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PubMed1997

A population-based case-control study of the CYP2D6 and GSTT1 polymorphisms and malignant brain tumors.

Previous studies of associations of metabolic polymorphisms with the occurrence of malignant brain tumors have suggested that there is a significantly increased risk of development of adult gliomas in individuals who carry a poor metabolizer CYP2D6 variant allele and the GSTT1 null genotype. To investigate this further, a population-based case control study of adult glioma in the San Francisco Bay area was conducted. Patients (n = 188) diagnosed with brain tumors and controls (n = 166) were enrolled using random digit dialing and were frequency matched for age, ethnicity and gender. Genotyping for the polymorphisms was performed using standard PCR-based techniques. The analysis of the data was restricted to Caucasians because the prevalence of these traits is known to vary by ethnicity. No overall association of either the GSTT1 null genotype or CYP2D6 homozygous variant PM genotype was observed with the occurrence of brain tumors. However, when stratified by histopathologic subtype, there was a significantly increased risk for oligodendroglioma associated with the GSTT1 null genotype, with an OR of 3.2 (95% CI 1.1-9.2). These data suggest that the GSTT1 polymorphism may play a role in the development of a subset of malignant brain tumors in adults, and indicate the need for further studies.

Adult↗
PubMed2003

Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma.

AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

DNA, Complementary↗
PubMed2001

Dose-image optimisation in digital radiology with a direct digital detector: an example applied to pelvic examinations.

In diagnostic radiology increasing attention has been focused on dose reduction while maintaining a clinically good image quality. With the use of digital detectors balancing dose vs image quality is done differently than in film-screen radiography, since dose and image brightness are uncoupled in digital imaging. In this study a new direct digital detector (flat-panel detector) was used in a dose-image optimisation of a simulated pelvic examination. X-ray images were taken with a direct digital detector (DDD), of the pelvic of a phantom using varying tube current (varying stochastic noise). The entrance surface dose was measured for each image. These images were scored by two radiologists according to EU guidelines. A dose comparison was made with an older PCR system (storage phosphor plates). With decreasing tube current the noise in the images increased and the image with the lowest dose and still acceptable image quality was identified. The results showed that the entrance surface dose using the DDD decreased from 1.4 mGy (PCR value) to 0.48 mGy (DDD standard settings). Through the optimisation the dose could be further decreased to 0.24 mGy while still maintaining an acceptable image quality. A substantial dose reduction was obtained with this new direct digital detector. This simple but efficient optimisation approach is easily applicable to other examinations and both DDD and storage phosphor plate detectors.

Humans↗
PubMed1997

Enhanced Reverse Transcriptase Polymerase Chain Reaction for Prostate-specific Antigen Combined With Needle Biopsy Results: A Superior Predictor of pT3 Disease.

Background: Preoperative staging for prostate cancer underestimates the final pathology stage in approximately 40-50% of the cases. Previous work from our institution demonstrated that an enhanced reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate-specific antigen (PSA) enabled more accurate staging of presumably localized prostate cancer. The goal of the current study is to determine if needle biopsy results when combined with the RT-PCR for PSA assay are a better predictor of final pathology stage. Methods and Results: One hundred sixty-two men with needle biopsy-diagnosed prostate cancer had blood drawn for the RT-PCR for PSA assay before undergoing radical prostatectomy. Polymerase chain reaction primers specific for the PSA gene were run, along with appropriate controls. Tumor was characterized using the TMN staging system: organ confined (pT2a-c), capsular penetration (pT2a-b), seminal vesicle involvement (pT3c). Surgical margins and lymph nodes were also evaluated. Of the 162 patients, the majority had localized disease by digital rectal examination: T2 = 97%, and T3 = 3%. On needle biopsy, 48 cases (30%) had a Gleason score >/=7 and 35 cases (22%) had perineural involvement (PNI). The RT-PCR for PSA assay was positive in 50 patients (31%). Final pathology revealed 39% of patients had pT3 disease; none of the 162 patients had lymph node involvement. Statistical analysis revealed that a Gleason score >/=7 had 81% specificity and 46% sensitivity in predicting pT3 disease (odds ratio 3.6). The presence of PNI on needle biopsy was 89% specific and 38% sensitive in predicting pT3 disease (odds ratio, 4.9). The RT-PCR for PSA assay was 89% specific and 62% sensitive in predicting pT3 disease (odds ratio, 13.0). All 14 cases with both RT-PCR for PSA and PNI positivity had pT3 disease. Logistic regression analysis demonstrated the independent predictive strength of PNI on needle biopsy, Gleason score >/=7, and RT-PCR for PSA positivity for identifying pT3 disease; their combined odds ratio was more than 180. Conclusions: Using the RT-PCR for PSA assay in conjunction with needle biopsy results increases the predictive strength for pT3 disease in patients with presumed organ-confined prostate carcinoma.

