Genetic and structural analysis of the Bacteroides conjugative transposon CTn341.

The genetic structure and functional organization of a Bacteroides conjugative transposon (CTn), CTn341, were determined. CTn341 was originally isolated from a tetracycline-resistant clinical isolate of Bacteroides vulgatus. The element was 51,993 bp long, which included a 5-bp coupling sequence that linked the transposon ends in the circular form. There were 46 genes, and the corresponding gene products fell into three major functional groups: DNA metabolism, regulation and antibiotic resistance, and conjugation. The G + C content and codon usage observed in the functional groups suggested that the groups belong to different genetic lineages, indicating that CTn341 is a composite, modular element. Mutational analysis of genes representing the different functional groups provided evidence for the gene assignments and showed that the basic conjugation and excision genes are conserved among Bacteroides spp. A group IIA1 intron, designated B.f.I1, was found to be inserted into the bmhA methylase gene. Reverse transcriptase PCR analysis of CTn341 RNA showed that B.fr.I1 was functional and was spliced out of the bmhA gene. Six related CTn-like elements were found in the genome sequences of Bacteroides fragilis NCTC9343 and Bacteroides thetaiotaomicron VPI5482. The putative elements were similar to CTn341 primarily in the tra and mob regions and in the exc gene, and several appeared to contain intron elements. Our data provide the first reported sequence for a complete Bacteroides CTn, and they should be of considerable benefit to further functional and genetic analyses of antibiotic resistance elements and genome evolution in Bacteroides.

Limitations of the use of single base changes in the p53 gene to detect minimal residual disease of breast cancer.

BACKGROUND/AIMS: Peripheral blood progenitor cell (PBPC) transplantation is frequently used in the treatment of malignant diseases, but contamination of the graft by tumour cells is a real concern and may lead to disease relapse. The feasibility of applying heterogeneous single base genetic changes as tumour specific markers to detect minimal residual disease in PBPC harvests was studied, using the p53 gene and breast cancer as models. METHODS: Tumour tissues from 51 patients with cellular aliquots from PBPC harvests available were studied. Thirty eight patients had metastatic disease or were at high risk of metastasis, and 13 had high risk stage II/III disease with four or more involved axillary lymph nodes. Tumour DNA was screened for p53 mutations in exons 5 to 9, using denaturing gradient gel electrophoresis, followed by sequencing. Based on sequence information, allele specific primers were designed for each mutation and the non-radioisotopic, amplification refractory mutation system (ARMS) was used to screen DNA from PBPC harvests for minimal residual disease. Attempts were made to optimise each system, based on parameters determined using the T47D breast cancer cell line with a confirmed point mutation in codon 194. RESULTS: Twelve different somatic mutations were found, two of which could not be sequenced. The remainder were point mutations. Only five of the 10 ARMS systems were successfully optimised, and minimal residual disease detection sensitivities ranged from one copy of tumour DNA in 10(2) to 10(3) copies of wild-type DNA. Using ARMS, three of five patients and eight of 12 of their PBPC harvests showed minimal residual disease. CONCLUSIONS: These results suggest that the use of single base genetic changes in minimal residual disease detection is relatively insensitive and is limited to a small number of patients and to certain mutations. In addition, it is labourious and therefore unlikely to play an important role in clinical practice.

Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system.

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.

Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene.

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.

Genetic structure and transcriptional analysis of a mobilizable, antibiotic resistance transposon from Bacteroides.

Tn4555 is a 12.1-kb Bacteroides antibiotic resistance transposon representative of a novel class of transmissible genetic elements that can be transferred by resident conjugative tetracycline resistance transposons (Tc(r)-elements) but are not capable of self-transfer. Previously it was shown that Tn4555 transposes by a site-specific recombination mechanism that utilizes a circular intermediate. This circular form is induced by tetracycline and it also is the substrate for conjugation. To better understand the mechanism of transposition, the entire nucleotide sequence of Tn4555 was determined and a set of genes potentially involved in transposition was identified. The transposon was 12,105 bp including a variable 6-bp coupling sequence associated with one of the transposon termini. The element had a 44.3% G + C composition and nine potential protein coding regions were observed, eight of which were encoded on the forward strand. Two putative transposition genes were found. The int gene product had significant C-terminal homology to the lambda family of integrases and the xis gene product was similar to several excisionase proteins encoded by both plasmids and conjugative transposons. The mobA mobilization gene and cfxA beta-lactamase gene of Tn4555 had been previously identified, and the remaining five open reading frames had no significant matches with sequences in the available databases. Northern hybridization analysis revealed that all Tn4555 genes except for orf-9 were expressed and two sets of genes, tnpA, int and xis, orf-5, orf-6 were organized in operons. None of the genes seemed to be induced significantly by the addition of tetracycline to cultures. Although a small 0.4-kb xis-specific transcript appeared in tetracycline-treated cultures it was not clear if this was due to an induction or if it was a specific degradation product.

