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Biomedical subjects

L De Wit

Publications and source records attributed to L De Wit.

At least 19 recordsLinked to original sources
PubMed2005

A mother-based intervention trial for osteoporosis prevention in children.

OBJECTIVE: To assess whether a lifestyle intervention delivered to mothers might impact on osteoporosis preventive behaviors in their children. METHODS: We performed a 2-year randomized controlled trial of individualized bone mineral density feedback with either an osteoporosis information leaflet, or small group education, in a population-based sample of 354 mothers from Southern Tasmania, Australia in 2000-02. Main outcomes were maternal report of calcium intake and physical activity change in their children. RESULTS: Receiving small group education was associated with mothers' report of increasing children's calcium intake (odds ratio 2.3, 95% confidence interval 1.4, 3.8), as was low t-score feedback (odds ratio 2.0, 95% confidence interval 1.2, 3.3). Mothers who increased their own physical activity were more often reported increasing both physical activity (odds ratio 2.7, 95% confidence interval 1.5, 5.0) and calcium intake in their children (odds ratio 2.2, 95% confidence interval 1.3, 3.7). Mothers who commenced calcium supplements more often reported increasing children's calcium intake (odds ratio 2.6, 95% confidence interval 1.0, 6.7) but not physical activity. CONCLUSIONS: Both bone mineral density feedback and small group education delivered to mothers are effective at inducing maternally reported osteoporosis preventive behavior change in their children. These results require confirmation by studies with objective outcome measures.

Adult↗
PubMed2005

Effects of bone density feedback and group education on osteoporosis knowledge and osteoporosis self-efficacy in premenopausal women: a randomized controlled trial.

In this 2-yr randomized controlled trial, we examined the effect of bone mineral density feedback and two different educational interventions (an osteoporosis information leaflet and group-based behavioral education [OPSMC]) on osteoporosis knowledge and self-efficacy in 470 women aged 25-44 yr. Osteoporosis knowledge increased across all intervention groups. Women receiving the OPSMC had a greater increase in both short (beta = +1.33, 95% confidence interval [CI] = 0.72-1.94) and long-term (beta = +0.64, 95% CI = 0.0034-1.25) osteoporosis knowledge, compared to those receiving the leaflet. In contrast, a low T-score was associated with a significant increase in long-term (beta = +0.66, 95% CI = 0.0034-1.25) but not short-term (beta = +0.57, 95% CI = -0.036 to 1.17) osteoporosis knowledge, compared to a normal T-score. Changes in osteoporosis self-efficacy were not associated with either low bone mineral density or receiving the OPSMC but were negatively associated with number of children (beta = -0.9, 95% CI = - 1.4 to -0.3) and working more than 20 h per week (beta = -2.7, 95% CI = -4.6 to -0.8). In conclusion, both the OPSMC and bone density feedback increased osteoporosis knowledge but not self-efficacy over 2 yr. Women with children or who worked full time have decreased osteoporosis self-efficacy, suggesting that this group should be a specific target for future interventional strategies.

Adult↗
PubMed2000

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated.

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.

Animals↗
PubMed2000

Cloning of the gene encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis.

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687-2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage lambdagt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.

Animals↗
PubMed1999

Spiral computed tomography for preoperative staging of potentially resectable carcinoma of the pancreatic head.

BACKGROUND: Pancreatic cancer is often locally invasive. Preoperative staging attempts to identify patients suitable for resection, in order to minimize unnecessary operations. The aim of this study was to assess the improved imaging provided by spiral computed tomography (CT) in the preoperative staging of potentially resectable pancreatic head carcinoma. METHODS: In 56 consecutive patients with pancreatic head carcinoma spiral CT findings were correlated prospectively with operative and histopathological findings. Criteria for irresectability at CT were infiltration of the peripancreatic fat and vascular ingrowth grade D, on a scale from A to F. RESULTS: At operation 27 (48 per cent) of 56 tumours were irresectable. Small metastases were found in seven patients (12 per cent). Ingrowth (adherence) to the portal or mesenteric vein was present in 19 patients (34 per cent). The sensitivity and specificity of CT for irresectability were 78 and 76 per cent respectively. Resection rates with a vascular margin free of tumour were 100 per cent for grade A, 63 per cent for grade B, 44 per cent for grade C, 15 per cent for grade D and 0 per cent for grade E, with a predictive value for ingrowth of 88 per cent for grades D or higher. The resectability rate was 11 per cent (one of nine) when infiltration of the anterior peripancreatic fat was present and 67 per cent when infiltration was absent (P < 0.01). CONCLUSION: Spiral CT with thin slices seems to improve detection of distant metastases and vascular ingrowth in patients with pancreatic head carcinoma.

Humans↗
PubMed1998

Initial experiences with the retroperitoneal approach for endoscopic nephrectomy with the patient in the prone position.

Retroperitoneal endoscopic nephrectomy with the patient in the prone position was performed in 12 patients. Indications for this procedure were end-stage kidneys with ureteropelvic junction stenosis or distal ureteric obstruction, nonfunctional kidneys with drug resistant renin-mediated hypertension, and distal ureter malignancy. The retroperitoneal area was exposed using an open surgical technique in combination with the use of a liquid-filled dissection balloon. Removal of kidney tissue was performed with a morcellator through one of the ports. On average, the operating time was 210 min (range 160-480 min) to complete a one-sided nephrectomy. No major complications occurred. Mean hospital stay in this series was 6.6 days, and the follow-up period was uncomplicated in all cases. Retroperitoneal endoscopic nephrectomy with the patient in the prone position is an acceptable alternative to open nephrectomy in selected indications.

