Biomedical subjects
Publications and source records attributed to D Wall.
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Post-transplant lymphoproliferative disease (PTLD) is a well-known complication of solid organ transplantation. While this disorder can often be controlled by decreasing immunosuppression, it is frequently difficult to control the resultant rejection. This case exemplifies this dilemma. To solve this problem, cyclosphosphamide (600 mg/m2) and prednisone (2 mg/kg times 5 days) were given every 3 weeks to simultaneously treat PTLD and provide immunosuppression.
The clinical course of a 6-year-old boy with adrenoleukodystrophy (ALD) who underwent allogeneic stem-cell transplantation during an early clinical stage is described. Twenty-three months after transplant, he remains neurologically stable, but with moderate neurological sequelae; the serum very long chain fatty acid profile has improved, but not normalized. The indications, mechanism of action, and complications of bone marrow transplantation in ALD are discussed briefly, along with other potential therapies.
Type IV pili (Tfp) mediate the movement of bacteria over surfaces without the use of flagella. These movements are known as social gliding in Myxococcus xanthus and twitching in organisms such as Pseudomonas aeruginosa and Neisseria gonorrhoeae. Tfp are localized polarly. Type IV pilins have a signature N-terminal domain, which forms a coiled-coil with other monomer units to polymerize a pilus fibre. At least 10 more proteins at the base of the fibre are conserved; they are related to the type II secretion system. Movements produced by Tfp range from short, jerky displacements to lengthy, smooth ones. Tfp also participate in cell-cell interactions, pathogenesis, biofilm formation, natural DNA uptake, auto-aggregation of cells and development. What is the means by which Tfp bring about the movement of cells?
The Myxococcus xanthus sglA1 spontaneous mutation was originally isolated because it allowed dispersed cell growth in liquid yet retained the ability to form fruiting bodies. Consequently, most of today's laboratory strains either contain the sglA1 mutation or were derived from strains that carry it. Subsequent work showed that sglA was a gene for social gliding motility, a process which is mediated by type IV pili. Here sglA is shown to map to the major pil cluster and to encode a 901-amino-acid open reading frame (ORF) that is homologous to the secretin superfamily of proteins. Secretins form a channel in the outer membrane for the transport of macromolecules. The closest homologs found were PilQ proteins from Pseudomonas aeruginosa and Neisseria gonorrhoeae, which are required for type IV pili biogenesis and twitching motility. To signify these molecular and functional similarities, we have changed the name of sglA to pilQ. The hypomorphic pilQ1 (sglA1) allele was sequenced and found to contain two missense mutations at residues 741 (G-->S) and 762 (N-->G). In addition, 19 independent social (S)-motility mutations are shown to map to the pilQ locus. In-frame deletions of pilQ and its downstream gene, orfL, were constructed. pilQ is shown to be essential for pilus biogenesis, S-motility, rippling, and fruiting body formation, while orfL is dispensable for these processes. The pilQ1 allele, but not the DeltapilQ allele, was found to render cells hypersensitive to vancomycin, suggesting that PilQ1 alters the permeability properties of the outer membrane. Many differences between pilQ1 and pilQ+ strains have been noted in the literature. We discuss some of these observations and how they may be rationalized in the context of our molecular and functional findings.
Social gliding motility of Myxococcus xanthus requires polar type IV pili. Tgl mutants lack pili and lack social motility. However, both defects can be rescued phenotypically, but not genotypically, when tgl+ donor and tgl- recipient cells make physical contact with each other. What is the cellular and molecular basis of this transfer of phenotype, which is called stimulation? Stimulation does not occur in liquid nor in soft (0.5%) agar; however, on a more firm surface (1.0% agar) cells stimulate each other efficiently. Microscopy revealed that cells placed on 1.0% agar readily became aligned whereas they remained poorly aligned on 0.5% agar. It was observed, moreover, that stimulation is greatly reduced when donor and recipient cells lack the ability to move because of mutations in any of several different motility genes. Microscopy showed that motile cells became highly aligned, and can even align nonmotile cells. Using a stimulation assay based on the assembly of pili, a strong correlation was found between conditions that promote cell alignment and the speed or extent of stimulation. Because pili are assembled only at the end(s) of a cell, and a parallel alignment of cells in contact brings them end-to-end, we would suggest that end-to-end contacts are important for stimulation of pilus assembly.
Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed extraction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.