Nurse practitioner support for transfusion in patients with haematological disorders in hospital and at home.

We prospectively studied the effects of dedicating a nurse to manage the provision of blood product support in a hospital haematology unit and at home to 45 patients with haematological disorders requiring regular transfusion. During the study 335 home blood tests, 65 home platelet transfusions and 155 hospital transfusions were managed by the nurse who organized the whole transfusion process, made home visits for blood tests and platelet transfusions and arranged hospital visits for red cell transfusions. Two hundred clinic visits and 65 day hospital attendances were avoided. The nurse-led service resulted in a significant reduction in the waiting time from admission to transfusion and in the total length of in-patient stay. The importance of and satisfaction with different aspects of the care of the transfusion process assessed by a ranking questionnaire showed improved satisfaction scores for all aspects of care. Preference for home blood sampling instead of hospital increased from 24% before to 100% after intervention. We have shown that a dedicated transfusion nurse provides a quality service between hospital and home that is greatly appreciated by patients requiring regular transfusions.

A novel 8-bp insertion in codon 281 of p53 in a patient with acute lymphoblastic leukaemia and 2 separate leukaemic clones. Mutations in brief no. 219. Online.

We report a novel p53 insertion in a case of aggressive acute lymphoblastic leukaemia in a 16 year old male, in which 2 separate leukaemic clones were previously identified by T-cell receptor sigma and immunoglobulin heavy chain gene rearrangement studies. Initial p53 mutation screening of blast cells from 29 patients with acute leukaemia by PCR-denaturing gradient gel electrophoresis showed that 2 had a silent codon 213 polymorphism and only the index case had a somatic mutation identified to be an 8 bp insertion in codon 281 (5'CCGGGGGG-3'). This insertion was associated with the second, more aggressive clone which underwent clonal evolution and became resistant to cytotoxic chemotherapy. With an allele-specific primer in PCR, we were able to demonstrate the presence of this clone as a minority at disease presentation, and in 2 of 3 collections of peripheral blood progenitor cells.

Vitronectin inhibits the thrombotic response to arterial injury in mice.

Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.

Factors influencing maternal behavior in the hubb/hubb mutant mouse.

We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants.

Effects of radiographic contrast agents on thrombin formation and activity.

Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on thrombin formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited factor X activation by the tissue factor-factor VIIa complex more potently than ioversol (53 +/- 3.7, 43.0 +/- 1.9, and 26.5 +/- 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of prothrombinase function, inhibiting thrombin formation by >75% at contrast concentrations of 0.6% (p <0.005). Ioversol inhibited prothrombinase to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of thrombin generation in plasma despite having the least effect on fibrin polymerization. Contrast agents inhibited binding of thrombin to fibrin, with ionic agents producing a more potent effect than ioversol (p <0.02). However, contrast agents did not inhibit thrombin-mediated platelet activation, had only a minor effect on inhibition of thrombin by antithrombin III, and did not affect thrombin-hirudin interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit thrombin formation and function -- i.e. inhibition of tissue factor-dependent factor Xa generation, inhibition of the prothrombinase complex, and inhibition of thrombin binding to fibrin. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.

Fas antigen (CD95) expression in peripheral blood progenitor cells from patients with leukaemia and lymphoma.

Fas antigen (CD95) is a cell surface receptor belonging to the tumour necrosis factor/nerve growth factor superfamily and is able to induce apoptosis when triggered by its' natural ligand or an anti-Fas antibody. Fas expression is low on CD34+ bone marrow (BM) progenitor cells, but is increased by various cytokines in vitro. We investigated Fas expression on CD34+ cells from 39 peripheral blood progenitor cell (PBPC) harvests and from 5 normal BM harvests by dual colour flow cytometry to determine if Fas expression was altered during mobilisation. By including calibrated microbeads during flow cytometry, we quantified the number of Fas antigen molecules per cell. A low percentage of PBPC (22%) and normal BM (23%) CD34+ cells expressed Fas antigen. Fas expression varied on CD34+ cells from different diseases and the highest expression was found in ALL (52%). There was a significant three fold increase in the number of Fas molecules/cell expressed on CD34+ cells (PBPC 6,230 molecules/cell, BM 2,236; p = 0.0003). This level of expression was considerably less than that for CD3/CD19 lymphocytes (33,095 molecules/cell) and CD14 monocytes (47,467 molecules/cell) in the PBPC harvest. In conclusion, mobilisation including the use of growth of factors, has minimal effect on CD34 progenitor cell Fas expression.

