causes Control ; 12(7): 607-14, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11552708

ABSTRACT

OBJECTIVE: The purpose of this study was to determine whether vitamin D receptor (VDR) gene polymorphisms influence risk of colorectal adenoma. METHODS: Polymorphisms in the 5' and 3' ends of the VDR gene were genotyped for 373 colorectal adenoma cases and 394 controls. RESULTS: Overall, there was no significant association between the 5' (FokI) or the 3' (BsmI) polymorphisms and adenoma risk. However, risk of large (>1 cm) adenomas decreased with increasing copies of the FokI f allele (p = 0.04). Compared to the FF genotype, odds ratios for the Ff and ff genotypes were 0.79 (95% CI 0.44-1.41) and 0.32 (95% CI 0.11-0.91), respectively. FokI genotype was more strongly related to large adenoma risk among subjects with low dietary calcium intake (ORFf=0.48; 95% CI 0.17-1.3; ORff=0.21: 95% CI 0.04-1.3), low dietary vitamin D intake (ORFf=0.25; 95% CI 0.09-0.69; ORff= 0.22; 95% CI 0.04-1.2), or dark skin color (ORFf=0.66; 95% CI 0.27-1.6; ORff=0.10; 95% CI 0.01-1.0). CONCLUSION: These results suggest that VDR FokI genotype influences development of colorectal adenomas. and that the effect may be modified by calcium and vitamin D status.


Subject(s)
Adenoma/genetics , Colorectal Neoplasms/genetics , Polymorphism, Genetic , Receptors, Calcitriol/genetics , Adenoma/ethnology , Adenoma/pathology , Aged , Case-Control Studies , Colorectal Neoplasms/ethnology , Colorectal Neoplasms/pathology , Female , Humans , Logistic Models , Male , Middle Aged , Odds Ratio , Polymerase Chain Reaction , Risk Factors , United States
13.
Dev Dyn ; 222(1): 52-70, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11507769

ABSTRACT

The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus ...FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus ...NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies contribute to the lysis of aggrecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting some diversity in the mode of aggrecan lysis during the excavation of a secondary ossification center.


Subject(s)
Bone Resorption , Cartilage/metabolism , Extracellular Matrix Proteins , Proteoglycans/metabolism , Tibia/enzymology , Aggrecans , Animals , Blotting, Western , Bone Marrow/metabolism , Chondrocytes/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epiphyses , Guanidine/metabolism , Immunoblotting , Immunoglobulin G/metabolism , Immunohistochemistry , Lectins, C-Type , Male , Matrix Metalloproteinases/metabolism , Microscopy, Electron , Microscopy, Immunoelectron , Models, Biological , Peptides/chemistry , Rats , Rats, Sprague-Dawley , Tibia/embryology , Time Factors
14.
Dev Dyn ; 222(1): 71-88, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11507770

ABSTRACT

In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-microm-thick band of matrix, referred to as "pre-resorptive layer." This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti-gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10-21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer-most likely through a cooperative attack leading to the disintegration of the collagen fibril framework.


Subject(s)
Bone Resorption , Cartilage/enzymology , Cartilage/physiology , Collagen/metabolism , Collagenases/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Biotinylation , Bone and Bones/embryology , Bone and Bones/ultrastructure , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Epiphyses/metabolism , Gelatin/metabolism , Immunoglobulin G/metabolism , Immunohistochemistry , Male , Matrix Metalloproteinase 13 , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/metabolism , Microscopy, Electron , Microscopy, Immunoelectron , Rats , Rats, Sprague-Dawley , Time Factors , Tissue Inhibitor of Metalloproteinase-1/pharmacology , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tissue Inhibitor of Metalloproteinase-2/pharmacology
15.
Mol Microbiol ; 38(1): 20-30, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11029687

ABSTRACT

The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined. C. trachomatis-infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml-1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM alpha-ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored. When chlamydiae were cultured in glucose concentrations greater than 1 mg ml-1, optimal growth and maximal glycogen accumulation occurred. In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant. When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C. trachomatis. This suggests that glycogen accumulation may not be essential for chlamydial survival. Reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed. Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability. Taken together, the data suggest that, unlike most free-living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.


Subject(s)
Carbon/metabolism , Chlamydia trachomatis/metabolism , Adenosine Triphosphate/metabolism , Culture Media , Glucose/metabolism , Glutamates/metabolism , Glycogen/metabolism , HeLa Cells , Humans , Reverse Transcriptase Polymerase Chain Reaction
16.
J Biol Chem ; 275(48): 38111-9, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-10984489

ABSTRACT

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.


