Biomedical subjects
R E Williams
Publications and source records attributed to R E Williams.
Degradation of nitrate ester and nitroaromatic explosives by Enterobacter cloacae PB2.
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Mutations in the palmitoyl-protein thioesterase gene (PPT; CLN1) causing juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposits.
A subtype of neuronal ceroid lipofuscinosis (NCL) is well recognized which has a clinical course consistent with juvenile NCL (JNCL) but the ultrastructural characteristics of infantile NCL (INCL): granular osmiophilic deposits (GROD). Evidence supporting linkage of this phenotype, designated vJNCL/GROD, to the INCL region of chromosome 1p32 was demonstrated (pairwise lod score with D1S211 , Z max = 2.63, straight theta = 0.00). The INCL gene, palmitoyl-protein thioesterase (PPT ; CLN1), was therefore screened for mutations in 11 vJNCL/GROD families. Five mutations in the PPT gene were identified: three missense mutations, Thr75Pro, Asp79Gly, Leu219Gln, and two nonsense mutations, Leu10STOP and Arg151STOP. The missense mutation Thr75Pro accounted for nine of the 22 disease chromosomes analysed and the nonsense mutation Arg151STOP for seven. Nine out of 11 patients were shown to combine a missense mutation on one disease chromosome with a nonsense mutation on the other. Mutations previously identified in INCL were not observed in vJNCL/GROD families. Thioesterase activity in peripheral blood lymphoblast cells was found to be markedly reduced in vJNCL/GROD patients compared with controls. These results demonstrate that this subtype of JNCL is allelic to INCL and further emphasize the correlation which exists between genetic basis and ultrastructural changes in the NCLs.
Biochemical characterization of recombinant fusions of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein. Implications in biological activity.
The physiological response to endotoxin (lipopolysaccharide (LPS)) can be regulated by two closely related LPS-binding proteins, LPS-binding protein (LBP), which potentiates LPS' inflammatory activity via interaction with the monocytic antigen CD14, and bactericidal/permeability-increasing protein (BPI), which neutralizes LPS. Both proteins bind LPS with high affinity sites in their N-terminal domains, whereas interaction between LBP and CD14 is dependent upon the LBP C-terminal domain. We have created fusions of the N- and C-terminal domains from each protein and compared the functional activities and pharmacokinetics of these fusions, the individual N-terminal domains, and the parent proteins. The N-terminal domains of BPI and LBP bound lipid A with their characteristic apparent affinity constants, regardless of the C-terminal fusion partner. In addition, the C-terminal domain of LBP allowed transfer of LPS to CD14 in conjunction with either N-terminal LPS binding domain. Proteins containing a BPI N-terminal domain had greater heparin binding capacities in vitro and were cleared more rapidly from the plasma of whole animals. Taken together, these data better define how closely related proteins such as BPI and LBP can have opposing effects on the body's response to LPS.
Loci for classical and a variant late infantile neuronal ceroid lipofuscinosis map to chromosomes 11p15 and 15q21-23.
The childhood neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative disorders characterised by progressive visual failure, neurodegeneration, epilepsy and the accumulation of an autofluorescent lipopigment in neurones and other cells. Three main subtypes have been identified according to age of onset, clinical features and ultrastructural morphology. These are infantile NCL (INCL; CLN1), classical late infantile NCL (LINCL; CLN2) and juvenile NCL (JNCL; CLN3). Several atypical forms of late infantile NCL (LINCL) have also been described including a Finnish variant LINCL (CLN5). The CLN2 gene has been excluded from the CLN1, CLN3 and CLN5 loci. A genome search was initiated using a homozygosity mapping strategy in five classical LINCL and two variant LINCL consanguineous families. A common region of homozygosity was identified on chromosome 11p15 in two of the classical families. Analysis of a further 33 classical LINCL families supported linkage in this region (Zmax = 3.07 at theta = 0.06 at D11S1338). A common region of homozygosity was also observed on chromosome 15q21-23 in the two variant LINCL families. Extension of the analysis to include a further seven families of identical ultrastructural phenotype established linkage to this region (Zmax = 6.00 at theta = 0.00 at D15S1020).
The impact of gunshot wounds on an orthopaedic surgical service in an urban trauma center.