Journal Article↗
PubMed2006

Automation of fluorescent differential display with digital readout.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Automation↗
PubMed2002

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets.

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Alleles↗
PubMed2005

[HOXD13 polyalanine tract expansion in synpolydactyly: mutation detection and prenatal diagnosis in a large Chinese family].

OBJECTIVE: Synpolydactyly (SPD, MIM 186000), also known as syndactyly type II, is a dominantly inherited limb malformation with incomplete penetrance and variable expressivity. Polyalanine tract expansion in HOXD13 has been shown to be the disease-causing mutation in SPD. The present study was designed to identify mutation in HOXD13 and to provide prenatal diagnosis, in a large Chinese SPD family consisting of 54 individuals. METHODS: The proband and 4 other affected individuals in the family were evaluated physically and radiologically to ascertain the SPD phenotype. Genomic DNA was extracted from peripheral blood samples obtained from 18 family members (9 affected and 9 unaffected), and from amniotic fluid and chorionic villus samples obtained from the proband during her two consecutive pregnancies. With the use of a pair of specific primers, a fragment of 161bp was amplified by polymerase chain reaction (PCR) to cover the imperfect GCN triplet repeat sequence in exon 1 of HOXD13 encoding the 15-residue polyalanine tract. The PCR products were detected by agarose gel electrophoresis, and sequenced after cloning into pMD18T vector. To confirm prenatal diagnosis, haplotype analysis was also performed by allele-typing three microsatellite markers, including the intronic CA repeats in HOXD13. RESULTS: Digital and radiographic findings indicated a typical SPD phenotype in the family. These included 3/4 finger syndactyly and 4/5 toe syndactyly with an extra digit in the syndactylous web. Unilateral finger syndactyly in the proband, unilateral toe syndactyly in 2 individuals, bilateral brachydactyly of the fifth toes in 1 individual, and clinodactyly of the fifth fingers in 4 individuals were also observed, indicating variable expressivity. Gel electrophoresis of the PCR products showed an additional longer fragment in all 9 affected individuals but not in the unaffected ones. Sequence analysis of the longer fragment revealed a 9-alanine expansion. The expansion was detectable in DNA from the amniotic fluid and chorionic villus samples. Furthermore, haplotype analysis ruled out potential contamination of the maternal DNA. These suggested that the two fetuses carried the same polyalanine expansion. CONCLUSION: HOXD13 polyalanine expansion was detected in a large Chinese family with SPD and prenatal diagnosis of two affected fetuses was achieved. This is the first report on prenatal diagnosis of SPD by detecting the HOXD13 polyalanine expansion in the Han population of the Chinese mainland.

Base Sequence↗
PubMed2005

High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population.

We have developed a new high-throughput, high-resolution genotyping method for the detection of alleles at the human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual-laser system to quantitate fluorescently labeled oligonucleotides attached to color-coded microbeads. In order to detect the HLA alleles with a frequency of more than 0.1% in the Japanese population, we created 48 oligonucleotide probes for the HLA-A locus, 61 for HLA-B, 34 for HLA-C, and 51 for HLA-DRB1. The accuracy of the PCR-SSOP-Luminex method was determined by comparing it to the nucleotide sequencing method after subcloning into the plasmid vector using 150 multinational control samples obtained from the International HLA DNA Exchange University of California Los Angeles. In addition, we performed the PCR-SSOP-Luminex method for HLA allele typing on DNA samples collected from 1,018 Japanese volunteers. Overall, the genotyping method exhibited an accuracy of 85.91% for HLA-A, 85.03% for HLA-B, 97.32% for HLA-C, and 90.67% for HLA-DRB1 using 150 control samples, and 100% for HLA-A and -C, 99.90% for HLA-B, and 99.95% for HLA-DRB1 in 1,018 Japanese samples. The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution in the Japanese population. It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist.

