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D J Johnson

Publications and source records attributed to D J Johnson.

At least 19 recordsLinked to original sources
PubMed2003

Characterizing the influence of structure and route of exposure on the disposition of dithiocarbamates using toluene-3,4-dithiol analysis of blood and urinary carbon disulfide metabolites.

Differences in the toxicities observed for dithiocarbamates have been proposed to result from the influence of nitrogen substitution, oxidation state, and route of exposure. To better characterize the fate of dithiocarbmates in vivoas a function of structure and route of exposure, rats were administered equimolar doses of carbon disulfide (CS2), N-methyldithiocarbamate, pyrrolidine dithiocarbamate, N,N-diethyldithiocarbamate, or disulfiram daily for five days, either po or ip, and sequential blood samples obtained. Protein dithiocarbamates formed by the in vivo release of CS2, parent dithiocarbamate, and protein-bound mixed disulfides were assessed in plasma and hemolysate by measuring toluene trithiocarbonate generated upon treatment with toluene-3, 4-dithiol (TdT). To aid in determining the bioavailability of CS2 from the administered dithiocarbamates, the urinary CS2 metabolites, 2-thiothiazolidine-4-carboxylic acid (TTCA) and 2-thiothiazolidin-4-ylcarbonylglycine (TTCG), were also determined. The levels of TdT-reactive moieties detected depended upon both the compound administered and the route of exposure. Parent dithiocarbamates, with the exception of disulfiram, were eliminated from blood within 24 h; but protein associated TdT-reactive moieties persisted and accumulated with repeated exposure, regardless of the route of exposure. N-Methyldithiocarbamate demonstrated the greatest potential to produce intracellular globin modifications, presumably through its unique ability to generate a methylisothiocyanate metabolite. Urinary excretion of TTCA and TTCG was more sensitive than TdT analysis for detecting dithiocarbamate exposure, but TdT analysis appeared to be a better indicator of in vivo release of CS2 by dithiocarbamates than were urinary CS2 metabolites. These data suggest that CS2 is a more important metabolite, following oral exposure, than are other routes of exposure, e.g., inhalation or dermal. In addition, data also suggest that acid stability, nitrogen substitution, and route of exposure are important factors governing the toxicity observed for a particular dithiocarbamate.

Administration, Oral↗
PubMed2001

Can costs be measured and predicted by modeling within a cooperative clinical trials group? Economic methodologic pilot studies of the radiation therapy oncology group (RTOG) studies 90-03 and 91-04.

PURPOSE: To (1) measure radiation therapy costs for patients in randomized controlled clinical trials, (2) compare measured costs to modeling predictions, (3) examine cost distributions, and (4) assess feasibility of collecting economic data within a cooperative group. METHODS: The Radiation Therapy Oncology Group conducted economic pilot studies for two Phase III studies that compared fractionation patterns. Expected quantities of Current Procedural Terminology (CPT) codes and relative value units (RVU) were modeled. Institutions retrospectively provided procedure codes, quantities, and components, which were converted to RVUs used for Medicare payments. Cases were included if the radiation therapy quality control review judged them to have been treated per protocol or with minor variation. Cases were excluded if economic quality review found incomplete economic data. RESULTS: The median and mean RVUs were within the range predicted by the model for all arms of one study and above the predicted range for the other study. CONCLUSION: The model predicted resource use well for patients who completed treatment per protocol. Actual economic data can be collected for critical cost items. Some institutions experienced difficulty collecting retrospective data, and prospective collection of data is likely to allow wider participation in future Radiation Therapy Oncology Group economic studies.

Brain Neoplasms↗
PubMed2001

Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM(-)) and G1948D (CRM(r)).

In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM(-) and CRM(r) variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.

Animals↗
PubMed2000

Bcl-2 accelerates multistep prostate carcinogenesis in vivo.