OBJECTIVE: To determine the prevalence of gunshot wound related orthopaedic injuries in an urban trauma center and outline the socioeconomic background of this patient population. DESIGN: Retrospective study conducted from January 1, 1994, through December 30, 1994. SETTING: University-affiliated level 1 trauma center. PATIENTS: Strict inclusion and exclusion criteria were established. INCLUSION CRITERIA: All patients were admitted through the emergency room with a gunshot wound for which the orthopaedic surgery service was consulted. The study group consisted of 284 patients. EXCLUSION CRITERIA: Those individuals excluded from the study were patients with an orthopaedic injury who died during or before attempts at resuscitation in the emergency room and patients treated on an outpatient basis. MAIN OUTCOME MEASURES: Orthopaedic and nonorthopaedic diagnoses, etiology, procedures performed, number of hours from admission to the first surgical procedure, average daily hospital census, drug and alcohol screen results, and patient financial status. RESULTS: The orthopaedic service was consulted on 284 patients admitted with gunshot wounds. This group comprised 24% of all orthopaedic admissions, 33% of the average daily orthopaedic census, and 14% of all orthopaedic surgery cases performed. Ninety-four percent were African American and 87% were male, with a mean age of 27 years. Approximately half were tested for alcohol and/or drugs, 45% of whom were positive for alcohol and 65% for drugs. Only 4% of the patients were privately insured. CONCLUSIONS: During the period of this study, gunshot wound injuries required more orthopaedic trauma resources than any other single diagnosis.
Biceps femoris and rump fat as additional ultrasound measurements for predicting retail product and trimmable fat in beef carcasses.
One hundred ninety-eight steers of Angus and Hereford breeding were evaluated ultrasonically for fat thickness over the 12-13th rib (UFAT), fat thickness over the rump (URUMP), 12-13th longissimus muscle area (UREA), and depth of the biceps femoris (UROUND) before slaughter. Carcass measurements associated with the USDA yield grade were also obtained. Carcasses were fabricated into closely trimmed (.32 cm fat), boneless subprimals. Regression procedures were used to predict weight and the percentages of retail product and trimmable fat. Final weight (FINALWT) accounted for most of the variation when predicting kilograms of retail product and trimmable fat, with R2 values of .836 and .435, respectively. As single predictors URUMP and UFAT accounted for most of the variation when predicting the percentages of retail product and trimmable fat with R2 values of .244 and .220, respectively. Adding URUMP to equations that included FINALWT, UREA, and UFAT increased R2 values for percentage of retail product from .175 to .318 and for weight of retail product from .847 to .865, whereas the addition of UROUND did not appreciably increase R2 values for the same models. Adding URUMP and UROUND to the model of FINALWT, UREA, and UFAT to predict kilograms and the percentage of trimmable fat increased R2 values from .530 to .610 and from .254 to .360, respectively. Models using live-animal measurements to predict weight and the percentage of retail product gave R2 values equal to models using the actual measurements found in the USDA Yield Grade equation.
The labial melanotic macule: a review of 79 cases.
Seventy-nine patients presented to the pigmented lesion clinic between 1983 and 1996 with labial melanotic macules. We have followed up these patients for up to 13 years and 3 months (mean 6 years, 3 months). We present evidence that this is a benign entity. The appropriate management is reassurance and discharge, with the advice to return only if the lesion grows or darkens. This did not occur in any of our patients during the time of their follow-up.
Diagnosis of lysosomal storage disorders: evaluation of lysosome-associated membrane protein LAMP-1 as a diagnostic marker.
Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathologies, will be a key factor in the development of effective therapies for many of these disorders. Newborn screening offers a potential mechanism for the early detection of these disorders. From studies of both normal and LSD-affected human skin fibroblasts we identified the lysosome-associated membrane protein LAMP-1 as a potential diagnostic marker. We have developed a sensitive method for the quantification of this protein with a time-resolved fluorescence immunoassay. A soluble form of LAMP-1 was observed in plasma samples, and determination of 152 unaffected individuals gave a median value of 303 micrograms/L with the 5th and 95th percentile at 175 and 448 micrograms/L respectively. Plasma samples from 320 LSD-affected individuals representing 25 different disorders were assayed. We observed that 17 of the 25 disorder groups tested had > 88% of individuals above the 95th percentile of the control population, with 12 groups having 100% above the 95th percentile. Overall, 72% of patients had LAMP-1 concentrations above the 95th percentile of the unpartitioned control population. We suggest that LAMP-1 may be a useful marker in newborn screening for LSDs.