Multiple myeloma in three siblings.

There have been 53 reported instances of myeloma occurring in more than one family member but in only three of these reports have three siblings been affected (Alexander & Benninghoff 1967; Maldonado & Kyle 1974; Horwitz et al. 1985; Crozes-Bony et al. 1995). We report a family where three siblings had an unequivocal diagnosis of myeloma made over a period of six years. The paraprotein isotype was IgG kappa in two of the siblings and kappa light chain only in the remaining sibling. The importance of these cases lies in the fact that they are highly suggestive of a genetic predisposition to the development of myeloma. Because of the high prevalence of p53 abnormalities in certain types of familial cancer screening for p53 mutation in exons 5-8 was performed by denaturing gradient gel electrophoresis (DGGE) on DNA extracted from the bone marrow of the three siblings. Although a p53 mutation was identified in one of the siblings it was felt to represent a somatic as opposed to a germline mutation.

Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma.

Quantifying progenitor cells in peripheral blood stem cell (PBSC) harvests by flow cytometric enumeration of CD34+ cells does not account for cell viability. Cell membrane asymmetry in early apoptosis exposes phosphatidylserine on the cell surface. This can be detected by staining with annexin V FITC. Apoptosis in 30 autologous PBSC harvests mobilised by cyclophosphamide + G-CSF or standard chemotherapy + G-CSF was analysed immediately after collection by dual-colour flow cytometry with CD34 PE and annexin V FITC. Harvests contained a median of 3.4 x 10(6)/kg (range 0.3-91.8) CD34+ cells. Of these 87.6% (range 30-96.5) were annexin V-. In 10% of harvests more than 50% of CD34+ cells were apoptotic. Differences in PBSC mobilisation or collection could not explain the variation in annexin V binding. Cyclophosphamide + G-CSF significantly increased the yield of CD34+ cells but did not increase apoptosis. Comparison of consecutive harvests showed no difference in the numbers of CD34+ cells collected but found a significant decrease in apoptotic CD34+ cells through multiple collections. Analysis of annexin V binding in PBSC harvests is a simple flow cytometry technique which gives additional information on the status of CD34+ progenitor cells.

Induction of CYP1A2 activity by carbamazepine in children using the caffeine breath test.

AIMS: Carbamazepine is a known enzyme inducer. The aim of this study was to determine whether carbamazepine induces the metabolism of caffeine in children. METHODS: Children due to receive carbamazepine for epilepsy were recruited into the study. The caffeine breath test was carried out prior to the administration of carbamazepine and after a minimum of 2-3 weeks therapy. Five children were studied and they received 200-600 mg carbamazepine daily. RESULTS: The mean values of the 2 h cumulative labelled carbon dioxide were 3.47% before and 7.65% during carbamazepine. There was a significant increase in the percentage labelled caffeine exhaled as carbon dioxide during the administration of carbamazepine (Student's paired t-test, P < 0.05). CONCLUSIONS: The results suggest that carbamazepine induces the metabolism of caffeine by the CYP1A2 pathway in the children studied.

Comparative adhesion of human haemopoietic cell lines to extracellular matrix components, bone marrow stromal and endothelial cultures.

We used flow cytometry to characterize cell adhesion molecule expression of the human haemopoietic cell lines KG1a, K562, HL-60, NALM-6 and CEM. A 51chromium labelling assay was used to study the adhesion of these cell lines to extracellular matrix components and to bone marrow stromal and endothelial cultures. Both adhesion molecule expression and functional binding behaviour varied between cell lines. All five cell lines expressed the integrins alpha4beta1 and alpha5beta1 and all adhered to fibronectin. However, differences in intensity of expression of these integrins failed to correlate with extent of fibronectin adhesion. Inhibition experiments demonstrated that adhesion of KG1a to fibronectin was completely inhibited by divalent cation chelation and partially inhibited by RGDS peptides and chondroitinase ABC, suggesting that both alpha4beta1 and alpha5beta1 as well as CD44 were responsible for this interaction. Adhesion to bone marrow stromal and endothelial layers was superior to that to purified extracellular matrix components and was partially inhibited by divalent cation chelation. RGD peptides and anti-alpha4 monoclonal antibody also partially inhibited KG1a adhesion to bone marrow endothelium. Discordance between cell adhesion molecule expression and adhesive behaviour suggest that current phenotypic descriptions remain incomplete and reinforce the need for complementary functional binding studies.