DNA Primers↗
PubMed2004

Spinal cord involvement in the nonhuman primate model of Lyme disease.

Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7-24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHP's examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHP's, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.

Animals↗
PubMed2003

Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis.

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

Bacteria↗
PubMed2002

Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve.

BACKGROUND: Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. METHODS AND RESULTS: To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor (Cbfa-1)]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin. CONCLUSIONS: These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis-like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin.

Alkaline Phosphatase↗
PubMed2004

Platelets adhering to the vascular wall mediate postischemic leukocyte-endothelial cell interactions in retinal microcirculation.

PURPOSE: Recent evidence suggests that platelets play a major role in ischemia-reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo. METHODS: Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia-reperfusion injury. CONCLUSIONS: This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Acridine Orange↗
PubMed2005

Ulcerative cutaneous mycobacteriosis due to Mycobacterium ulcerans: report of two Mexican cases.

We report two patients from Central Mexico, with ulcerated cutaneous lesions containing acid-fast bacilli (AFB) and ultimately diagnosed as Mycobacterium ulcerans disease. The first patient had a long history (11 years) of disease involving multiple lesions of both upper and lower extremities. Histopathological changes included necrosis of the subcutaneous tissue with large numbers of extracellular AFB. Cultures at 32 degrees C were "positive for mycobacteria," but were not further identified. The polymerase chain reaction for M. ulcerans performed on skin bopsies was positive. The lesions improved after treatment with rifampin and isoniazid (INH) for one month, followed by ethambutol and streptomycin. The second case followed trauma to the right hand, which spread over 2 years to the right upper extremity, the back, and both legs, with a loss of digits and metacarpal bones of the right hand. The histopathological findings were similar to the first case, including presence of AFB. PCR for M. ulcerans on extracts of skin biopsies was positive. Rifampin, INH, pyrazinamide, and levofloxacin resulted in marked improvement of the ulcer; ethambutol and streptomycin were later used, also. We report these cases because they are rare (approximately 6 previous cases were reported from Mexico), and both are unusually disseminated. They are significant in alerting the medical community to M. ulcerans infection, which is still active in Mexico, and the treatment used has not been reported previously.

Adult↗
PubMed2005

Myocardial osteopontin expression is associated with collagen fibrillogenesis in human dilated cardiomyopathy.

BACKGROUND: Osteopontin (OPN), an extracellular matrix (ECM) protein, plays an important role in myocardial remodeling by promoting collagen synthesis and accumulation in experimental animal models. AIMS: We hypothesized that OPN could be expressed in myocardial tissues and contribute to collagen accumulation and myocardial dysfunction in human dilated cardiomyopathy (DCM). METHODS AND RESULTS: Endomyocardial biopsy tissues were obtained from 51 patients with DCM and 15 controls by right ventricular endomyocardial biopsy. OPN, collagen types I (Col I) and III (Col III) mRNA levels were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The cellular source of OPN was analyzed using immunohistochemistry and in situ hybridization. Myocardial collagen volume fraction (CVF) was determined by digital planimetry. OPN, Col I and Col III mRNA levels were higher in DCM patients than in controls (P<0.01). OPN mRNA levels were positively correlated with Col I levels and CVF in DCM patients (OPN vs. Col I: r=0.60, P<0.01; OPN vs. CVF: r=0.52, P<0.001). Immunostaining of OPN was present in cardiomyocytes from DCM patients. In situ hybridization identified cardiomyocytes as the major source of OPN mRNA transcription in DCM patients. OPN and Col I mRNA levels were highly expressed in the DCM subgroup with large left ventricular (LV) end-systolic diameter (LVESD > or = 54.5 mm) or low LV ejection fraction (LVEF < 29.5%). There was a weak positive correlation between OPN mRNA levels and LV end-systolic diameter (r=0.39, P<0.01). Levels of OPN mRNA were also negatively correlated with LV ejection fraction (r=-0.43, P<0.01). CONCLUSIONS: These results suggest that OPN may play a pivotal role in the development of Col-I-induced cardiac fibrosis and dysfunction in human DCM.