Tooth wear in orthodontically treated patients.
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IL-12 induces human T cells secreting IL-10 with IFN-gamma.
A clear differentiation of Th1 and Th2 cytokine-secreting subsets in humans has not yet been defined. To further examine cytokine-directed differentiation of human T cell responses to both exogenous and autoantigens, we generated 346 short term T cell lines at limiting dilutions from six normal individuals to tetanus toxoid and myelin basic protein in the presence of IL-2 with or without the addition of IL-12 and anti-IL-4 mAb. T cell lines were examined for [3H]thymidine incorporation and cytokine secretion of IFN-gamma, IL-4, and IL-10. After culture in the presence of IL-12 and anti-IL-4 mAb, the predominant T cell response to Ag stimulation was simultaneous secretion of IL-10 and IFN-gamma. The concomitant secretion of IL-10 and IFN-gamma by T cells was confirmed by stimulating lines in the absence of APCs with plate-bound anti-CD3 mAb after two rounds of Ag-specific stimulation. Moreover, IL-12 enhanced IL-10 and IFN-gamma production in a myelin basic protein-reactive T cell clone, demonstrating that a differentiated T cell clone could be induced to secrete both cytokines. The addition of a neutralizing anti-IFN-gamma Ab to cultures with IL-12 and anti-IL-4 mAb during the generation of tetanus toxoid-reactive lines had no effect on the induction of IL-10 and IFN-gamma secretion, indicating that IL-12 and not IFN-gamma was responsible for the induction of this subset of T cells. Thus, in human T cells, IL-12 induces concomitant secretion of IL-10 and IFN-gamma.
Expression and characterization of cysteine-modified variants of an amino-terminal fragment of bactericidal/permeability-increasing protein.
rBPI23 is a biologically active, recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI). While rBPI23 is readily purified from culture supernatants of Chinese hamster ovary (CHO)-K1 transfectants, it is heterogeneous, consisting of monomer and disulfide-linked dimer, characteristics due presumably to the presence of three cysteines within the molecule. We have examined the role of these cysteines in rBPI23 expression, function, and dimer formation by mutating their codons to alanine (C132A), serine (C135S), or alanine (C175A) and expressing analogues of N-terminal fragments ("variants") lacking one, two, or all three cysteines in permanently transfected CHO-K1 cells. We also expressed a variant in which serine 18 was changed to cysteine (S18C), as found in both bovine and rabbit BPI. The C132A variant was readily secreted and purified as a homogeneous, stable monomeric protein species. The C135S and S18C variants were produced as mixtures of monomer and dimer; the C135S variant was poorly secreted, difficult to purify, and unstable on storage. In contrast, the C175A variant and those lacking any two or all three cysteines were expressed but not secreted. Purified rBPI23 and the C132A and S18C variants had comparable bactericidal and lipopolysaccharide (LPS) binding activities and were similarly effective at neutralizing LPS-induced tumor necrosis factor synthesis by THP-1 cells; the purified C135S variant lacked all activities. From these studies with CHO-K1 transfectants, we conclude that (i) cysteines 135 and 175 are both necessary for efficient secretion of a biologically active N-terminal BPI fragment, presumably through the formation of a disulfide bond, (ii) cysteine 132 is responsible for dimer formation, and (iii) only the C132A modification yields a stable, biologically active, N-terminal BPI fragment (designated rBPI21) that is free of dimeric species.
Comment on condylar movement and mandibular rotation during jaw opening.
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The effect of prostatectomy on symptom severity and quality of life.
