Role for homodimerization in growth deregulation by E2a fusion proteins.

The oncogenic transcription factor E2a-Pbx1 is expressed in some cases of acute lymphoblastic leukemia as a result of chromosomal translocation 1;19. The early observation that E2a-Pbx1 incorporates transcriptional activation domains from E2a and a DNA-binding homeodomain from Pbx1 inspired a model in which E2a-Pbx1 promotes leukemogenic transformation of lymphoid progenitor cells through transcriptional induction of target genes defined by the Pbx1 portion of the molecule. However, the subsequent demonstration that the only known DNA-binding module on the molecule, the Pbx1 homeodomain, is dispensable for the induction of lymphoblastic lymphoma in transgenic mice called into question the contribution made by the Pbx1 portion. In this study, we have used a domain swap approach coupled with a fibroblast-based focus formation assay to evaluate further the requirement for PBX1-encoded peptide elements in growth deregulation by E2a-Pbx1. No impairment of focus formation was observed when the entire Pbx1 portion was replaced with DNA-binding/dimerization domains derived from yeast transcription factor GAL4 or GCN4. Furthermore, replacement of Pbx1 with tandem FKBP domains that mediate homodimerization in the presence of a synthetic ligand led to striking growth deregulation exclusively in the presence of the dimerizing agent. N-terminal elements encoded by E2A, including the AD1 transcriptional activation domain, were required for dimerization-induced focus formation. We conclude that transcriptional target genes defined by heterologous C-terminal DNA-binding modules are not required in growth deregulation by E2a fusion proteins. We speculate that interactions between N-terminal E2a elements and undefined proteins that could function as components of a transcriptional coactivator complex may be more important.

Use of flexible intermediate and intensive care to reduce multiple transfers of patients.

OBJECTIVE: To test an alternative flexible approach to traditional fixed intermediate and intensive care to minimize transfers of patients. METHODS: Patients admitted to a 28-bed nursing unit with intermediate care potential and a 12-bed intensive care unit at a 300-bed teaching community hospital were studied. The group included 524 patients with a discharge diagnosis code for mechanical ventilation. During eight 3-week cycles, 1073 transfers of patients were tabulated. A plan-do-study-act method was used to improve weaning from mechanical ventilation and reduce the number of inappropriate days in intensive care. Admissions and transfers to the 2 units for all patients during the eight 3-week cycles were compared over time. Length of stay and mortality were noted for all patients treated with conventional and noninvasive ventilation. RESULTS: Direct admissions to the flexible intermediate unit increased with no overall change in admissions to the intensive care unit. Fewer patients needed conventional ventilation, and more in both units were treated with noninvasive ventilation. The median number of transfers per patient treated with mechanical ventilation decreased from 1.94 to 1.20. Length of stay and mortality also decreased among such patients. Some cost savings were attributable to the decrease in the number of transfers. Transfers out of the hospital directly from the intensive care unit increased from 2.24% to 4.43%. CONCLUSIONS: In a community teaching hospital, flexible care policies decreased the number of in-hospital transfers of patients treated with mechanical ventilation.

Incidence of deep venous thrombosis associated with femoral venous catheterization.

OBJECTIVE: To determine in adult medical patients the incidence of deep venous thrombosis (DVT) resulting from femoral venous catheterization (FVC). METHODS: A prospective, observational study was performed at a 420-bed community teaching hospital. Heparin-coated 7-FR cm femoral venous catheters were inserted unilaterally into a femoral vein. Each contralateral leg served as a control site. Age, gender, number of FVC days, DVT risk factors, administration of DVT prophylaxis, and DVT formation and site were tabulated for each patient. Venous duplex sonography was performed bilaterally on each patient within 7 days of femoral venous catheter removal. RESULTS: Catheters were placed in 29 men and 13 women. Femoral DVT was identified by venous duplex sonography in 11 (26.2%) of the FVC legs and none (0%) in the control legs. Posterior tibial and popliteal DVT was identified in both the FVC and control legs of 1 patient. DVT formation at the site of FVC insertion was highly significant (p = 0.005). There were no statistically significant associations with age (p = 0.42), gender (p = 0.73), number of DVT risk factors (p = 0.17), number of FVC days (p = 0.89), or DVT prophylaxis (p = 0.99). CONCLUSION: Placement of femoral catheters for central venous access is associated with a significant incidence of femoral DVT as detected by venous duplex sonography criteria at the site of femoral venous catheter placement. Physicians must be aware of this risk when choosing this vascular access route for adult medical patients. Further studies to assess the relative risk for DVT anf its clinical sequelae when using the femoral vs other central venous catheter routes are indicated.

