Biomedical subjects

R B Johnson

Publications and source records attributed to R B Johnson.

At least 19 recordsLinked to original sources

PubMed2006

Synergism between nifedipine and cyclosporine A on the incorporation of [35S]sulfate into human gingival fibroblast cultures in vitro.

BACKGROUND AND OBJECTIVE: We assessed the effects of cyclosporine A and nifedipine on the in vitro incorporation of [(35)S]sulfate into gingival fibroblast cell cultures derived from responder and nonresponder subjects who had received an organ transplant followed by a therapeutic regimen using a combination of those drugs. MATERIAL AND METHODS: Gingival fibroblasts were isolated from responder and nonresponder subjects and maintained in vitro. Prior to cell harvest, gingival interleukin-1beta concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Cells were untreated or exposed to either 10(-7)-10(-10) m nifedipine or 100-500 ng/ml cyclosporine A. Incorporation of [(3)H]proline or [(35)S]sulfate into the cell cultures was determined by liquid scintillation analysis. In addition, the effects of 400 ng/ml cyclosporine A + 10(-7) m nifedipine and 400 ng/ml cyclosporine A + 10(-10) m nifedipine on incorporation of [(35)S]sulfate into the cell cultures was determined. Data were compared by factorial analysis of variance (anova) and a posthoc Tukey's test. RESULTS: Gingiva from responders contained significantly more interleukin-1beta than gingiva from nonresponders (p < 0.01). The cell cultures derived from responders incorporated significantly more [(35)S]sulfate than those derived from nonresponders following exposure to either cyclosporine A or 10(-7) m nifedipine. In addition, the exposure of fibroblasts derived from gingival overgrowth to either 400 ng/ml cyclosporine A + 10(-7) m nifedipine or 400 ng/ml cyclosporine A + 10(-10) m nifedipine significantly increased or decreased, respectively, the incorporation of [(35)S]sulfate into the cultures. CONCLUSION: The therapeutic combination of cyclosporine A and nifedipine could be a significant risk factor for gingival overgrowth in subjects susceptible to either agent. The mechanism for overgrowth could include edema secondary to increased sulfated-glycosaminoglycan (sGAG) synthesis by fibroblasts, but further investigation is required.

Analysis of Variance↗

PubMed2005

Interleukin-18 concentrations and the pathogenesis of periodontal disease.

BACKGROUND: Interleukin-18 (IL-18) is reported as an important regulatory cytokine in non-oral inflammation. Our objective was to compare the concentrations of IL-18 within diseased and healthy human gingiva with concentrations of other T(H)1 and T(H)2 cytokines to determine possible effects of IL-18 on gingival inflammation. METHODS: Gingival biopsies were obtained prior to routine tooth extraction. Gingiva was grouped by the depth of the adjacent gingival sulcus: < or =3 healthy (featuring no bleeding on probing) and > or =3 mm diseased (featuring bleeding on probing). Diseased gingiva was subdivided into 3, 4 to 6 and >6 mm groups. Gingival interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12, IL-18, and interferon (IFN)-gamma concentrations were assessed by enzyme linked immunosorbent assay (ELISA). Data were compared by factorial analysis of variance and the Pearson's correlation test. RESULTS: Concentrations of IL-2, IL-4, IL-6, IL-10, IL-18, and IFN-gamma adjacent to 4 to 6 mm diseased sites were greater than adjacent to < or =3 mm healthy sites (P <0.001). IL-12 concentrations were lower within diseased than within healthy gingiva (P <0.001). IL-6 and IL-18 concentrations were greater adjacent to >6 mm sites compared to healthy sites (P <0.001); the concentrations of the other cytokines (except IL-12) were similar to healthy sites. IL-6 and IL-18 concentrations were positively correlated, and IFN-gamma and IL-12 negatively correlated, with the adjacent gingival sulcular depth. CONCLUSIONS: Periodontal inflammation may not successfully resolve because of accumulation of IL-6 and IL-18, and decreased concentrations of IL-12, within diseased gingiva. Because of the highly significant correlation between IL-18 concentration and gingival sulcular depth, IL-18 may be a useful target for either preventive or palliative therapy for periodontitis.