The impact of bcl-2 proto-oncogene expression on the pathogenesis and progression of prostate cancer was examined in a transgenic mouse model. Probasin-bcl-2 transgenic mice were crossed with TRAMP (TRansgenic Adenocarcinoma Mouse Prostate) mice that express the SV40 early genes (T/t antigens) under probasin control. Prostate size, cell proliferation, apoptosis, and the incidence and latency of tumor formation were evaluated. The double transgenic, probasin-bcl-2 X TRAMP F1 (BxT) mice exhibited an increase in the wet weight of the prostate. This was associated with an increase in proliferation, attributable to T/t antigens, and a decrease in apoptosis attributable to bcl-2. The latency to tumor formation was also decreased in the BxT mice compared to the TRAMP mice. The incidence of metastases was identical in both the TRAMP and BxT mice. Lastly, the incidence of hormone-independent prostate cancer was reduced in the BxT mice compared to the TRAMP mice. Together, these results demonstrate that bcl-2 can facilitate multistep prostate carcinogenesis in vivo.

Adenocarcinoma↗
PubMed2000

Validation of a reverse transcriptase multiplex PCR test for the serotype determination of U.S. isolates of bluetongue virus.

Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants primarily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemiology of the virus, but current methods are cumbersome. A single-tube multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previously developed for the serotype determination of U.S. BTV isolates, was evaluated. The determination of serotype was based on the size of the resultant amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a closely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates originating from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of the isolates were determined by both a virus neutralization (VN) procedure and the mRT-PCR. Comparison of the mRT-PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and was shown to be more reliable and specific than the VN assay.

Animals↗
PubMed2000

The impact of bcl-2 expression and bax deficiency on prostate homeostasis in vivo.

Prostatic glandular epithelial cells undergo apoptosis in response to androgen-deprivation. The molecular determinants of androgen-responsiveness in these cells are incompletely understood. Recent evidence suggests that bcl-2 gene family members may be important in this context. We used the probasin promoter to target a human bcl-2 transgene specifically to the prostate in order to assess its impact on conferring resistance to androgen withdrawal in, otherwise sensitive, prostatic glandular epithelial cells in vivo. We examined the contribution of bax to mediating androgen-responsiveness in prostatic glandular epithelial cells using bax knockout mice. The histologic appearance of the prostates from probasin-bcl-2 transgenic mice or bax-/- mice did not differ from those of control littermates. There was no evidence of hyperplastic or neoplastic growth. There was no difference between probasin bcl-2 transgenic mice, bax-/- mice, and control littermates in steady-state levels of apoptosis. Following castration our findings suggest that both bax and bcl-2 may each contribute to the androgen-responsiveness of prostatic glandular epithelial cells. It is apparent from these results, however, that bax is not required to mediate cell death in prostatic glandular epithelial cells following castration. A comparison between the apoptotic indices in the ventral prostate from the probasin-bcl-2 and bax-/- mice following castration suggests that the presence of bcl-2 may be a more important indicator of androgen-sensitivity than a deficiency of bax.

Androgen-Binding Protein↗
PubMed2000

Routine, single-item screening to identify abusive relationships in women.

BACKGROUND: Abusive relationships are associated with several demographic factors and many clinical problems in women. However, practices often do not screen for abuse. METHODS: This is a descriptive study of 1526 women aged 19 to 69 years who completed a health survey in 31 office practices. The 53-item survey included a question designed to screen for an abusive relationship. Our analysis compared self-reported measures of symptoms (N = 13) and functional limitations (n = 6) of women who had abusive relationships with those who did not. We also examined the utility of using a constellation of clinical problems to identify risk for abuse. RESULTS: Women in abusive relationships were more likely to be poor (37% vs 14%; P < .001) and young (87% were younger than 51 years versus 69% of those who were not in such relationships; P < .001). They had twice as many bothersome symptoms (3.1 vs 1.7; P < .001) and functional problems (1.6 vs 0.8; P < .001). Approximately 40% (36/89) of low-income women with emotional problems were at risk for abuse versus only 6% (64/1025) of women with adequate financial resources and no emotional problems. However, because so many women were at low risk, almost twice as many in this group (n = 64) reported abusive relationships than in the high-risk group (n = 36). CONCLUSIONS: Women in abusive relationships have many symptoms and functional limitations. However, symptoms and clinical problems provide insufficient clues for abuse. It is better just to ask. A single-item screening question appears adequate for this purpose.