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Proteins associated with poly-beta-hydroxybutyrate (PHB) granules were purified from four Acinetobacter strains isolated from modified activated sludge treatment plants. Four predominant proteins of 64 kDa, 41 kDa, 38 kDa and 13 kDa were identified. N-terminal amino acid sequencing of the 64-kDa and 13-kDa proteins from Acinetobacter RA3849 identified these proteins as the products of the phaCAc and phaPAc (formerly designated ORF1) genes, respectively. The expression of the 13-kDa protein (referred to as GA13) is shown to be required for the accumulation of large amounts of PHB in a recombinant Escherichia coli strain.

Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes.

The polyhydroxyalkanoic acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaAAc (encoding a beta-ketothiolase), phaBAc (encoding an acetoacetyl coenzyme A reductase), and phaCAc (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly-beta-hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaBAc, an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.

Clinical features and natural history of Beckwith-Wiedemann syndrome: presentation of 74 new cases.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome with variable expression. To define the range and frequency of complications in BWS, we have studied a cohort of 76 affected patients (two previously reported). The most frequent complications were macroglossia (97%), abdominal wall defect (80%) and birth weight or postnatal growth > 90th centile (88%). Other common features were ear creases/pits (76%), facial naevus flammeus (62%), nephromegaly (59%) and hypoglycaemia (63%). Rarer complications included hemihypertrophy (24%), moderate/severe developmental delay (4%), congenital heart defects (6.5%), polydactyly (4%), neoplasia (4%) and cleft palate (2.5%). Pre-term labour occurred in 53% and polyhydramnios in 33% of BWS pregnancies. The six deaths all occurred in babies born pre-term, three of whom had major congenital abnormalities. Five patients (6.5%) from four kindreds had an unequivocal family history of BWS, but 15 of 68 apparently sporadic cases had a relative with possible BWS (minor features only). Incomplete penetrance may lead to familial BWS being underdiagnosed.

Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located.

The polyhydroxyalkanoic acid (PHA) synthase gene (phaCAc) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-beta-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phaCAc revealed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaCAc revealed that the PHA synthase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organism for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.

Differentiation of polyphosphate and poly-beta-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge.

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.

Palliation of bone metastases in prostate cancer. Hemibody irradiation or strontium-89?

The palliation of bone pain is a common clinical problem once metastatic prostate cancer has escaped from hormonal control. This retrospective study compares the results of treatment using hemibody irradiation (HBI) at the Royal Marsden Hospital (27 cases) with isotope therapy using the bone-seeking isotope strontium-89 (89Sr) at Southampton General Hospital (51 cases). Prior to analysis patients were matched for potential prognostic factors (performance status, bone scan extent of disease, age, histology and duration of hormone response) to minimize the effect of treatment selection bias. Pain control assessed at 3 months was similar for HBI and matched 89Sr cases, with 63% and 52% respectively showing some benefit. Median survival was similar for these groups at 20 and 21 weeks respectively. The unmatched 89Sr group, which had more favourable prognostic factors, had a better outcome with 96% showing improvement in pain and with a median survival of 59 weeks. Subsequent univariate analysis demonstrated that performance status and extent of disease on bone scan were of overriding importance in determining outcome. Transfusion requirements were higher for the HBI group than for the matched 89Sr group (50% and 25% respectively) but other bone marrow toxicity was similar. Despite routine anti-emetic therapy 37% of patients treated with HBI had some nausea or vomiting. Although expensive, 89Sr appears as effective a treatment option as HBI. Response is most likely with either approach when patients have a good performance status and a limited extent of disease.