Adolescent↗

PubMed2004

Effect of strain on bone nodule formation by rat osteogenic cells in vitro.

The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation. Fetal rat calvarial cells experienced a Flexercell regimen within either FLEX I (deformable) or FLEX II (non-deformable) substrates. Cells in FLEX I plates were exposed to growth medium only; those in FLEX II plates were exposed to either growth medium only, or growth medium + 10(-7) M IGF-1. Cell numbers were assessed from 1 to 6 days. Other cells were exposed to the Flexercell regimen (-2 kPa, 0.05 Hz) for 1-3 (Group 1), 3-6 (Group 2), 1-9 (Group 3) or 10-15 (Group 4) days and were maintained, at other times, under standard conditions. After 21 days, nodules were counted within each well and within the compression, <999, 1000-4900, 5000-9999, 10,000-14,999 and 15,000-25,000 microstrain regions of the FLEX I membrane. Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups (P<0.001). The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 (P<0.001), but not different from control or IGF-1 groups. Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions. Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.

Animals↗

PubMed2004

The ovariectomized sheep as a model for human bone loss.

The aim of this study was to determine the effects of ovariectomy on the trabeculae of ovine iliac bone, with a view to the further development of a model for human osteoporosis. Seven ovariectomized (OVX) and five control Dorset ewes were observed for one year. Iliac crest bone samples were obtained at the time of ovariectomy ("time 0") and again when the animals were killed 12 months later. At "time 0" and at 3-month intervals thereafter samples of blood and urine were collected for the assay of serum 17beta-oestradiol and osteocalcin (OC) and urinary deoxypyridinium (D-PYR). One year after ovariectomy, trabecular bone volume and thickness were reduced (P<0.05) and trabecular separation was increased (P<0.05) as compared with the controls. In OVX sheep, serum 17beta-oestradiol concentrations were significantly lower at the end of the experiment than at "time 0", while serum OC and urinary D-PYR concentrations were significantly higher (P<0.001). The results suggest that the OVX sheep is a valid model for changes in trabecular bone architecture associated with oestrogen deficiency, especially in women experiencing early menopause.

Animals↗

PubMed2004

Effects of multiple dentinal lesions on the rat pulp.

The purpose of this study was to compare the effects of single and multiple dentinal lesions on the dental pulps of rats. 3H-proline was used to measure pulpal reactionary dentin deposition, and 3H-thymidine was used to measure pulpal cell proliferation by radioautography. Data were compared by factorial analysis of variance and a post-hoc Tukey test. A significant increase in radiolabeled collagenous proteins within reactionary dentin and pulpal cell labeling indices were evident coincident to single dentinal injuries, compared with controls (p < 0.001). After double injuries, the radiolabeled collagenous proteins within reactionary dentin and the pulpal labeling indices remained significantly greater than in controls (p < 0.01); however, each was significantly less than after single injuries (p < 0.05). These data suggest that pulpal cell proliferation and collagenous protein deposition into reactionary dentin are decreased subsequent to a previous dentinal injury. Thus, the healing potential of the dental pulp could be decreased by recent tooth cavity preparation.

Analysis of Variance↗

PubMed2004

Interleukin-11 and IL-17 and the pathogenesis of periodontal disease.

