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J King

Publications and source records attributed to J King.

At least 145 records · Page 8Linked to original sources
PubMed1997

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.

Antibodies, Monoclonal↗
PubMed1997

Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.

Human nasopharyngeal carriage of Streptococcus pneumoniae constitutes the major natural reservoir of pneumococci and is thought to be the prelude to virtually all pneumococcal disease. If carriage could be greatly reduced, pneumococcal transmission and disease could be largely eliminated. To facilitate the studies of mechanisms important in carriage and to identify immunogens that can elicit protection against carriage, we characterized an adult mouse model of nasopharyngeal carriage. Non-anaesthetized mice were inoculated intranasally with pneumococci in 10 microl of fluid. Nasopharyngeal carriage was observed with strains of capsular types 3, 4, 6A, 6B, 14, 19, and 23. Carriage was stable over time, and the numbers of pneumococci carried were relatively independent of inoculation dose; findings which indicate that the recovery of pneumococci from 1 day to 2 weeks post inoculation was dependent on colonization, rather than just temporary contamination. To ensure carriage in the largest percentage of mice, without causing sepsis or death, inoculations of 10(7) colony forming units (cfu) should be used. In this model, carriage was generally observed without concomitant bacteremia or sepsis and carriage was observed even with strains that were avirulent when injected i.v. The model should be useful for the identification of protection-eliciting antigens, since intranasal immunization with heat-killed pneumococci or lysates of pneumococci protected against carriage.

Animals↗
PubMed1997

Arabidopsis dihydropteroate synthase: general properties and inhibition by reaction product and sulfonamides.

Dihydropteroate synthase (DHPS) (EC 2.5.1.15) was extracted from leaves of Arabidopsis thaliana and purified 21-fold by ion-exchange chromatography. This enzyme preparation was then characterized for several of its properties. Michaelis-Menten constants for the substrates, p-aminobenzoic acid and dihydropteridine diphosphate were estimated to be 2.5 and 91 microM, respectively. In an optimized assay, the reaction product, dihydropteroic acid, competitively inhibited the enzyme activity with a Ki of 81 microM. However, neither dihydropteroic acid nor tetrahydrofolate, products further downstream in the biosynthetic pathway inhibited enzymatic activity. This appears to be the first report of product inhibition of DHPS from a higher plant. The relative inhibitory properties of several sulfonamides, analogues of p-aminobenzoic acid, were also examined. The substitutions on the amide nitrogen of the sulfonamides influenced the degree of inhibition; thus I50 values for the inhibition of the DHPS activity by sulfanilamide, sulfacetamide and sulfadiazine were estimated to be 18.6, 9.6 and 4.2 microM, respectively. The competitive pattern of inhibition was shown in experiments with sulfadiazine.

Arabidopsis↗
PubMed1997

Disulphide-bonded intermediate on the folding and assembly pathway of a non-disulphide bonded protein.

The trimeric parallel beta-coil P22 tailspike contains eight cysteines per chain, but lacks disulphide bonds in the native state, in both the crystalline and solution forms. However, cysteines in a folding intermediate are reactive with thiol blocking reagents, which prevent further productive folding both in vivo and in vitro. The in vivo refolding yield was independent of the availability of metal ions, but was sensitive to redox potential. Isolation by nondenaturing gel electrophoresis of the protrimer intermediate, a trimeric folding intermediate that precedes the fully folded trimer in the in vivo and in vitro pathways, revealed the presence of interchain disulphide bonds. Incubation of the isolated protrimer with reducing agents generated the native trimer. The formation of beta-sheets with interdigitated strands from different subunits in the native trimer may require the transient disulphide bonds for proper alignment. To our knowledge this is the first report of a disulphide bond present in a folding intermediate of a non-disulphide bonded protein.

Cysteine↗
PubMed1997

Use of a new vitamin C-deficient diet in a depletion/repletion clinical trial.

To conduct an inpatient study on the recommended dietary allowance (RDA) for vitamin C, we developed a unique vitamin C-deficient diet using a nutrient database and selective menus. Fourteen different menus were developed offering > 300 items with 0-2.4 mg vitamin C per serving. During the 4-6 mo volunteers were hospitalized, daily dietary vitamin C was restricted to < or = 5.0 mg. The mean daily dietary vitamin C intake was < 3.9 mg for the seven study subjects. With concurrent supplementation, the diet provided > or = 85% of the RDA for 17 essential nutrients. Within 3 wk of admission the diet induced vitamin C deficiency as indicated by plasma concentrations, which decreased from 23 +/- 6.9 to 6.9 +/- 2.0 mumol/L. Daily intake of vitamin C and five other nutrients was determined by nutrient database analyses. Mean energy, protein, the iron were 105-185% of the RDA and total and saturated fat were 32% and 10% of energy, respectively. Weight and nutritionally relevant indexes remained normal. Dietary adherence, calculated by the number of days with < or = 5.0 mg vitamin C per total study days, was 88-98% per repletion dose. Computer analyses of menu selections permitted individual preferences to be met while restricting vitamin C intake to < or = 5.0 mg/d. There were no complications from the diet during the depletion and repletion phase. With this diet, ascorbic acid pharmacokinetics for escalating doses could be determined in healthy volunteers.