Strontium-89 chloride for pain palliation in prostatic skeletal malignancy.

In a multi-centre study strontium-89 was shown to be effective in relieving bone pain from prostatic carcinoma in patients who had failed conventional therapies. Of 83 patients assessed at 3 months, following the administration of a dose of at least 1.5 MBq/kg, 75% derived benefit and 22% became pain free. Symptomatic improvement usually occurred within 6 weeks and continued for between 4 and 15 months (mean 6 months). Based on the dose estimation part of this study the recommended dose of strontium-89 is 150 MBq. Toxicity was low, provided platelet levels were above 100 x 10(9) l-1 at the time of treatment. Repeat treatments with strontium-89 may be given at intervals of not less than 3 months. Strontium-89 is administered intravenously on an out-patient basis with no special radiological protection precautions.

ELISA for identification of blood meal source in black flies (Diptera: Simuliidae).

A modified ELISA, in which a biotinylated second antibody and a streptavidin-biotinylated peroxidase complex are used, has been developed to identify the blood meal sources of black flies. The modified assay is sensitive enough to identify correctly 100% of blood meals at 73 h postingestion and 80% of blood meals at 116 h postingestion in black flies held in the field at ambient temperatures.

A prospective, randomised double-blind crossover study to examine the efficacy of strontium-89 in pain palliation in patients with advanced prostate cancer metastatic to bone.

The palliative efficacy of strontium-89 chloride has been evaluated in a prospective double-blind crossover study comparing it with stable strontium as placebo in 32 patients with prostate cancer metastatic to bone. Response was assessed 5 weeks after each treatment. 26 patients were evaluable. Complete pain relief was only reported following strontium-89 injection. Statistical comparison between placebo and strontium-89 showed clear evidence of a therapeutic response to strontium-89 compared with only a limited placebo effect (P less than 0.01).

Polyphosphate production by strains of Acinetobacter.

Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA3123. No correlation was found between the polyphosphate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.

Regulation of isofunctional enzymes in Pseudomonas alcaligenes mutants defective in the gentisate pathway.

The regulation of the inducible set of gentisate pathway enzymes used by Pseudomonas alcaligenes (P25X1) has been studied in strains derived from mutant strains of P25X1 that had lost the constitutive enzymes that degrade m-cresol, 2,5-xylenol and 3,5-xylenol. The enzyme, 3-hydroxybenzoate 6-hydroxylase II, that catalyzes the oxidation of 3-hydroxybenzoate to gentisate is substrate- and product-induced while gentisate dioxygenase II is substrate induced. Neither 3-hydroxybenzoate nor gentisate could induce the synthesis of maleylpyruvate hydrolase II and fumarylpyruvate hydrolase II. The results suggest that the structural genes encoding these four inducible enzymes and malepylpyruvate hydrolase I (a constitutive enzyme) exist in at least four operons. There is strict induction specificity of expression of this inducible set of gentisate pathway enzymes. 3-Hydroxy-4-methylbenzoate failed to induce whilst 3-hydroxybenzoate and 3-hydroxy-5-methylbenzoate served as inducers of 6-hydroxylase II. Degradation of 2,5-xylenol is mediated by constitutive enzymes whereas the inducible set of enzymes are responsible for the metabolism of m-cresol and 3,5-xylenol.

Driving pressure and arterial carbon dioxide tension during high-frequency jet ventilation in postoperative patients.