BACKGROUND: Interleukin (IL)-11 and IL-17 are cytokines that modulate the inflammatory process and have not been assessed within normal or inflamed gingival tissues. Our purpose was to compare concentrations of human IL-11 and IL-17 within healthy and diseased human gingiva to determine their possible role in the initiation or progression of periodontal diseases. METHODS: Biopsies from healthy (non-hemorrhagic gingiva adjacent to a < or = 3 mm gingival sulcus) and diseased gingiva (hemorrhagic gingiva adjacent to a > or = 3 mm periodontal pocket) were studied. IL-11, IL-17, RANTES, and IL-6 concentrations were assessed within solubilized gingival biopsies by enzyme-linked immunosorbent assay. Data were compared by factorial analysis of variance and a post-hoc Tukey honestly significant difference (HSD) test. Regression analysis and partial correlation analysis (adjusted for sample weight) were also used to determine correlations between the variables. RESULTS: Interleukin-11 concentrations were highest within gingiva adjacent to 3 mm diseased pockets (P < 0.001), and IL-17 concentrations were highest at 4 to 5 mm sites compared to other sites (P < 0.001). Gingival concentrations of both cytokines were significantly lower in gingiva adjacent to a > or = 6 mm pocket. RANTES concentrations were significantly greater in gingiva adjacent to > or = 6 mm pockets than in tissues derived from other sites (P < 0.001). IL-11, IL-6, and RANTES concentrations were significantly correlated with sulcular depth. CONCLUSIONS: Gingival concentrations of IL-11 and IL-17 are different in diseased gingiva adjacent to 3, 4 to 5, and > or = 6 mm pockets, suggesting that their concentrations change as a consequence of the progression of gingivitis to periodontitis and that both cytokines could have a significant role in this progression. These data may be useful for the design of procedures for prevention of periodontal disease.

Adolescent↗

PubMed2003

Sensitivity, specificity and accuracy of stress SPECT myocardial perfusion imaging for detection of coronary artery disease in the distribution of first-order branch vessels, using an anatomical matching of angiographic and perfusion data.

We sought to investigate the utility of stress single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) for the identification of coronary artery disease (CAD) in the distribution of first-order branch vessels. We evaluated 135 consecutive patients with coronary angiography and stress SPECT MPI. We anatomically matched angiography and SPECT to assess the sensitivity, specificity and accuracy of SPECT MPI for the detection of CAD in the distribution of first-order branches. Subgroup analysis for stress test performance and previous coronary artery bypass grafting (CABG) was also performed. The sensitivity, specificity and accuracy of stress SPECT MPI for the detection of CAD in the distribution of first-order branch vessels were all 67%. For isolated branch vessel CAD, stress SPECT MPI had a sensitivity of 44%. In patients without CABG, the sensitivity, specificity and accuracy for the detection of CAD in the distribution of first-order branch vessels were 71%, 67% and 68%, compared with 60%, 67% and 64% for patients with CABG. The sensitivity for isolated branch vessel CAD was 50% for patients without CABG, but only 29% for patients with CABG. The sensitivity and specificity for CAD in the distribution of branch vessels were similar for all patients for all stress test modalities and heart rate response (sensitivity, 64-69%; specificity, 61-69%). Stress SPECT MPI offers intermediate sensitivity, specificity and accuracy for the detection of CAD in the distribution of first-order coronary artery branch vessels. However, for isolated branch vessel CAD, stress SPECT has a lower sensitivity, particularly in patients with previous CABG.

Coronary Angiography↗

PubMed2002

Effect of estrogen deficiency on skeletal and alveolar bone density in sheep.

BACKGROUND: This study provides a longitudinal assessment of changes in alveolar and skeletal bone mineral density (BMD) in ovariectomized animals. METHODS: Following ovariectomy (OVX) (n = 6) or sham-operation (n = 6) intraoral radiographs were made at 4-month intervals and serum 17-beta-estradiol, osteocalcin, and interleukin (IL)-6, urinary deoxypyridinium, and salivary IL-6, deoxypyridinium, and osteocalcin concentrations were evaluated. Twelve months after surgery, animals were sacrificed and the mandible and radius/ulna removed. Bones were sectioned and radiographed. Mean BMD and cortical thicknesses were calculated from each region. RESULTS: OVX animals had a progressive decrease in serum 17-beta-estradiol, increased serum osteocalcin and IL-6, urinary deoxypyridinium and salivary IL-6, osteocalcin and deoxypyridinium (P < 0.001), suggesting that they were becoming osteoporotic. The BMD of the radius/ulna and mandibular alveolar bone was significantly reduced in OVX animals (P < 0.05 and P < 0.001, respectively). Reduced alveolar bone BMD became evident in OVX animals 6 months after surgery and became more severe during the subsequent 6 months. Alveolar crestal height was also significantly reduced in OVX animals (P < 0.001). These biochemical and density changes preceded a significant reduction in serum 17-beta-estradiol, which occurred between 4 and 8 months following surgery. CONCLUSIONS: Serial measurements of alveolar BMD predicts loss of skeletal BMD in OVX sheep. Changes in alveolar BMD precede estrogen deficiency, suggesting that early signs of reduced BMD may be detected in peri-menopausal women. The presence of biomarkers of bone metabolism within saliva and their correlation with reduced BMD suggests that saliva could be used as an adjunct screening method for assessment of skeletal bone density.