Adult↗
PubMed1997

Analysis of the mechanism of action of anti-human interleukin-6 and anti-human interleukin-6 receptor-neutralising monoclonal antibodies.

Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.

Animals↗
PubMed1997

A serine/threonine protein kinase from Mycobacterium tuberculosis.

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.

Amino Acid Sequence↗
PubMed1997

Cloning of the antigen 85A from Mycobacterium gordonae and its use for the specific PCR identification of these mycobacteria.

The complete nucleotide sequence of 85A antigen of Mycobacterium gordonae was determined. This gene encodes 339 amino acids, including 43 amino acids for the signal peptide, followed by a mature protein of 296 amino acids. A polymerase chain reaction (PCR) assay for the rapid detection of M. gordonae DNA using two pairs of oligonucleotide primers, derived from our sequence, is described. This one-step PCR has been used successfully to amplify 38 strains of M. gordonae. Conversely, the primers did not amplify DNA from any of the 25 mycobacterial species tested. The results suggest that this PCR assay could be a good alternative to existing commercial assays for the specific identification of M. gordonae on early culture on solid medium or on early BACTEC broth culture.

Amino Acid Sequence↗
PubMed1995

[Laparoscopic splenectomy revisited].

We present a new technique for laparoscopic splenectomy which allows manual control of endoscopic dissection. After induction of pneumoperitoneum and insertion of an endoscope to check the absence of any contraindications, an incision is made for insertion of the hand and forearm. An airtight system allowing insertion of the hand while maintaining the pneumoperitoneum is fixed around the incision. A second port is inserted in the left flank. The procedure is then performed with laparoscopic instruments while the left hand allows for spleen mobilisation and easier exposure of the structures to be dissected and divided. Eight patients with a mean age of 51.3 years have been operated on for Idiopathic Thrombocytopenia. The average duration of the procedure was 90 min (80-130 min). No postoperative complication occurred. The mean duration of stay was 3.8 days (3-7 days). These results of this technique are encouraging. The procedure is quicker and safer than an exclusive laparoscopic approach. The incision allows removal of the intact spleen. Other applications of this system can be envisaged.

Adult↗
PubMed1994

Rapid diagnosis of cytomegalovirus lung infection by DNA amplification in bronchoalveolar lavages.

We used polymerase chain reaction (PCR) to detect cytomegalovirus (CMV) deoxyribonucleic acid (DNA) in the bronchoalveolar lavages (BAL) of 16 CMV-infected patients with active disease. We also tested PCR on a control group of 20 patients including latently infected patients without evidence of active CMV infection. Results were compared with those of CMV culture and of a rapid method of diagnosis which detects CMV in BAL cells by nucleic acid hybridization. PCR allowed the diagnosis in 93% of the actively infected patients compared to 73% for the CMV culture. Among the 20 control patients without evidence of active CMV infection, PCR was negative in all the 24 BAL tested. Hybridization on BAL cells with the CMV probe detected nine out of 10 actively infected patients, but the specificity of the test was only 68.5%. In our experience, PCR appears to be at least as sensitive as CMV culture, it provides results faster and it performs better than the detection of the virus by hybridization on BAL cells. Only active CMV infection was detected with the PCR conditions used in our study. This suggests that the PCR can be applied to bronchoalveolar lavage fluids as a rapid method to detect CMV lung infection.

AIDS-Related Opportunistic Infections↗
PubMed1994

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.

ATP-Binding Cassette Transporters↗
PubMed1994

Nucleotide sequence of the 85B-protein gene of Mycobacterium bovis BCG and Mycobacterium tuberculosis.

We have cloned and sequenced the genes coding for the 85B antigen from M. bovis BCG and M. tuberculosis. Within this gene, the only difference in sequence between M. bovis BCG and M. tuberculosis corresponds, respectively, to a C-->T yielding a Leu-->Phe replacement at position 100 of the mature 85B protein. Therefore as we described previously for the 85A gene, there is also very little variation between these two species within the 85B gene.

Acyltransferases↗
PubMed1993

Strong promoter activity of human and rat islet amyloid polypeptide/amylin gene constructs in mouse beta cells (beta TC 3).

Islet amyloid polypeptide (IAPP)('amylin') is co-produced with insulin in pancreatic beta cells and is the formative polypeptide of pancreatic amyloid in patients with type 2 (non-insulin-dependent) diabetes mellitus. Islet amyloid and type 2 diabetes occur in man, but not in rat. To study transcription regulation of IAPP gene expression in man and rat, luciferase reporter constructs containing different portions of the upstream region of both IAPP genes were expressed in transfected cells. Both the human and the rat IAPP gene constructs revealed higher promoter activity in beta cells (particularly in beta TC3 cells) than in non-beta cells. In both IAPP genes potential transcription elements, with homology to insulin gene transcription elements, were identified. beta TC3 cells provide a good model system in which to study regulation of human and rat IAPP gene expression.

Amyloid↗
PubMed1992

Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85.

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to the Mycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained for Mycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA from Mycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.

Antigens, Bacterial↗
PubMed1991

Internal deletions in human interleukin-6: structure-function analysis.

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.

A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294-amino-acid mature protein. Comparison with previously described genes for the 30-kDa antigen (the alpha antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome.

Amino Acid Sequence↗