Subject(s)
Chlamydia trachomatis/enzymology , Ribonucleotide Reductases/genetics , Amino Acid Sequence , Base Sequence , Chlamydia trachomatis/drug effects , Cloning, Molecular , DNA Primers , Drug Resistance, Microbial/genetics , Hydroxyurea/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolism , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
17.
Cancer Epidemiol Biomarkers Prev ; 9(7): 657-63, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10919734

ABSTRACT

A common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene, where a cytosine at nucleotide 677 is replaced by a thymine (677C-->T), is associated with enzyme thermolability and a reduction in the conversion of 5,10-methyltetrahydrofolate (5,10-MTHF) into 5-methyltetrahydrofolate. We assessed the association between homozygosity for the MTHFR 677CT genotype (TT) and colorectal adenoma risk in a large sigmoidoscopy-based case-control study of members of a prepaid health plan in Los Angeles. MTHFR genotype was determined for 471 cases and 510 age-, sex-, clinic-, and sigmoidoscopy-date-matched controls. Information on RBC and plasma folate levels were analyzed for 331 cases and 350 controls. When compared with the presence of at least one wild-type allele (CT/CC), the odds ratio (OR) for the TT genotype was 1.19 [95% confidence interval (CI), 0.77-1.76] after adjusting for race and the matching factors. Compared with those in the lowest quartiles of RBC and plasma folate and a wild-type allele, adenoma risk was increased for TT homozygotes in the lowest folate quartiles (genotype: OR, 2.04 and 95% CI, 0.6-7.0; OR, 1.84 and 95% CI, 0.6-7.0 for RBCs and plasma folate, respectively) and decreased in TT homozygotes in the highest quartiles (genotype: OR, 0.82 and 95% CI, 0.32-2.10; OR, 0.65 and 95% CI, 0.22-1.95, respectively). There was also a significant interaction between TT genotype and the increased adenoma risk associated with alcohol. These data are consistent with an interaction between MTHFR genotype and folate availability.


Subject(s)
Adenoma/genetics , Colorectal Neoplasms/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Polymorphism, Genetic , Adenoma/etiology , Aged , Case-Control Studies , Colorectal Neoplasms/etiology , Cytosine/metabolism , Female , Genotype , Humans , Loss of Heterozygosity , Male , Methylenetetrahydrofolate Reductase (NADPH2) , Middle Aged , Point Mutation , Risk Assessment , Sigmoidoscopy , Thymine/metabolism
18.
AJNR Am J Neuroradiol ; 21(5): 939-40, 2000 May.
Article in English | MEDLINE | ID: mdl-10815672

ABSTRACT

A patient with a rare variation of the fronto-orbital artery is presented. In this case, the fronto-orbital artery arises from the A1 segment of the contralateral anterior cerebral artery. The angiographic findings in this case are illustrated and discussed. The importance of recognizing this variant is related to the planning of surgery or endovascular therapy in the anterior cerebral artery region.


Subject(s)
Arteriovenous Malformations/diagnostic imaging , Cerebral Angiography , Frontal Lobe/blood supply , Orbit/blood supply , Adult , Humans , Intracranial Aneurysm/diagnostic imaging , Intracranial Aneurysm/surgery
19.
J Drug Target ; 7(6): 453-69, 2000.
Article in English | MEDLINE | ID: mdl-10758915

ABSTRACT

Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid:plasmid DNA (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanolamine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphatidylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid:pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.


Subject(s)
Genetic Therapy , Lipids/administration & dosage , Lung/metabolism , Transfection , Animals , Drug Stability , Excipients/pharmacology , Female , Lipids/chemistry , Mice , Mice, Inbred BALB C
20.
Dev Dyn ; 215(3): 190-205, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10398530

ABSTRACT

In order to determine which proteinases mediate the resorption of endochondral cartilage in the course of long bone development, a novel assay called "histozymography" has been developed. In this assay, frozen sections of tibial head from 21-day-old rats are placed for 4 hr at room temperature on light-exposed photographic emulsion (composed of silver grains embedded in gelatin). We report a localized but complete digestion of emulsion gelatin facing two tissue sites which are, therefore, presumed to contain an active proteinase. One of the sites is localized at the growth plate surface forming the epiphysis/metaphysis interface. The other consists of small patches located within the epiphysis at the edge of the marrow space. Both sites are engaged in the resorption of endochondral cartilage. In both sites, inhibitor tests have established that the involved proteinase is a gelatinase. Furthermore, the use of neutralizing antibodies against gelatinase A or B have demonstrated that only those that are specific for the latter block the reaction. That gelatinase B is present in the two sites has been confirmed by light microscopic immunohistochemistry. Finally, when immunoelectron microscopy is used for fine localization of the cartilage structures that form the epiphysis/metaphysis interface, the enzyme is detected within the 0.5-microm thick edge of the cartilage, and outside the cartilage, it is present in debris composed of type II collagen-rich fibrils in various states of digestion. It is concluded that gelatinase B attacks the edge of an endochondral cartilage and helps to solubilize the type II-collagen-rich fibrillar framework, which is then released as debris for further digestion. This final step opens the way to invasion by capillaries, thereby making possible the replacement of cartilage by bone. Dev Dyn 1999;215:190-205.


Subject(s)
Bone Development/physiology , Cartilage/growth & development , Collagenases/analysis , Epiphyses/enzymology , Tibia/growth & development , Animals , Collagen/metabolism , Emulsions , Enzyme Inhibitors/pharmacology , Epiphyses/growth & development , Frozen Sections , Gelatin/metabolism , Male , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Microscopy, Immunoelectron , Neovascularization, Physiologic , Rats , Rats, Sprague-Dawley , Tibia/blood supply
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