Ascorbic Acid↗
PubMed1997

The use of the bladder neck support prosthesis in combined genuine stress incontinence and detrusor instability.

The Bladder Neck Support Prosthesis (BNSP) was used in 21 women with combined genuine stress incontinence (GSI) and detrusor instability (DI). Outcomes included frequency volume charts, pad tests, voiding cystometry and quality of life scores, up to the sixth month. Of the 21 recruits, 5 never wore the BNSP home, leaving 16 participants. A further 2 did not reach week 4 because of poor efficacy or inability to fit the device. In the 14 who reached week 4, the median number of leaks/day declined from 4.3 to 1.0 (p = 0.002). Median pad test loss fell from 53 to 7 mL (p = 0.012). Cystometry showed an increase in maximum bladder capacity (p < 0.05) and a modest reduction in severity of detrusor instability, with no evidence of outflow obstruction. Three further women discontinued because of poor efficacy (2) or a poorly fitting device (1), leaving 11 of 16 participants (69%) at week 8, when median pad test loss fell to 2 mL. The BNSP is a useful option in patients with the unfortunate combination of an unstable bladder and an incompetent urethra, but requires careful fitting and attention to detail.

Aged↗
PubMed1997

Semen characteristics in HIV-1 positive men and the effect of semen washing.

We have undertaken an analysis of semen from HIV infected men with regard to sperm counts and motility, non-spermatozoal cells, and viral nucleic acid. Regression analysis showed that sperm concentration and motility were positively associated with blood CD4 cell count. By contrast, non-spermatozoal cell concentration (round cells) was inversely related to CD4 count. Extracellular HIV RNA was detected in the majority of semen samples and proviral DNA in a minority. Percoll gradient washing of 12 semen samples yielded six samples containing adequate sperm concentration for analysis. This washing procedure reduced prewash extracellular RNA to below detectable limits in all cases; proviral DNA present in two of the six prewash samples was also reduced to below detectable limits after washing. We conclude that semen washing before artificial insemination may reduce the risk of HIV transmission from an infected man to an uninfected woman. However, further evidence from prospective analyses of such an approach is required.

CD4 Lymphocyte Count↗
PubMed1997

The effect of prepubertal diabetes duration on diabetes. Microvascular complications in early and late adolescence.

OBJECTIVE: To define the significance of prepubertal diabetes duration in the development of diabetic microvascular complications in adolescents. RESEARCH DESIGN AND METHODS: Study A compares complications in 38 prepubertal (PreP) and 140 pubertal (Pub) subjects of the same age (10-14 years) and diabetes duration (3-12 years) to determine if the absence of puberty itself confers a lower risk of complications. Study B examines the importance of prepubertal and pubertal diabetes duration in 193 older adolescents (ages 15-22 years) with prepubertal onset of diabetes. Retinopathy status was assessed using stereoscopic fundus photography of seven fields per eye. Albumin excretion rate (AER) was assessed by three consecutive overnight urine collections, using a polyclonal radioimmunoassay. RESULTS: In study A, there were no significant differences between the PreP and Pub groups for retinopathy (27 vs. 29%, P = 0.8) or differences in elevated AER (17 vs. 31%, P = 0.1). In study B, longer prepubertal diabetes duration improved the prediction for retinopathy over postpubertal duration alone (P < 0.0005). No relationship with duration was found for elevated AER (> 7.5, > 15, and > 30 micrograms/min). CONCLUSIONS: Prepubertal subjects with diabetes did not have less retinopathy or elevated albumin excretion compared with pubertal subjects of the same age. Prepubertal diabetes duration is significantly related to the presence of retinopathy in adolescents.

Adolescent↗
PubMed1997

Integrated primary health care. Opportunities for psychiatric-mental health nurses in a reforming health care system.

As changes in our health care system evolve, making the transition to greater parity of mental and physical health is paramount to move health care in the direction of prevention and health promotion. Although parity is the goal, the most feasible path to reaching it may lie in relinking mental health to physical health in managed care models of primary health care. This article identifies emerging directions in the mental health field and points to new opportunities for advancing the practice of psychiatric-mental health nurses.

Health Care Reform↗
PubMed1997

Advanced practice psychiatric-mental health nursing in a community-based nurse-managed primary care program.

In the evolving health care environment, advanced practice opportunities for psychiatric-mental health nurses may lie in managed care models of community oriented primary health care. As providers, care managers, health promoters and educators, and client advocates with family systems expertise, psychiatric-mental health advanced practice specialists are a necessary component of all primary health care. This article describes the development of a community-based primary care practice that integrates physical and mental health care on the same site.

Adult↗
PubMed1997

Computed tomography changes following cryotherapy for hepatic cancer.

Encouraging survival and tumour marker results have been described in patients where the focally destructive technique, hepatic cryotherapy, is used to treat primary and secondary hepatic malignancy. Radiology allows assessment of the cryotherapy procedure and follow-up treatment. This paper aims to review and describe the appearance of hepatic cryotherapy by CT.

