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Biomedical subjects

A Lee

Publications and source records attributed to A Lee.

At least 325 records · Page 18Linked to original sources
PubMed1998

Tolerance and breath hydrogen excretion following ingestion of maltitol incorporated at two levels into milk chocolate consumed by healthy young adults with and without fasting.

Little is known about the gastrointestinal effects of ingesting maltitol in chocolate. This study was designed to determine whether it leads to increased gastrointestinal symptomatology and if that symptomatology is dose related. It was also designed to discover whether breath hydrogen excretion in response to maltitol is dose related. In a double-blind, crossover study, 20 healthy volunteers aged 18-24 y ingested 100 g chocolate containing 40 g sucrose, 10 g sucrose plus 30 g maltitol or 40 g maltitol after fasting (abstinence from food and liquids from 2200 h on the night before chocolate consumption) and not fasting. There was no difference in symptomatology between fasting and nonfasting periods, and consumption order had no effect on symptomatology. Relative to ingestion of sucrose, 30 g maltitol caused no significant difference in symptoms, but 40 g resulted in more mild borborygmi (P < 0.05) and mild flatulence (P < 0.01) but not moderate or severe symptoms. Neither 30 nor 40 g maltitol caused significantly greater laxation than sucrose ingestion (P > 0.05). In a separate study, 10 healthy volunteers aged 18-24 y ate the same test products before breath H2 testing; 40 g maltitol in chocolate caused a greater total breath H2 excretion compared with 30 g maltitol (P < 0.05) or sucrose (P < 0.01). Total breath hydrogen excretion was also greater with 30 g maltitol compared with sucrose (P < 0.05). This dose-related response was consistent with the lower symptomatology after ingestion of 30 vs. 40 g maltitol. We have shown that 30 g maltitol in chocolate causes no significant symptomatology in young adults; however, 40 g caused mild borborygmi and flatus but no increased laxation. An increased breath H2 response indicates colonic fermentation of this polyol.

Adolescent↗
PubMed1998

Animal models for host-pathogen interaction studies.

There is no model of Helicobacter pylori infection that exactly mimics the human diseases. In a particular, there are no good models of ulceration or gastric adenocarcinoma. Patterns of gastritis induced in the animals tend to be lymphocytic and lack the neutrophil infiltration typical of H. pylori infection in the adult. However, the animal models are starting to provide valuable information with respect to factors involved in the colonisation of the gastric mucosa and the importance of host factors in the development of gastric atrophy, as well as making possible the screening of potential therapeutic agents and vaccine candidates. Models include gnotobiotic piglets, primates, cats, dogs, ferrets and a range of rodents. Recent advances in the mouse models mean that they will allow us to dissect bacterial host interactions in a novel manner due to the availability of a wide range of immunological reagents and numerous mutant or transgenic strains.

Animals↗
PubMed1998

The use of a video to convey preanesthetic information to patients undergoing ambulatory surgery.

UNLABELLED: In a prospective, randomized, single-blind trial, we assessed the effectiveness of a preoperative video as a source of additional patient information before ambulatory surgery. One hundred twenty-seven patients were allocated to either treatment (video) or control (nonvideo) groups. Of the 127, 17 (13%) patients correctly answered all process, risk, and misconception statements using a questionnaire. Overall, the video group was 2-16 times more likely to recall all knowledge questions correctly than the nonvideo group after adjusting for previous general anesthesia experience, state (how one feels at the moment), and trait (how one generally feels) anxiety levels (relative risk 6.36, 95% confidence interval 2.01-15.82). The predictors of correct risk knowledge were those who had a video intervention (relative risk 7.12, 95% confidence interval 3.70 to 10.07) and low trait anxiety scores (relative risk 5.88, 95% confidence interval 1.69 to 25.00). A video could be an important additional component of the preoperative interview, but anesthesiologists will still need to provide patient-specific information. IMPLICATIONS: This study randomly allocated adults to see a video about anesthesia before scheduled ambulatory surgery. The video group had better recall of information. The video was a useful adjunct to routine preoperative consultations.

Adolescent↗
PubMed1998

A comparison between the Fick method and indirect calorimetry for determining oxygen consumption in patients with fulminant hepatic failure.