Cardiomyopathy, Dilated↗
PubMed2003

Dose-dependent effects of strontium on osteoblast function and mineralization.

BACKGROUND: Strontium-ranelate is now being used in the treatment of osteoporosis in elderly patients. As the majority of these patients already have a decreased renal function they are at an increased risk for accumulation of the element. Recent findings from epidemiologic studies in dialysis patients and experimental data obtained in a chronic renal failure (CRF) rat model established a dose-related multiphasic effect of strontium (Sr) on bone formation. To confirm these in vivo findings an in vitro set-up, consisting of primary rat osteoblast cultures, was applied. Sr was added to the culture medium at concentrations of 0, 0.5, 1.0, 2.0, 5.0, 20, and 100 microg/mL, respectively. METHODS: Calcium incorporation (index of mineralization) and alkaline phosphatase activity were determined in the medium during the culture period, while at the end of the experiment, nodule formation (mineralized + unmineralized area) was quantified using a digital imaging system. mRNA synthesis of various osteoblast specific genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared to the control group (0 microg/mL Sr), a significantly reduced nodule formation in the presence of an intact mineralization was found for the lowest 0.5 and 1 microg/mL Sr doses, suggesting an impaired in vitro osteoblast differentiation. Both nodule formation and mineralization were normal for the 2 and 5 microg/mL doses. For the highest Sr doses (20 and 100 microg/mL) a reduced mineralization was observed in the presence of an intact nodule formation indicating an inhibitory effect on the hydroxyapatite formation. The alkaline phosphatase activity reflected the multiphasic pattern of the nodule formation while the calcium incorporation corresponded with the pattern of nodular mineralization. No variations in cell proliferation were found. RT-PCR revealed that Sr interfered with the osteoblast at the level of the mRNA synthesis of several relevant genes. CONCLUSION: Using the proposed in vitro model we confirmed the multiphasic effect of Sr on bone formation previously demonstrated in a CRF rat model. The data presented allow us to suggest that at low concentrations Sr interferes with the bone formation at the level of cell differentiation, whereas at high concentrations the disturbed mineralization in the presence of an intact nodule formation is indicative for a physicochemical interference of Sr with the hydroxyapatite formation.

Alkaline Phosphatase↗
PubMed2006

The 460Trp allele of alpha-adducin increases carotid intima-media thickness in young adult males.

BACKGROUND: The 460Trp allele of the alpha-adducin gene (ADD1), which is involved in a form of salt-sensitive hypertension, has been associated with patterns of target organ damage. OBJECTIVES: As carotid artery intima-media thickness (IMT) largely depends upon unknown genetic factors, besides being associated to conventional risk factors, we tested the association of the 460Trp allele of ADD1 with IMT in a well-characterized sample of young healthy normotensive subjects, to assess the role of ADD1 polymorphism without overlapping effects of age or already elevated blood pressure. METHODS: Anthropometric measurements, blood pressure (BP), and carotid artery wall IMT (high-resolution sonography and digitalized morphometry) were obtained in 420 healthy normotensive Caucasian university students. Genotypes for ADD1 were detected by automated genomic polymerase chain reaction (PCR). RESULTS: ADD1 genotypes were evenly distributed between genders. IMT was significantly larger in carriers of the 460Trp allele of ADD1, while a significant gender x ADD1 interaction (P = 0.02) demonstrated that IMT was increased only in males carrying the 460Trp allele (P < 0.001). No significant association was found in females. CONCLUSIONS: The 460Trp allele of ADD1 contributes substantially to increase carotid IMT, in a male hormonal milieu only, at least in the young age range.

Adult↗
PubMed2003

Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease.