Airborne fungi have been postulated as a cause of symptoms among office workers. Using the MAST chemiluminescent system, this study evaluated 36 IgG and 36 IgE antibody levels in 47 office workers from an area with elevated airborne fungal concentrations and 44 office workers from an otherwise similar area with lower airborne fungal exposure. No difference was found in IgG antibody to fungi between the lower and higher exposure areas, but high IgG antibody to one or more of the fungi studied was detected in 67% of all the workers tested. IgE antibody to one or more antigens was detected in 40% of the participants. Workers who reported atopic symptoms (sneezing, runny nose, and itchy eyes) or "sick building" symptoms (any three of the following temporally related to work: headache, fatigue, stuffy nose, irritated eyes, or sore throat) were more likely to have one positive IgE antibody test. Type I hypersensitivity to aeroallergens besides fungi may play a role in some symptoms reported by some participants in this office building.
Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.
BACKGROUND: The hypothesis being tested in this paper is that endotoxin levels in donors and in recipients during liver transplantation influences postoperative outcome. METHODS: Endotoxin levels in systemic venous and portal venous blood were measured using in 46 adult donors and 44 adult recipients (47 liver transplants) during the period 1992-95. Endotoxin was measured using a modification of the Limulus amoebocyte lysate (LAL) assay. RESULTS: In the donor, systemic endotoxin levels were above normal levels at 10.0 +/- 1.3 pg/mL from the start and rose to 15.8 +/- 2.9 pg/mL after dissection of the hilar structures, but fell to 10.6 +/- 0.8 pg/mL just prior to the removal of the liver (control = 7.8 pg/mL). The mean portal venous endotoxin levels were 18.2 +/- 3.4 pg/mL after dissection of the hilar structures and 12.6 +/- 0.9 pg/mL after cannulation of the portal vein. In the recipients, the highest level in the portal venous blood occurred at the end of the anhepatic phase (46.5 +/- 6.7 pg/mL). The systemic venous samples in the recipients were elevated to start with, but fell rapidly to 19.3 +/- 1.5 pg/mL 24 h postoperatively, and to 13.2 +/- 1.0 pg/mL by day 7. The endotoxin concentrations were higher in recipients who developed complications. CONCLUSIONS: Endotoxin is elevated throughout the recipient transplantation procedure and up to 7 days postoperatively. High levels of endotoxin at induction, the anhepatic phase and at certain time points correlated with patients who developed postoperative complications.
While there have been reports on changes in the renin-angiotensin system and angiotensin II (AT) receptors in diabetes, there is no agreement on the nature of these changes. This study has characterised specific AT receptors in the heart, kidney, liver and adrenal glands of the streptozotocin (STZ)-diabetic rat using radioligand binding studies with the radioligand 125I-[Sar1, Ile8]-angiotensin II. Left ventricular AT receptor density increased by 135% 4 weeks after treatment and by 206% 12 weeks after treatment; in the liver, AT receptor density increased by 476% (4 weeks) and 263% (12 weeks) and in the adrenal gland by 236% (4 weeks) and 109% (12 weeks). In contrast, renal AT receptor density decreased by 49% (4 weeks) and 36% (12 weeks). Competition-displacement assays with losartan, an AT1-selective ligand, showed that the proportion of AT receptor subtypes remained unchanged. STZ treatment decreased plasma angiotensinogen by 72% (4 weeks) and 67% (12 weeks) and increased plasma renin concentration after 12 weeks; plasma renin activity and aldosterone concentrations remained unchanged. Treatment with human insulin (5 U/day) attenuated changes in plasma angiotensinogen and AT receptor density except in the left ventricle. We conclude that there are major changes in AT receptors in the STZ-diabetic rat that are tissue-specific and time-dependent. Plasma angiotensinogen and renin secretion change in directions that result in the maintenance of plasma renin activity and aldosterone concentration.