OBJECTIVE: To compare the Fick method of determining oxygen consumption (VO2) with a gas exchange method in a group of patients in whom the cardiac output and mixed venous oxygen saturation values were consistently high. DESIGN: A prospective, observational study. SETTING: A ten-bed intensive therapy unit at a university teaching hospital. PATIENTS: Seventeen patients suffering from fulminant hepatic failure who required ventilatory support and invasive hemodynamic monitoring. All patients were sedated and paralyzed throughout the study period. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: VO2 was determined simultaneously by indirect calorimetry and by the Fick method five or six times in each patient over a 5-hr period after resuscitation with fluids and, if clinically indicated, norepinephrine infusion. The agreement between the methods was poor (limits of agreement +19 to -101 mL/min/m2) and the Fick method consistently underestimated gas exchange measurements (mean bias 41 mL/min/m2). The bias varied widely, both between and within individual patients. The reproducibility of the Fick-derived VO2 was worse than the indirect calorimetry measurements, indicating that the dispersion of data attributable to measurement error was greater with the Fick method. CONCLUSIONS: Under clinical conditions, the agreement between Fick calculations and indirect calorimetry measurements of VO2 in hyperdynamic patients with fulminant hepatic failure was extremely poor. The reproducibility of Fick calculations was less than the reproducibility derived by gas exchange measurements because of the large measurement errors that may occur with the Fick method when the cardiac output is large and the arterial-venous oxygen content difference is small. Fick calculations systematically underestimate gas exchange measurements. The Fick method is inaccurate and unreliable when an estimation of VO2 is required in patients with this hemodynamic pattern.

Adult↗
PubMed1998

Trauma patients with multiple extremity injuries: resource utilization and long-term outcome in relation to injury severity scores.

BACKGROUND: Trauma patients with multiple extremity injuries (MEI) make heavy demands on hospital resources and face long-term difficulties in rehabilitation, yet the literature contains little about their treatment as a distinct group. METHODS: In this study, a cohort of 54 patients with MEI, all treated at a Level I trauma center, was compared with a trauma control (TC) group that had major injuries not focused at the extremities (but excluding patients with neurologic sequelae of head or spinal cord injuries). Demographic features, primary measures reflecting utilization of hospital resources, return-to-employment and productivity data, and health-related quality of life scores (Medical Outcomes Study 36-Item Short Form Health Survey [SF-36]) were compared. RESULTS: Although mean Injury Severity Scores (ISS) for the MEI and TC groups were almost identical (16.2 and 17.4, respectively), the patients with MEI had a mean hospital stay almost twice as long (25 vs. 13 days) and had double the resource intensity weight compared with the TC group. After discharge, the trend of the MEI group was to greater long-term disability, based on SF-36 scores, and lower "return to productivity" figures. The ISS did not predict the greater demands on resources made by the MEI group relative to our TC group. Main injury severity scores for the extremities were more predictive than the ISS for length of hospital stay and the SF-36 concepts at the 2-year follow-up evaluation. CONCLUSION: The study emphasizes the need for injury scoring systems that better predict the needs of patients with MEI and that will serve as a basis for equitable funding of trauma centers.

Adolescent↗
PubMed1998

Comparing generic and disease-specific measures of physical and role functioning: results from the Veterans Health Study.

OBJECTIVES: This study compared the performance of generic measures of Medical Outcome Study Short Form 36-Item Health Survey physical functioning and role limitations with disease-specific measures of physical functioning and role limitations using specific disease attributions for chronic lung disease, chronic low back pain, and osteoarthritis of the knee. METHODS: Data were analyzed from the Veterans Health Study among patients receiving Veteran's Administration ambulatory care. Patients identified as having one of the three study conditions were included in the study (n = 932). RESULTS: The study revealed that the generic physical functioning and role limitations scales had higher correlations with other generic SF-36 scales, whereas disease-specific attribution measures had larger R2 values in explaining variability in symptom-based disease severity and larger t statistic values in discriminating the impacts of patients taking medications and having surgery. CONCLUSIONS: The generic measures of physical functioning and role limitations were more applicable in assessing a broad array of health-related quality-of-life issues, whereas disease-specific measures of physical functioning and role limitations were more useful in evaluating clinical management and limitations associated with specific disease conditions. The results of the study suggest that the use of disease-specific attribution assessments was more cost-efficient than the development of new disease-specific instruments. Disease-specific attribution could be used to complement generic measures in assessing patient outcomes.

Activities of Daily Living↗
PubMed1998

Seamless health care for chronic diseases in a dual health care system: managed care and the role of family physicians.