Adult↗
PubMed1999

Crystallization and preliminary X-ray analysis of active site-inhibited human coagulation factor VIIa (des-Gla).

Human coagulation factor VIIai that lacks the Gla domain (residues 1-44) has been prepared, purified, and crystallised. First, recombinant factor VII was activated to form factor VIIa, the active site was then inhibited with 1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone, and finally the Gla domain was removed by chymotryptic digestion, yielding factor VIIai (des-Gla). After further purification single crystals suitable for x-ray analysis were obtained by vapour diffusion. Crystals of factor VIIai (des-Gla) belong to the tetragonal space group P41212 or P43212 with unit cell dimensions a = b = 94.85 A, c = 114.30 A, contain one molecule per asymmetric unit, and diffract to 2.3-A resolution when exposed to synchrotron radiation.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Crystal structure of active site-inhibited human coagulation factor VIIa (des-Gla).

Factor VIIa (FVIIa) is a crucial haemostatic protease consisting of four distinct domains termed the Gla, epidermal growth factor-1 (EGF-1), EGF-2, and protease domains (from N- to C-terminus). The crystal structure of human FVIIa inhibited at the active site with 1, 5-dansyl-Glu-Gly-Arg-chloromethyl ketone and lacking the Gla domain has been solved to a resolution of 2.28 A. The EGF-2 and protease domains were well resolved, whereas no electron density for the EGF-1 domain was observed, suggesting a flexible arrangement or disorder within the crystal. Superposition of the protease domain of the present structure with that previously resolved in the tissue factor (TF)/FVIIai complex revealed that although overall the domain structures are similar, the EGF-2 domain is rotated by 7.5 degrees relative to the protease domain on binding TF. A single cleavage in the protease domain was found, between Arg315 and Lys316 (chymotrypsin numbering 170C-170D) in a FVII-specific insertion loop: this cleavage appeared to be essential for crystallisation. Insertion of the heavy chain N-terminal Ile153 is essentially identical in the two structures, as is the geometry of the active site residues and the inhibitor C-terminal arginine residue. Some differences are seen in the cleaved loop, but changes in TF-contact residues are generally minor. This structure supports the hypothesis that TF binding enables spatial domain arrangements in the flexible FVIIa molecule necessary for procoagulant function and furthermore that active site occupancy induces FVIIa active conformation via N-terminal insertion.

Amino Acid Sequence↗
PubMed1999

Streamlining nutritional care for the physician's office.

OBJECTIVE: Nutritional care needs are overlooked in clinical practice. We review nutritional needs and describe an approach for improving nutritional care in clinical practice. DESIGN: Data from a controlled trial and several population cohorts. SETTING: Primary care practices and a population survey in New Hampshire and Vermont, USA. SUBJECTS: The controlled trial involved 1651 persons aged 70+years. The cohorts include information from 1879 persons aged 12+. INTERVENTION: All patients completed standard surveys which included information about nutritional needs. 22 practices participated in the trial. RESULTS: The higher the BMI, the less healthy the population. 15 30% of patients report problems or concerns with eating/weight and nutrition. Patients with problems or concerns are often bothered by other health and social problems. Patients who have productive interactions with clinicians have improved nutritional care and are more likely to report help with eating problems (68% vs 86%; Odds ratio 5.0 (95% CI: 0.9-27.0). CONCLUSIONS: Nutritional issues are common and complex. A productive provider-patient interaction can improve the nutritional care of patients. Essential elements for a productive interaction include an informed, educated patient and a provider (or clinical team) prepared to assess and manage the broad range of issues that are important to the patient. Technology facilitates necessary feedback between patient and provider.