To achieve normocarbia during conventional mechanical ventilation, ventilator settings are determined initially on the basis of body weight. The best ventilator settings for CO2 elimination during high-frequency jet ventilation (HFJV) have not been so clearly defined. A recent study has suggested that eucarbia will be obtained with HFJV when tidal volume (VT) per kg of body weight is kept within a narrow, well-defined range. In the same study, a "bench test" demonstrated that VT was directly proportional to the jet ventilator driving pressure (DP). The goal of our study was to confirm this recommended VT/kg to obtain eucarbia and to determine whether the relation observed between VT and DP in the laboratory was true clinically. We studied 14 patients admitted to the ICU for postoperative support. We determined a good correlation between DP and VT/kg (r = .811, p less than .001) for the group as a whole and a good inverse correlation between DP or VT/kg and PaCO2 for most individual patients; however, there was a poor inverse correlation between DP or VT/kg and PaCO2 for the group as a whole, due to wide patient-to-patient variation in the efficiency of jet ventilation. We conclude that there is no universal formula for setting jet ventilator DP or VT/kg to affect normocarbia in humans.

Synchronous versus nonsynchronous high-frequency jet ventilation: effects on cardiorespiratory variables and airway pressures in postoperative patients.

In order to compare the differences of high-frequency jet ventilation (HFJV) synchronized with the cardiac cycle (sync) to that nonsynchronized with the cardiac cycle (async), ten stable postoperative ICU patients, without heart failure, in sinus rhythm were ventilated randomly in either mode. The async mode was HFJV at 100 cycle/min, while the sync mode was HFJV triggered by the R-wave of the ECG tracing. The heart rate ranged between 64 and 127 beat/min. Synchronization was studied at one of two periods, sync 0 and sync 60. Sync 0 consisted of inspiration triggered by the R-wave, with jet ventilation occurring early in systole; sync 60 represented a 60% delay of the time between the succeeding R-waves, with jet ventilation occurring in mid-diastole. There was no significant difference in the cardiorespiratory data when async was compared to either sync 0 or sync 60. Therefore, in these patients without heart failure, the selection of async vs. either sync mode appeared to have neither adverse nor beneficial hemodynamic effects.

Use of monoclonal antibodies to investigate a possible role of thyroglobulin in the pathogenesis of Graves' ophthalmopathy.

One possible mechanism for Graves' ophthalmopathy is that the progressive orbital inflammation is initiated by formation of thyroglobulin (Tg)-anti-Tg immune complexes at sites of Tg binding to extraocular muscle membranes. In this study monoclonal antibodies (MCAB) against human Tg were used as probes (1) to identify Tg in eye muscle membranes prepared from normal subjects and (2) to measure binding of human Tg and Tg-anti-Tg immune complexes to eye muscle membranes. Reactivity of anti-Tg MCAB with Tg, thyroid, and eye muscle membranes was determined by binding of [125I]anti-Tg monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA), and the indirect immunofluorescence technique. Seven membrane fractions, prepared by differential sucrose gradient centrifugation, were used. Whereas [125I]anti-Tg MCAB bound to all thyroid membrane fractions tested, no [125I]anti-Tg bound to eye muscle membranes. Similarly, reactivity of anti-Tg MCAB with eye muscle membranes was not demonstrated in ELISA or immunofluorescence tests. Although Tg-anti-Tg immune complexes bound to thyroid membranes, such complexes did not bind to eye muscle membranes. Significant binding of [125I]human Tg to eye muscle or thyroid membranes was not demonstrated for any membrane preparation. On the other hand moderate, but significant, binding to skeletal muscle was shown. Similar results were found using an ELISA. Binding of [125I]anti-Tg-Tg complexes of [125I]Tg to thyroid and eye muscle membranes was not affected by the presence of normal human serum, phosphate ions, pH, or incubation temperature, conditions claimed by others to be critical for Tg and Tg-anti-Tg immune complex binding. Since Tg is not present in normal human eye muscle a major role of Tg, or Tg-anti-Tg immune complexes, in the pathogenesis of Graves' ophthalmopathy appears to have been excluded by these findings.

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000.

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus.

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.