The transfer origin for Bacteroides mobilizable transposon Tn4555 is related to a plasmid family from gram-positive bacteria.

Conjugal transfer of Bacteroides mobilizable transposon Tn4555 was examined with an Escherichia coli-based assay system. It was shown that mobilization required the cis-acting oriT(Tn) region and that the Tn4555 mobA(Tn) gene and RK231 must be present in trans. With alkaline agarose gel electrophoresis and filter blot hybridizations, it was shown that at oriT(Tn) there was a site- and strand-specific cleavage event that was dependent on mobA(Tn). The 5' end of this cleavage site was mapped by primer extension, and the nucleotide sequence surrounding the site had homology to a family of oriT nick sites found in mobilizable plasmids of gram-positive bacteria. Removal of the nick site by deletion of 18 bp surrounding the site resulted in a significant loss of transfer activity.

Enhanced drug metabolism in young children with cystic fibrosis.

The effect of cystic fibrosis on caffeine metabolism was studied in young children using the caffeine breath test. Eight children with cystic fibrosis aged 2-6 years and nine age matched controls were studied on a single occasion, and the cumulative percentage of labelled caffeine exhaled as carbon dioxide measured over two hours. This was significantly higher in the patients with cystic fibrosis than in controls, suggesting an increase in the CYP1A2 metabolic pathway in the former. The fact that these were young children with minimal lung and liver disease suggests that enhanced drug metabolism in children with cystic fibrosis is hereditary rather than secondary to lung and liver damage.

Effects of chemotherapy-induced testicular damage on inhibin, gonadotropin, and testosterone secretion: a prospective longitudinal study.

To investigate the role of inhibin in the control of follicle-stimulating hormone (FSH) secretion, we have measured levels of immunoreactive inhibin (ir-inhibin), inhibin B, Pro-alpha C containing inhibins, FSH, luteinizing hormone (LH), and testosterone in twelve men with hematological malignancies before, during, and after chemotherapy. Inhibin B levels fell significantly by 1 month from a mean +/- SE baseline level of 273.2 +/- 32.8 pg/mL, reaching a nadir of 52.6 +/- 15.3 pg/mL at 4 months (P < 0.0001). FSH levels increased within the first month from a baseline level of 3.9 +/- 0.6 IU/L, reaching a peak level of 22.4 +/- 3.3 IU/L at 4 months (P < 0.0001). FSH and inhibin B were significantly and inversely correlated (r = 0.69, P < 0.0001). Pro-alpha C containing inhibin levels increased significantly (P < 0.05) at 3 months and were significantly and positively correlated with FSH (r = 0.38, P = 0.002). LH levels increased significantly but to a much lesser extent than FSH, the increase becoming evident only 4 months after treatment commenced (P < 0.03). Levels of ir-inhibin and testosterone remained unchanged throughout the study. These data provide strong support to the hypothesis that inhibin B is the physiologically important form of inhibin in men, negatively regulating FSH secretion at the pituitary. Furthermore, they suggest that FSH stimulates inhibin alpha-subunit secretion by the testis.

CHOP-based chemotherapy is as effective as alternating PEEC/CHOP chemotherapy in a randomised trial in high-grade non-Hodgkin's lymphoma. Scotland and Newcastle Lymphoma Group.

The aim of this study was to test whether survival for patients with high-grade non-Hodgkin's lymphoma (NHL) can be improved with a non-cross-resistant regimen as compared to a CHOP-based regimen. This is a multicentre study comprising 325 adult patients, median age 58 years, with high-grade non-Hodgkin's lymphoma: patients of any age and performance status were eligible provided they were able to receive the drugs in the regimens. Patients were randomised to either B-CHOP-M (bleomycin, cyclophosphamide, doxorubicin, vincristine, prednisolone and methotrexate) or PEEC-M (methylprednisolone, vindesine, etoposide, chlorambucil and methotrexate) alternating with B-CHOP-M. At a median follow-up of 9 years, there was no significant difference in overall survival or disease-free survival between the two arms. Toxicities for the two regimens were equivalent. This study confirms that for relatively unselected patients with high-grade non-Hodgkin's lymphoma, an alternating multidrug regimen does not improve upon the results obtained with B-CHOP-M.