The recombinant N-terminal 107-amino acid polypeptide fragment 2-108 of the DnaJ molecular chaperone of Escherichia coli, which contains the J-domain (residues 2 to 76) and the Gly/Phe-rich region (residues 77 to 108), was uniformly labeled with nitrogen-15 and carbon-13. The complete NMR solution structure of the J-domain was determined with the program DIANA on the basis of 682 nuclear Overhauser enhancement (NOE) upper distance limits and 180 dihedral angle constraints. It contains three well-defined helices comprising residues 6 to 10, 18 to 32 and 41 to 57, and a fourth helix, consisting of residues 61 to 68, which is well defined as a regular secondary structure but for which the location relative to the remainder of the molecule is not precisely determined. The helices II and III form an antiparallel helical coiled-coil. Helix I is approximately parallel to the plane defined by the helices II and III and runs from the carboxy-terminal end of the helix III to the center of helix II. Helix IV is positioned near the carboxy-terminal end of helix III and is on the same side of the coiled coil as helix I, but it is oriented approximately perpendicular to the plane of the helices II and III. This novel alpha-protein topology leads to formation of a hydrophobic core involving side-chains of all four helices. A strong correlation is seen between the extent of sequence-conservation of hydrophobic residues in the family of J-domain homologues, and the structural organization of the hydrophobic core in these proteins. The residues which have key roles for the specificity of the interaction of DnaJ-like proteins with their corresponding Hsp70 counterparts are located on the outer surfaces of the helices II and III, and in the loop connecting these two helices. Measurements of backbone amide proton exchange rates, 15N spin relaxation times and heteronuclear 15N {1H} NOEs provided additional insights into local conformational equilibria and internal rate processes in DnaJ(2-108). In the Gly/Phe-rich region, which is poorly ordered in the NMR solution structure and does not form a globular core, the polypeptide segment 90 to 103 differs from the segments 77 to 89 and 104 to 108 by reduced local flexibility. Considering that this same segment shows sequence conservation with corresponding segments in the Gly/Phe-rich regions of other DnaJ-like proteins, its reduced flexibility may be directly linked to the formation of the ternary DnaJ-DnaK-polypeptide complex.
DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.
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An in vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patient's stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelof's syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.
PURPOSE: To determine the maximum-tolerated dose of cyclophosphamide (CTX) when administered sequentially with melphalan 60 mg/m2/d for 3 days, followed by autologous bone marrow rescue (ABMR), in children with recurrent or progressive malignant brain tumors, and to make preliminary observations on efficacy. PATIENTS AND METHODS: Nineteen patients between the ages of 2 and 21 years were enrolled and 18 were assessable for effects of therapy. CTX was administered to seven patients at 750 mg/m2/d for 4 days, to five patients at 975 mg/m2/d, to three patients at 1,200 mg/m2/d, and to three patients at 1,500 mg/m2/d. All patients received ABMR. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used in 15 patients. Toxicity, response to therapy, time to progression, and survival and monitored. RESULTS: The median time to a granulocyte count more than 500/dL was 19 days (range, 11 to 39), and for a platelet count more than 50,000/dL was 33 days (range, 16 to 60). Four heavily pretreated patients (22%) died of transplant-related complications. No dose-limiting, non-hematologic toxicities were defined for the study. Seven of 18 patients (39%) had a complete response (CR) or a partial response (PR). These included four patients with medulloblastoma (CR and three PRs), two with germinomas (two CRs), and one with ependymoma (one CR). The estimated 1-year survival rate was 39% (SE 12%). CONCLUSION: CTX, at a maximum total dose of 6,000 mg/m2, administered sequentially with melphalan and followed by ABMR was tolerable in children with recurrent brain tumors who had not been heavily pretreated. Responses were seen in patients with medulloblastoma and germinomas. Further trials in children with chemosensitive tumors, with minimal residual disease, are planned.
BACKGROUND: There is concern about the morale of general practitioner registrars. There may be stress-provoking factors that could be avoided or minimized. AIMS: The aims of the study were to assess the sources of stress and job satisfaction of general practitioner registrars, to compare registrars' job satisfaction with that of established principals using a recently published survey and to identify registrars' usual responses to stress. METHOD: A postal questionnaire survey was sent to all 143 general practitioner registrars in the West Midlands Region. The main measures were: self-rating scales of stresses associated with work and training; the Warr, Cook and Wall job satisfaction scale; and self-reported responses to stress. RESULTS: A total of 118 (83%) general practitioner registrars responded. The most potent sources of stress were family-job conflict, working for the Membership of the Royal College of General Practitioners, patients' unrealistic expectations and disruption of social life. Registrars practised good coping responses to stress. Registrars in this study had significantly greater job satisfaction than general practitioner principals in a 1993 survey for three out of 10 items measured (responsibility given, hours of work and the job as a whole) and significantly worse scores for three items (recognition for good work, rate of pay and variety of work). CONCLUSIONS: Registrars have additional stresses to those of established principals because they need to study for examinations, learn new tasks in general practice and carry out their service commitments at a stage in life when many are newly married or have a young family. Training in stress management for general practitioner registrars is recommended.
Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, delta 32, P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such as an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. delta 32, RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (lambda P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.