Neither private nor state run health care systems are perfect. Although there is increasing evidence that Health Maintenance Organizations (HMOs) provide comparable care at lower cost, HMOs tend to select healthy patients. The dual health care system in Hong Kong spends about 3.9 per cent of GDP, with health indices among the best in the world. Hong Kong still faces the problem of escalating health care expenditure. One should take advantage of the dual health care system to evolve a new paradigm for a primary-led seamless health care service. The Diabetes Centre of a university teaching hospital together with the University of Community and Family Medicine has started a structured shared care programme in diabetes mellitus, involving general practitioners in both the private and public sectors integrating the primary and secondary care, and the private and public sectors. This programme starts to develop an infrastructure for providing quality care at an affordable cost for a large pool of patients with chronic disease. Unlike other "managed care schemes", this one is not run by profit-oriented companies, but by health professionals with an interest in providing best possible care at an affordable cost. The "disease management" approach needs a care delivery system without traditional boundaries; and a continuous improvement process which develops and refines the knowledge base, guidelines and delivery system.

Chronic Disease↗
PubMed1998

Gastrin release and gastric acid secretion in the rat infected with either Helicobacter felis or Helicobacter heilmannii.

Helicobacter pylori infection in humans has been shown to be associated with changes in gastric physiology, including exaggerated basal and meal-stimulated gastrin levels. This has been suggested to be due to the direct effects of the bacterium through inflammation and its urease enzyme. The gastric bacteria Helicobacter felis and Helicobacter heilmannii colonize the antrum of rats in large numbers and induce no significant inflammatory response. Thus, the direct effect of Helicobacter infection on gastric physiology, independent of gastritis, could be studied. Basal, freely fed and stimulated acid and gastrin levels were recorded from animals infected with H. felis, H. heilmannii or uninfected controls over a 30 week period. No significant difference was found between freely fed gastrin over 7 weeks or fasting gastrin over 24 weeks or basal and stimulated acid over 30 weeks between all three groups. Triple therapy did not alter gastrin or acid output. The antrum of all Helicobacter-infected rats was well colonized; triple therapy cleared H. felis but not H. heilmannii. Very little inflammation was seen in control or Helicobacter-infected animals. In conclusion, Helicobacter-induced effects on gastric physiology are unlikely to be due to direct bacterial effects, but are best explained by other factors (i.e. inflammatory damage).

Animals↗
PubMed1998

A novel method of extracting plasmid DNA from Helicobacter species.

BACKGROUND: Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. MATERIALS AND METHODS: Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. RESULTS: High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. CONCLUSION: This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.

Amino Acid Sequence↗
PubMed1998

Intestinal tuberculosis: clinicopathologic analysis and diagnosis by endoscopic biopsy.

OBJECTIVES: Tuberculosis is still an important cause of granulomatous colitis in developing countries. If we can diagnose tuberculosis using endoscopic biopsy material, clinicians can avoid invasive diagnostic procedures and needless operations. For this purpose, we evaluated clinical manifestations, pathological findings, and diagnostic methods in endoscopically biopsied intestinal tuberculosis patients. METHODS: From January 1991 to December 1996, 42 patients with intestinal tuberculosis were endoscopically examined and tissue culture, immunohistochemical stain, Ziehl-Neelsen stain, and polymerase chain reaction in fresh and fixed tissue were applied. The pathological findings were analyzed and compared with the results of the other diagnostic methods. RESULTS: In tuberculosis patients, transverse ulcers with surrounding hypertrophic mucosa and multiple erosions were usual colonoscopic findings. The granulomas were found in 74% of the cases. The positivity ranged from 30-45%. There were no significant differences in the positivity among those diagnostic methods (p > 0.05). The positivity of Ziehl-Neelsen stain in fixed tissue was higher in the group having granulomas and it was reversed in PCR (p < 0.05). The increasing number of biopsy particles raised the positivity of Ziehl-Neelsen stain and PCR in fixed tissue (p < 0.05). CONCLUSIONS: Transverse ulcers were the most characteristic colonoscopic finding and granulomas were frequent pathological findings in intestinal tuberculosis. Higher positivity and reliable results were found in tissue culture, Ziehl-Neelsen stain, and polymerase chain reaction. To increase the diagnostic rate, the endoscopist should take enough tissue and deep biopsy material from ulcer bases and diseased mucosae.

Adolescent↗
PubMed1998

Phylloquinone in gingival crevicular fluid in adult periodontitis.

Phylloquinone is a lipid soluble vitamin which is an absolute growth requirement for black-pigmented anaerobes, many of which are implicated in the aetiology of periodontal diseases. This cross-sectional study aimed to detect the levels of phylloquinone in GCF from healthy and diseased sites in subjects with adult periodontitis, in order to investigate further its potential role in the disease process. The sample consisted of eighteen patients with adult periodontitis. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each subject. GCF was sampled and the amount of phylloquinone in each sample was determined using reverse-phase high performance liquid chromatography coupled to electrochemical detection. The mean amount of phylloquinone in accumulated GCF from diseased sites was 406 pg/site and 80 pg/site from healthy sites (p=0.013). When the amounts of phylloquinone in GCF were expressed as concentrations the values were 228 ng/ml and 3350 ng/ml for diseased and healthy sites respectively (p=0.084). These findings suggest the levels of phylloquinone in GCF differs in periodontal health and disease in subjects with adult periodontitis. The total phylloquinone at diseased sites may provide the nutritional requirements favouring the growth of black-pigmented anaerobes.