OBJECTIVE: To determine the effectiveness of prostatectomy in reducing symptom severity and bother and in improving disease-specific and general quality of life. PATIENTS AND METHODS: A prospective, cohort study was performed in National Health Service and private hospitals in the Northern, Wessex, Mersey, and South-West Thames Health Regions which comprised 5276 men undergoing prostatectomy recruited by 101 of the 106 (96%) surgeons (specialist and non-specialist) performing prostatectomy during a 6-month period. Patients were assessed using the American Urological Association (AUA) Symptom Index Score, the AUA symptom bother score, disease-specific and generic quality-of-life scores, the occurrence of adverse events (urinary incontinence, erectile impotence and retrograde ejaculation) and three global (general) questions on the results of their treatment. The outcome was assessed 3 months after surgery. RESULTS: Prostatectomy was effective in reducing both symptoms (initial mean score 20.1 reduced to 7.4, P < 0.001) and symptom bother (initial mean score 14.4 reduced to 4.3, P < 0.001). Not all men experienced a good reduction in symptoms; 121 (3.9%) were worse, 301 (9.6%) were the same, and 721 (23%) experienced only slight improvement. The type of operation, grade of principal operator and use of pre-operative investigations were not associated with the extent of symptomatic improvement. Changes in symptom severity were highly correlated with changes in bothersomeness++ and disease-specific quality of life but not with generic quality of life. A third of men who were continent before surgery reported some incontinence 3 months later, although only 6% found it a problem. Two-thirds of men experienced retrograde ejaculation and 31% experienced some erectile impotence following surgery. CONCLUSIONS: Prostatectomy is effective in reducing symptoms in most men. Men who experience a substantial reduction in symptoms were more likely to report a favourable outcome. The study confirmed that approximately one-third of men reported an unfavourable result 3 months after their operation.
Cost of treating an infected total knee replacement.
Twenty consecutive cases of infected total knee replacements surgically treated between 1990 and 1993 in which complete clinical and financial data were available were reviewed to determine the resources required of the hospital and the surgeon. These results were compared with a similar analysis of a stratified, unselected sample 30 cases each of primary total knee arthroplasty and nonseptic revision total knee arthroplasty during the same period. Parameters compared between the infected total knee replacement, noninfected revision total knee replacement, and primary total knee replacement included number of surgical procedures performed, number of hospitalizations, number of hospital days, total operative time, and total intraoperative blood loss. Financial data included the amount charged and reimbursed by the hospital and by the surgeon. The actual hospital cost was estimated as well. Based on all parameters measured, surgical treatment of the infected total knee implant required 3 to 4 times the resources of the hospital and the surgeon compared with a primary total knee implant and approximately twice the resources of a nonseptic revision total knee implant. The reimbursement received resulted in an estimated net loss of approximately $15,000 per case to the hospital for the group as a whole, but approximately $30,000 per case per Medicare patient.
Peptide synthesis on chitin.
The use of chitin as a support for solid-phase peptide synthesis is described and illustrated by synthesis of four peptides, varying in length from 10 to 29 residues. Syntheses were performed in a continuous-flow peptide synthesizer, using Fmoc chemistry. A cleavable linker, p-[(R,S)-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-di methoxybenzyl]- phenoxyacetic acid, was attached to chitosan at the desired substitution level, and the complex acetylated to yield a linker substituted chitin. The effects of temperature, solvents and degree of linker substitution on the syntheses were studied. Acyl carrier peptide (ACP) synthesis studies indicated that temperature was the single most important parameter. Increasing the temperature of the synthesis from 20 to 55 degrees C resulted in an enormous improvement of this synthesis, with about 90% of the crude product being the correct peptide. Denaturing solvents, such as DMSO, could be used without significant effect on the flow properties of the support. The synthesis of one peptide was mainly improved by lowering the degree of substitution from 0.3 to 0.1 mmol/g, suggesting peptide aggregation was a problem in this case. The results of three syntheses on chitin were comparable with those obtained with a commonly used commercial support. This work shows that, under appropriate conditions, chitin can be utilized directly as a support for peptide synthesis.
T cell-targeted immunofusion proteins from Escherichia coli.
Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.
Bridging scientist and practitioner perspectives in clinical psychology.
Literature suggests that a complex and often hostile relationship exists between the science and practice of clinical psychology. Contributors to this conflict of viewpoints are reconsidered within the proposition that there are different roads to discovery and that there may be good reasons to keep the science and practice of clinical psychology somewhat separate. Results of a national survey of 325 psychologists are reviewed that support the view that psychological practitioners value research and consider their practices to be augmented by scientific findings. However, they are in need of vehicles of communication that will help them translate scientific findings into practice. Results suggest that practitioners do more to understand scientific findings than scientists do to understand the problems that face clinical practitioners. Ways to facilitate communication between and among these groups are considered.