Cryosurgery↗
PubMed1997

Ex vivo expansion of megakaryocyte progenitors: effect of various growth factor combinations on CD34+ progenitor cells from bone marrow and G-CSF-mobilized peripheral blood.

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34+ cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1); SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34+ cells yielded significantly higher numbers of CD41+ MK progenitors than BM CD34+ cells with any of the growth factor regimens assayed. PB CD34+ cells (2x10[5]) at day 0 generated 1.2 to 1.3x10(6) CD41+ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34+ cells produced 5x10(5) CD41+ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41+ cells were generated in cultures with combinations 2 and 3 (2.3x10[5] and 4.2x10[4], respectively). The addition of MGDF to cultures that were grown with combination 1 for 5 days increased the number of CD41+ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB-derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34+ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP+ cell expansion ex vivo, suggesting the feasibilty of MK expansion ex vivo in patients with prolonged thrombocytopenia posttransplantation. In summary, these data indicate that GCSF-mobilized CD34+ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34+ BMPCs. CD34+/GP+ MK progenitors may be an appropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

Adult↗
PubMed1997

High serum carcinoembryonic antigen concentration in patients with colorectal liver metastases is associated with poor cell-mediated immunity, which is predictive of survival.

BACKGROUND: Carcinoembryonic antigen (CEA) inhibits lymphocyte function and patients with cancer have lower cell-mediated immunity (CMI) than the normal population. To test this association an investigation was made of the relationship between CMI score, CEA level and survival. METHODS: CEA level, CMI score and other variables were compared with the survival time of 109 patients with colorectal liver metastases using Cox regression analysis. RESULTS: There was a significant association between CMI and CEA categories (P = 0.04, chi 2 test). The odds of patients with normal CMI having a CEA level in the upper quartile observed for all patients were 22 per cent of those of patients with depressed CMI (odds ratio 0.22 (95 per cent confidence interval (c.i.) 0.03-0.90), mid-P corrected). The median survival time of patients with normal CMI scores was 943 days compared with 488 days for those with depressed CMI (P = 0.03, log rank test; P = 0.04, Peto). The mortality risk of patients with normal CMI at entry to the study was, in the first 2 years of treatment, 40 per cent of that of patients with depressed CMI (95 per cent c.i. for relative risk 0.20-0.82, Cox regression). CONCLUSION: In patients with colorectal hepatic metastases, CMI is predictive of survival and a raised serum CEA level is associated with depressed CMI.

Adult↗
PubMed1996

Scaffolding mutants identifying domains required for P22 procapsid assembly and maturation.

Assembly of the icosahedral shells of the dsDNA bacteriophages, herpesviruses, and adenoviruses requires proteins not found in the mature virion, termed scaffolding proteins. The bacteriophage P22 precursor procapsid contains approximately 300 scaffolding molecules within a shell composed of 420 coat protein subunits. Though nonsense mutants are common, few mutants affecting the functions of the scaffolding protein have been recovered. We report here the isolation and characterization of new missense mutants unable to form infectious virions under restrictive conditions. These mutant scaffolding subunits were competent for protein folding and capsid assembly under restrictive conditions. Two mutants were defective in assembly into the procapsid of the portal complex, which serves as the channel through which DNA is packaged. These mutations may identify a region of the scaffolding protein required for interaction with the portal subunits. Two mutants in a different region of the sequence were impaired in scaffolding release from the procapsid both in vivo and in vitro. These mutations may identify a new domain required for scaffolding release. Scaffolding release appeared to be required for capsid expansion; in turn, scaffolding release seemed to depend upon the presence of a portal. This may help to order the pathway of events in phage maturation.

Bacteriophage P22↗
PubMed1996

Three-dimensional structure of scaffolding-containing phage p22 procapsids by electron cryo-microscopy.

The procapsids of bacterial viruses are the products of the polymerization of coat and scaffolding subunits, as well as the precursors in DNA packaging. Electron cryo-microscopy has been used to study the three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins. The scaffolding mutant structure has been resolved to 19 A resolution and agrees with the 22 A resolution wild-type procapsid reconstruction. Both procapsid reconstructions contain an outer icosahedral coat protein shell and an inner scaffolding protein core. The outer core protein forms a T = 7 icosahedral lattice with distinctive channels present at the centers of the pentons and hexons. In addition, the hexons display a prominent skew. Computational isolation of the skewed hexon shows the presence of a local 2-fold axis that reduces the number of unique conformations in the asymmetric unit to four at this resolution. We have classified the four unique subunits into three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface. In addition, at the inner surface of the coat protein, finger-like regions that extend towards the scaffolding protein core are present in two of the subunits. The finger-like regions suggest the presence of an ordered interaction between the inner coat protein and the scaffolding protein. However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry.

Bacteriophage P22↗
PubMed1996

Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of alpha-interferon-resistant or -intolerant patients with chronic myeloid leukemia.

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy-1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy-1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.

Adolescent↗