Adult↗
PubMed1998

Human mannose-binding protein inhibits infection of HeLa cells by Chlamydia trachomatis.

The role that collectin (mannose-binding protein) may play in the host's defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 microg/ml for TW-3 and UW-5, and at 6.25 microg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L2 was not affected by supplementation with complement or addition of an L2-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L2, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.

Bacterial Adhesion↗
PubMed1998

SdeK is required for early fruiting body development in Myxococcus xanthus.

Myxococcus xanthus cells carrying the Omega4408 Tn5lac insertion at the sde locus show defects in fruiting body development and sporulation. Our analysis of sde expression patterns showed that this locus is induced early in the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h of development. Further studies showed that expression of sde is induced as growing cells enter stationary phase, suggesting that activation of the sde locus is not limited to the developmental process. Because the peak levels of sde expression in both an sde+ and an sde mutant background were similar, we conclude that the sde locus is not autoregulated. Characterization of the sde locus by DNA sequence analysis indicated that the Omega4408 insertion occurred within the sdeK gene. Primer extension analyses localized the 5' end of sde transcript to a guanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequence in the -12 and -24 regions upstream of the sde transcriptional start site shows similarity to the sigma54 family of promoters. The results of complementation studies suggest that the defects in development and sporulation caused by the Omega4408 insertion are due to an inactivation of sdeK. The predicted amino acid sequence of SdeK was found to have similarity to the sequences of the histidine protein kinases of two-component regulatory systems. Based on our results, we propose that SdeK may be part of a signal transduction pathway required for the activation and propagation of the early developmental program.

Amino Acid Sequence↗
PubMed1998

Antitoxin-in-membrane and antitoxin-in-well assays for detection of toxigenic Corynebacterium diphtheriae.

The Elek culture plate precipitin test is routinely used for the detection of exotoxin from toxigenic strains of Corynebacterium diphtheriae. Recently, the World Health Organization standardized this test to ensure accuracy, reliability, and reproducibility. In this study, we further modified the standard Elek test by using the antitoxin-in-membrane (AIM) and antitoxin-in-well (AIW) approaches. In the AIM tests, each strain was stabbed and streaked backwards and away from a point approximately 7 mm from the edge of a sterile cellulose acetate-cellulose nitrate filter membrane disk (pore size, 0.45 microm; diameter, 25 mm) containing 25 IU of diphtheria antitoxin. For AIW tests, a central well (diameter, 5 mm) containing 9 microl of antitoxin (4.5 IU) was surrounded by eight equidistant stab-streaks of each strain placed 10 mm from the well. In both methods, precipitin bands of identity typically were noted after 24- and 48-h incubations at 37 degrees C. Both toxigenic and weak toxigenic strains gave clear and reproducible results. Compared with the standard Elek test, the AIM and AIW tests each use 50% less medium and 75 and 87% less antitoxin, respectively. AIM has the potential to test up to 14 isolates and AIW has the potential to test up to 24 isolates on the same plate. Furthermore, clearer positives were noted with weak toxigenic strains. In a blinded test of 209 verified C. diphtheriae isolates, a 99.5% agreement with the standard Elek test was obtained overall. Both modifications conserve reagents and medium, permit the simultaneous testing of a larger number of strains, and may be particularly suitable for reference laboratories or hospitals involved in diphtheria epidemic settings.

Antitoxins↗
PubMed1998

Family history as a predictor of poor long-term outcome in depression.

BACKGROUND: We investigated whether family history had prognostic significance in depression in a study which addressed some of the methodological shortcomings of previous studies. METHOD: We collected family history data on a consecutive series of 89 patients admitted with RDC major depression, blind to the outcome of the proband. This comprised 116, 283 and 120 first-degree relatives examined with the SADS-L, FH-RDC and case note data, respectively. The outcome of 74 of these probands (83%), previously categorised into four operationally defined groups, was then examined. RESULTS: A positive family history of severe psychiatric illness (i.e. a relative with a history of either a psychosis, hospitalised depression or suicide) was associated with poor outcome in the proband. This association persisted after controlling for variable family size, age structure and gender. As family history was correlated with neither Kendell's neurotic/psychotic index nor the proband's neuroticism score, an individual with high scores an all three would have a greatly increased chance of having a poor outcome. CONCLUSIONS: A family history of severe psychiatric illness in a first-degree relative may be useful as one of the vulnerability factors for predicting poor long-term outcome in depression.