Adolescent↗
PubMed1999

Building an idealized measurement system to improve clinical office practice performance.

The aim of this article is to introduce key concepts and approaches for building a better practice-based measurement. Physicians are being challenged to produce measurably higher quality of services, lower costs, and better clinical outcomes to remain viable. In the absence of provider-driven practice improvements and independent measurement systems, office practices will remain dependent on performance data from external forces as drivers of change. Three key questions will be addressed in our pursuit of better measurement systems for continuous improvement and competitive advantage: (1) What do we want a measurement system to tell us in the first place? (2) What might an idealized measurement system look like if we had one? (3) What are some of the challenges that office practices face in closing the gap between building an idealized measurement system and the current state of office practice measurement?

Data Collection↗
PubMed1998

Development of a head and neck companion module for the quality of life-radiation therapy instrument (QOL-RTI)

PURPOSE/OBJECTIVE: A review of available head and neck quality of life (QOL) instruments reveals them to inadequately address important radiation related side effects, or to be too cumbersome for routine use. The purpose of this study was to develop a head and neck disease specific module as a companion to the previously developed quality of life - radiation therapy instrument (QOL-RTI). The goal was to create a more complete, yet concise, head and neck site-specific module geared toward patients receiving radiation therapy for head and neck cancer. METHODS AND MATERIALS: This exploratory study included 34 consecutive patients undergoing definitive radiotherapy over a 6-7 week course (60-79.8 Gy). We developed and administered a 14-item questionnaire to all eligible patients treated with radiotherapy for head and neck cancer who were not already registered in another research study assessing quality of life (e.g., RTOG). During the treatment period, the QOL-RTI general tool and the head and neck (H&N) module were administered as follows: at baseline, at week four (for test-retest), and at the end of the treatment period. For validation purposes the QOL-RTI/H&N was compared to the functional assessment cancer tool head and neck (FACT-H&N) questionnaire. The FACT-H&N was administered one time at week 4, on the same day as the QOL-RTI/H&N. This report includes the treatment phase of the study (during the course of radiation). RESULTS: Mean age was 62 years (range 40-75). Internal consistency of the module was satisfactory (Chronbach's alpha = 0.85). Test-retest yielded a correlation coefficient of 0.90 (p < 0.001). Concurrent validity, established by comparing the module to the FACT/H&N , yielded a correlation coefficient of 0.85. Significant changes in quality of life scores during a course of radiation was noted for both general quality of life tool and the site specific module. For the head and neck module, the difference in the mean baseline (7.17) and end of treatment scores (4.20) was 2.94, or 42% change (p < 0.0001). A smaller, yet still significant, difference in scores was seen in the general QOL tool (22 % change, p = 0.001). Item analysis of the module revealed statistically significant (p < 0.05) worsening in quality of life scores in the following areas: pain in throat, swallowing difficulty (meat/bread and liquids), changes in mucous and saliva, changes in taste, difficulty chewing, trouble with coughing, and speech difficulties. Items that were not significant were pain in the mouth, and appearance. CONCLUSION: These initial results suggest that the H&N companion module to the QOL-RTI is a valid and reliable tool that is responsive to changes in QOL during a course of H&N radiation therapy. This tool differs from existing H&N tools by including specific assessments of mucous, saliva, taste, cough, and local pain in a concise format. Significant changes in QOL scores were noted in all of these items. Evaluation of the tool in the post-treatment period (follow-up) is ongoing.

Adult↗
PubMed1998

The solution structure of human coagulation factor VIIa in its complex with tissue factor is similar to free factor VIIa: a study of a heterodimeric receptor-ligand complex by X-ray and neutron scattering and computational modeling.