Alveolar Process↗

PubMed2001

Activation of human rhinovirus-14 3C protease.

The catalytic efficiency of human rhinovirus-14 (HRV14) 3C protease as a function of solvents and other regulators has been investigated using synthetic peptides as substrates. The proteolytic activity of HRV14 3C was found to be strongly stimulated by a series of anions in vitro and the activation was accompanied by changed Km, kcat, and increased kcat/Km values. A more than 72-fold increase in the 3C catalytic efficiency toward peptide substrates was observed in the presence of 0.8 M sodium sulfate. Several approaches, including size-exclusion chromatography and chemical cross-linking experiments, suggested that no oligomerization of the 3C enzyme occurred in the presence of activating anions. However, the anions did induce a significant conformational change of HRV14 3C protease, as revealed by circular dichroism spectrometry and tyrosine fluorescence analyses, which might contribute to 3C enzyme activation. Finally, the results obtained from 3C protease inhibitor studies suggested that the S1 specificity pocket of HRV14 3C was mainly affected by the activating anions. An induced-fit catalysis mechanism for viral proteases is discussed.

3C Viral Proteases↗

PubMed2001

Validation of a model of left ventricular segmentation for interpretation of SPET myocardial perfusion images.

Several models of left ventricular segmentation have been developed that assume a standard coronary artery distribution, and are currently used for interpretation of single-photon emission tomography (SPET) myocardial perfusion imaging. This approach has the potential for incorrect assignment of myocardial segments to vascular territories, possibly over- or underestimating the number of vessels with significant coronary artery disease (CAD). We therefore sought to validate a 17-segment model of myocardial perfusion by comparing the predefined coronary territory assignment with the actual angiographically derived coronary distribution. We examined 135 patients who underwent both coronary angiography and stress SPET imaging within 30 days. Individualized coronary distribution was determined by review of the coronary angiograms and used to identify the coronary artery supplying each of the 17 myocardial segments of the model. The actual coronary distribution was used to assess the accuracy of the assumed coronary distribution of the model. The sensitivities and specificities of stress SPET for detection of CAD in individual coronary arteries and the classification regarding perceived number of diseased coronary arteries were also compared between the two coronary distributions (actual and assumed). The assumed coronary distribution corresponded to the actual coronary anatomy in all but one segment (#3). The majority of patients (80%) had 14 or more concordant segments. Sensitivities and specificities of stress SPET for detection of CAD in the coronary territories were similar, with the exception of the RCA territory, for which specificity for detection of CAD was better for the angiographically derived coronary artery distribution than for the model. There was 95% agreement between assumed and angiographically derived coronary distributions in classification to single- versus multi-vessel CAD. Reassignment of a single segment (segment #3) from the LCX to the LAD territory further improved the model's fit with the anatomic data. It is concluded that left ventricular segmentation using a model with assumed coronary artery distribution is valid for interpretation of SPET myocardial perfusion imaging.

Coronary Angiography↗

PubMed2001

Efficacy of intracoronary or intravenous VEGF165 in a pig model of chronic myocardial ischemia.