Adolescent↗
PubMed1998

Glutathione reverses the growth abnormalities of skin fibroblasts from insulin-dependent diabetic patients with nephropathy.

Oxidative stress has been proposed as a possible pathogenic factor for diabetic complications. It is relevant in determining cell replicative capacity and life span, and in vitro antioxidant treatment is able to reverse the impaired proliferative activity of different cell types. It was recently demonstrated that cultured skin fibroblasts from insulin-dependent diabetic patients with nephropathy age prematurely and have a shorter life cell cycle. To test whether the growth phenotype of cells from patients with diabetic nephropathy was related to a lack of protection from oxidative stress, the effect of reduced glutathione (GSH) on cultured skin fibroblasts from 13 insulin-dependent diabetes mellitus (IDDM) patients with nephropathy (DN), 10 IDDM patients without kidney disease (D), and 10 nondiabetic control subjects (C), in normal (5 mM) glucose (NG) and high (22 mM) glucose (HG) medium was studied. After 6 to 8 passages, fibroblasts from DN showed impaired growth both in NG (mean +/- SD fold increase over baseline counts in DN 1.17 +/- 0.6 versus D 1.7 +/- 0.5 versus C 1.95 +/- 0.8; P = 0.04 by ANOVA) and in HG (mean +/- SD fold increase over baseline counts DN 1.16 +/- 0.41 versus D 1.89 +/- 0.66 versus C 2.24 +/- 0.9; P = 0.003 by ANOVA). GSH prevented the growth abnormalities of cells from DN restoring it to values similar to that of the other two groups (mean +/- SD fold increase over baseline counts NG +/- GSH: DN 1.68 +/- 0.9 versus D 1.78 +/- 0.49 versus C 1.99 +/- 0.7, P = 0.6; and in HG + GSH: DN 1.66 +/- 0.69 versus D 1.87 +/- 0.75 versus C 2.2 +/- 0.9, P = 0.3). Growth rates were not affected by the addition of GSH in fibroblasts from D and C. The treatment of fibroblasts from D and C with the inhibitor of the gamma-glutamylcysteine synthetase activity, L-buthionine-S,R-sulfoximine, resulted in growth impairment, and the addition to the culture medium of another antioxidant, superoxide dismutase, corrected the growth abnormalities in fibroblasts from DN. The impaired growth of cultured fibroblasts from IDDM patients with nephropathy is prevented by GSH and superoxide dismutase and is independent of prevailing glucose concentrations. This suggests that oxidative stress is an important mechanism of intrinsic cell dysfunction in these patients.

Adult↗
PubMed1998

Immunocytochemical localization and biochemical characterization of large proteoglycans in developing rat bone.

This study used biochemical and light and electron microscopic immunohistochemical methods to localize and characterize large hyaluronate-binding proteoglycans in the developing mandible of fetal rats at embryonic day 15 (Day 15) to Day 18 using a monoclonal antibody (MAb) 5D5. This antibody is derived from bovine sclera and specifically recognizes the core protein of large proteoglycan such as versican, neurocan and brevican, but not that of aggrecan. At the light microscopic level, MAb 5D5 moderately stained the extracellular matrices among osteoblasts at the centers of ossification in Day 15 mandible specimens. Weaker staining was observed in osteoblasts, whereas Meckel's cartilage lacked staining. Ultrastructural immunocytochemistry showed the presence of immunogold particles over unmineralized matrices among osteoblasts and their intracellular organelles. In Day 16 to 18 specimens, bone nodules were recognized in LR gold sections before immunostaining, but, after immunostaining, consistently appeared devoid of mineral crystals and were seen as a demineralized structure that had an electron dense periphery within which fine filamentous and granular material were present. The appearance of these structures was created by the demineralization of thin sections on grids during immunostaining. Specific immunogold staining was clearly seen over the demineralized structures corresponding to bone nodules. The majority of immunogold particles tended to localize inside of the structures. Bone proteins were extracted from fresh, Day 18 specimens with a three-step technique: 4 M guanidine HCl (GdnCl,-extract), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis after chondroitinase ABC digestion, showed that G1-extract gave a 5D5 reactive band greater than 400 kDa, whereas E-extract produced two major reactive populations of small molecular size with core proteins approximately 63 and 74 kDa. These results indicate that the large proteoglycan having smaller molecular weight is preferentially localized to bone nodules and may correlate with bone matrix mineralization.

Animals↗