Factor VIIa (FVIIa) is a soluble four-domain plasma serine protease coagulation factor that forms a tight complex with the two extracellular domains of the transmembrane protein tissue factor in the initiating step of blood coagulation. To date, there is no crystal structure for free FVIIa. X-ray and neutron scattering data in solution for free FVIIa and the complex between FVIIa and soluble tissue factor (sTF) had been obtained for comparison with crystal structures of the FVIIa-sTF complex and of free factor IXa (FIXa). The solution structure of free FVIIa as derived from scattering data is consistent with the extended domain arrangement of FVIIa seen in the crystal structure of its complex with sTF, but is incompatible with the bent, less extended domain conformation seen in the FIXa crystal structure. The FVIIa scattering curve is also compatible with a subset of 317 possible extended structures derived from a constrained automated conformational search of 15 625 FVIIa domain models. Thus, the scattering data support extended domain models for FVIIa free in solution. Similar analyses showed that the solution scattering derived and crystal structures of the FVIIa-sTF complex were in good agreement. An automated constrained search for allowed structures for the complex in solution based on scattering curves showed that only a small family of compact models gave good agreement, namely those in which FVIIa and sTF interact closely over a large surface area. The general utility of this approach for structural analysis of heterodimeric complexes in solution is discussed. Analytical ultracentrifugation data and the modeling of these data were consistent with the scattering results. It is concluded that in solution FVIIa has an extended or elongated domain structure, which allows rapid interaction with sTF over a large surface area to form a high-affinity complex.

Amino Acid Sequence↗
PubMed1998

Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function.

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

Amino Acid Substitution↗
PubMed1998

Release of carbon disulfide is a contributing mechanism in the axonopathy produced by N,N-diethyldithiocarbamate.

The neurotoxicity of N,N-diethyldithiocarbamate (DEDC) is established, although the mechanisms responsible for its neurotoxicity are not. Previous experiments have demonstrated that DEDC has the ability to produce CS2-mediated protein cross-linking in vitro and that DEDC releases CS2 in vivo. The release of CS2 with subsequent cross-linking of proteins presents a potential mechanism through which DEDC may exert its neurotoxicity. In the present study DEDC (3 mmol/kg po) was given to rats every other day for 8 and 16 weeks. At the end of each treatment period, erythrocyte spectrin, hemoglobin, and spinal cord neurofilament preparations were isolated and examined for cross-linking using polyacrylamide gel electrophoresis, reverse phase HPLC, and Western blot techniques, respectively. Additional rats were perfused and sections of the lumbar and cervical spinal cord and the muscular branch of the posterior tibial nerve were removed and examined by light and electron microscopy. Relative to controls, significant levels of cross-linking were observed in all the proteins examined at both 8 and 16 weeks of treatment. Morphological changes were not detected at 8 weeks, but at 16 weeks degenerated and swollen axons filled with disorganized masses of neurofilaments were present in the distal regions of the long tracts of the lumbar and cervical spinal cord and also in the muscular branch of the posterior tibial nerve. The ability of DEDC to covalently cross-link proteins in vivo and to produce axonal structural changes identical to those produced by CS2 is consistent with release of CS2 from DEDC being a contributing mechanism in DEDC-induced neurotoxicity.

Animals↗
PubMed1998

A computerized school-based health assessment with rapid feedback to improve adolescent health.

Adolescent health problems are often undetected in physicians' offices. The Dartmouth Primary Care Cooperative Information Project has developed a validated and reliable approach to identify adolescent health problems and initiate education in a school setting. A self-administered, anonymous, 26-item questionnaire was given to 204 students in a rural high school. Responses were scanned into PC-based software. Within one working day students were given individualized letters identifying their problem health issues as detected by the questionnaire and recommendations for education. Ninety-nine percent of students participated. Six weeks later 49% of a sample of 41 students reported reading the information and 50% planned to change behavior. This standardized, validated strategy of adolescent health assessment, feedback, and education was feasible for use in schools. The school responded to the data by employing a psychologist to address mental health needs.

Adolescent↗