OBJECTIVES: We sought to optimize vascular endothelial growth factor (VEGF) treatment for therapeutic angiogenesis in myocardial ischemia, we explored the efficacy of five different regimens. BACKGROUND: Although VEGF165 is one of the most potent pro-angiogenic growth factors, VEGF165 treatment for myocardial ischemia has been hampered by low efficacy and dose-limiting hypotension after systemic or intracoronary delivery. METHODS: This study evaluated the effect of intravenous or intracoronary rhVEGF165 in the presence or absence of nitric oxide (NO) synthase inhibition in a porcine model of chronic myocardial ischemia. Forty-two Yorkshire pigs with chronically occluded left circumflex coronary arteries were randomly assigned to receive 10 microg/kg of VEGF165: 1) rapid (40 min) intravenous VEGF165 0.25 microg/kg/min, 2) slow (200 min) intravenous VEGF165 0.05 microg/kg/min, 3) rapid intracoronary VEGF165 0.25 microg/kg/min, 4) rapid intracoronary VEGF165 0.25 microg/kg/min + nitro-L-arginine methyl ester hydrochloride (L-NAME) or 5) rapid vehicle infusion. RESULTS: Intracoronary and intravenous VEGF165 induced hypotension. Intracoronary VEGF-induced hypotension was blocked by L-NAME. Coronary angiography three weeks after treatment showed improvement in collateral index in both intracoronary groups but not the intravenous VEGF165 groups. Likewise, myocardial blood flow and microvascular function in the ischemic territory improved in both intracoronary groups but not in the intravenous groups. Global and regional myocardial function showed no significant improvements in any groups. CONCLUSIONS: Intracoronary infusion of VEGF165 significantly improves blood flow to the ischemic myocardium. Concomitant administration of L-NAME inhibits VEGF-induced hypotension while most likely preserving VEGF-induced angiogenesis. Intravenous infusion of VEGF165 was not effective in augmenting either myocardial flow or function in this model.

Animals↗

PubMed2001

In vitro effects of azithromycin on Salmonella typhi: early inhibition by concentrations less than the MIC and reduction of MIC by alkaline pH and small inocula.

To explain good clinical results of azithromycin in patients with typhoid fever, 10 strains of Salmonella typhi were grown in cation-adjusted Mueller-Hinton broth. MICs of azithromycin were 4-16 mg/L. At a sub-MIC of 2 mg/L, early inhibition of growth was shown at 2, 4 and 8 h of incubation, but at 24 and 48 h growth to turbidity occurred. At 4 mg/L, inhibition occurred up to 8 h, after which growth towards turbidity followed. Elongated curved bacilli formed in broth containing 4 mg/L after 24-48 h. Adjusting the pH of the broth with phosphate-citrate buffer to 7.5 and 8.0 caused reductions in MICs to 0.25-0.5 mg/L. Large inocula of 10(6) cfu/mL resulted in median MICs four- to six-fold greater than with inocula of 10(1)-10(3) cfu/mL. An inoculum of 10 bacteria per mL in broth at pH 7.5 resulted in an MIC of 0.13 mg/L. Clinical benefits in patients may occur because of early inhibition by sub-MIC concentrations of azithromycin, and due to lower MICs at alkaline pH and lower MICs with small inocula that may correspond to the low-grade bacteraemia in typhoid fever.

Anti-Bacterial Agents↗

PubMed2001

Survival of rotavirus antibody activity derived from bovine colostrum after passage through the human gastrointestinal tract.