Treponema have been implicated recently in the pathogenesis of digital dermatitis (DD) and contagious ovine digital dermatitis (CODD) that are infectious diseases of bovine and ovine foot tissues, respectively. Previous analyses of treponemal 16S rDNA sequences, PCR-amplified directly from DD or CODD lesions, have suggested relatedness of animal Treponema to some human oral Treponema species isolated from periodontal tissues. In this study a range of adhesion and virulence-related properties of three animal Treponema isolates have been compared with representative human oral strains of Treponema denticola and Treponema vincentii. In adhesion assays using biotinylated treponemal cells, T. denticola cells bound in consistently higher numbers to fibronectin, laminin, collagen type I, gelatin, keratin and lactoferrin than did T. vincentii or animal Treponema isolates. However, animal DD strains adhered to fibrinogen at equivalent or greater levels than T. denticola. All Treponema strains bound to the amino-terminal heparin I/fibrin I domain of fibronectin. 16S rDNA sequence analyses placed ovine strain UB1090 and bovine strain UB1467 within a cluster that was phylogenetically related to T. vincentii, while ovine strain UB1466 appeared more closely related to T. denticola. These observations correlated with phenotypic properties. Thus, T. denticola ATCC 35405, GM-1, and Treponema UB1466 had similar outer-membrane protein profiles, produced chymotrypsin-like protease (CTLP), trypsin-like protease and high levels of proline iminopeptidase, and co-aggregated with human oral bacteria Porphyromonas gingivalis and Streptococcus crista. Conversely, T. vincentii ATCC 35580, D2A-2, and animal strains UB1090 and UB1467 did not express CTLP or trypsin-like protease and did not co-aggregate with P. gingivalis or S. crista. Taken collectively, these results suggest that human oral-related Treponema have broad host specificity and that similar control or preventive strategies might be developed for human and animal Treponema-associated infections.

Animals↗
PubMed2000

Characteristics of HLA class I and class II polymorphisms in Rwandan women.

OBJECTIVE: To define HLA class I and class II polymorphisms in Rwandans. METHODS: PCR-based HLA genotyping techniques were used to resolve variants of HLA-A, B, and C to their 2- or 4-digit allelic specificities, and those of DRB1 and DQB1 to their 4- or 5-digit alleles. RESULTS: Frequencies of 14 A, 8 C, and 14 B specificities and of 13 DRB1 and 8 DQB1 alleles were >/=0.02 in a group of 280 Rwandan women. These major HLA factors produced 6 haplotypes extending across the class I and class II regions: A*01-Cw*04-B* 4501-DRB1*1503-DQB1*0602 (A1-Cw4-B12- DR15 - DQ6), A * 01 - Cw * 04 - B * 4901 -DRB1 * 1302-DQB1*0604 (A1-Cw4-B21-DR13-DQ6), A*30 - Cw*04 - B*15 - DRB1*1101 - DQB1*0301 (A19-Cw4-B15-DR11-DQ7), A*68-Cw*07-B* 4901-DRB1*1302-DQB1*0604(A28-Cw7-B21- DR13 - DQ6), A*30 - Cw*07 - B*5703 - DRB1* 1303-DQB1*0301(A19 - Cw7 - B17 - DR13 - DQ7), and A*74-Cw*07-B*4901-DRB1*1302-DQB1* 0604 (A19-Cw7-B21-DR13-DQ6), respectively. Collectively, these extended haplotypes accounted for about 19% of the total. Other apparent class I-class II haplotypes (e.g., Cw*17-B*42-DRB1*0302-DQB1*0402, Cw*06- B*58-DRB1*1102-DQB1*0301, and Cw*03- B*15-DRB1*03011-DQB1*0201) did not extend to the telomeric HLA-A locus, and other 3-locus class I haplotypes (e.g., A*68-Cw*04-B*15, A*74-Cw*04-B*15, and A*23-Cw*07-B*4901) completely or partially failed to link with any specific class II alleles. DISCUSSION: Frequent recombinations appeared to occur between the three evolutionarily conserved HLA blocks carrying the class I and class II loci. The HLA class I profile seen in Rwandans was not directly comparable with those known in the literature, although the class II profile appeared to resemble those in several African populations. These data provide additional evidence for the extensive genetic diversity in Africans.

Alleles↗