BACKGROUND: Rotavirus is a major cause of infectious diarrhea in infants and young children. Several studies have shown that hyperimmune bovine colostrum, derived from cows immunized with rotavirus, can prevent rotavirus diarrhea when given passively. The objective of this study was to determine whether colostral antibody activity survived transit through the gut by measuring the level of rotavirus antibody activity in the feces. METHODS: Hyperimmune colostrum containing different levels of rotavirus antibody was administered to 105 children attending nine Adelaide childcare centres. Subjects were asked to drink 100 ml of whole milk supplemented with colostrum 3 times a day, for a period of 6 days. Stool samples were collected from the subjects before, during, and after consumption of the study product. Rotavirus activity was determined using a novel virus reduction enzyme-linked immunosorbent assay (ELISA) and a virus neutralization assay. RESULTS: Rotavirus antibody activity was detected in 521 (86%) of 602 fecal specimens obtained during the study using the virus reduction ELISA. The antibody activity was detected as early as 8 hours after ingestion of hyperimmune colostrum and up to 72 hours after consumption had ceased. There was a strong relation (r = 0.81) between the titer of rotavirus antibody administered to subjects and the level of antibody activity detected in the feces. CONCLUSIONS: The results show that antirotavirus activity survived passage through the gut. Therefore, passive immunotherapy may be used to prevent or treat infectious diseases that affect the entire length of the gastrointestinal tract.

Antibodies, Viral↗

PubMed2001

Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus.

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.

Bacterial Proteins↗

PubMed2001

Biochemical characterization of signal peptidase I from gram-positive Streptococcus pneumoniae.

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and Escherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.

Amino Acid Sequence↗

PubMed2001

Leptin within healthy and diseased human gingiva.

BACKGROUND: Plasma leptin concentrations are reported to be elevated in patients with inflammatory diseases. There is no consensus concerning the biological mechanism for this phenomenon. To date, tissue leptin concentrations have not been assessed within normal or inflamed gingiva. The purpose of this study was to assess concentrations of human leptin within healthy and diseased gingiva to define its possible role in periodontal disease progression. METHODS: Healthy (non-hemorrhagic gingiva adjacent to a < or =3 mm gingival sulcus) and inflamed gingiva (hemorrhagic gingiva adjacent to a > or =3 mm periodontal pocket) were studied. Leptin, vascular endothelial growth factor (VEGF; to assess potential vascular expansion), and interleukin-6 (IL-6; to assess periodontal disease activity and severity) concentrations were assessed within solubilized gingival biopsies by enzyme-linked immunosorbent assay. Data were grouped by sulcular depth and compared by factorial analysis of variance, regression analysis, and Scheffé comparisons. RESULTS: Leptin concentrations were highest within gingiva adjacent to a < or =3 mm sulcus and progressively declined within gingiva adjacent to a > or =3 mm sulcus. VEGF concentrations were highest within gingiva adjacent to 4 to 6 mm pockets and nearly equivalent in healthy (< or =3 mm sulcus) and severely diseased gingiva (>6 mm sulcus). IL-6 was positively correlated and leptin negatively correlated with adjacent probing depth; IL-6 concentration was significantly higher and leptin significantly lower in gingiva adjacent to >6 mm pockets compared to sites adjacent to <6 mm pockets (P<0.001). CONCLUSIONS: Human leptin is present within healthy and marginally inflamed gingiva and decreases in concentration as the adjacent probing depth increases. When leptin concentrations decreased (> or =3 mm sulcus), VEGF concentrations increased, suggesting that leptin could be released from gingiva coincident to vascular expansion. Thus, gingiva, in addition to adipose tissue, could be a source of circulating leptin in patients with periodontal disease. This possibility requires further investigation.

Adolescent↗

PubMed2000

Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene has been expressed as a nonfusion protein in bacterial cells and purified to homogeneity using sequential chromatographic columns. The purified NS5B protein exhibited RNA-dependent RNA polymerase activity using poly(A) template and the K(m) and V(max) were determined as 8.4 microM and 1976 pmol/mg-min, respectively. This full-length NS5B protein exhibited much stronger binding affinity toward the 30-mer poly(G) than other homopolymeric RNAs of the same size. For the first time, we demonstrate that the HCV NS5B was able to bind various ribonucleotides. Using a panel of oligonucleotides varying in length, we studied the NS5B catalytic efficiency and proposed the size of the NS5B active site to be 8-10 nucleotides. The multifunctional nature of NS5B protein is also discussed and compared with other viral RNA polymerases.

Binding Sites↗

PubMed2000

De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase.

The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.

3' Untranslated Regions↗