The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

Haplotype uncertainty in association studies.

Inferring haplotypes from genotype data is commonly undertaken in population genetic association studies. Within such studies the importance of accounting for uncertainty in the inference of haplotypes is well recognised. We investigate the effectiveness of correcting for uncertainty using simple methods based on the output provided by the PHASE haplotype inference methodology. In case-control analyses investigating non-Hodgkin lymphoma and haplotypes associated with immune regulation we find little effect of making adjustment for uncertainty in inferred haplotypes. Using simulation we introduce a higher degree of haplotype uncertainty than was present in our study data. The simulation represents two genetic loci, physically close on a chromosome, forming haplotypes. Considering a range of allele frequencies, degrees of linkage between the loci, and frequency of missing genotype data, we detail the characteristics of genetic regions which may be susceptible to the influence of haplotype uncertainty. Within our evaluation we find that bias is avoided by considering haplotype probabilities or using multiple imputation, provided that for each of these methods haplotypes are inferred separately for case and control populations; furthermore using multiple imputation provides the facility to incorporate haplotype uncertainty in the estimation of confidence intervals. We discuss the implications of our findings within the context of the complexity of haplotype inference for larger marker rich regions as would typically be encountered in genetic analyses.

Non-Hodgkin's lymphoma and family history of hematologic malignancy.

Familial aggregation of non-Hodgkin's lymphoma, and the co-occurrence of non-Hodgkin's lymphoma and other hematologic malignancies within families, provide evidence for genetic or common environmental etiologies for these conditions. The authors analyzed the association between non-Hodgkin's lymphoma risk and family history of hematologic malignancy using a case-control study based in the United Kingdom. The study recruited patients diagnosed with lymphoma during 1998-2001. Results indicated an increased risk of non-Hodgkin's lymphoma for persons with a positive family history of any hematologic malignancy (odds ratio = 1.70, 95% confidence interval: 1.08, 2.69) and particularly of any lymphoma (odds ratio = 2.43, 95% confidence interval: 1.14, 5.19). The authors compared the number of hematologic malignancies among relatives reported by the cases and controls with that expected from the national rates of hematologic malignancy registered in the United Kingdom. Through these comparisons, the authors raise questions about the validity of self-reported family history of hematologic malignancy, especially regarding identification of specific types of hematologic malignancies. Given these reservations, they consider how future epidemiologic studies may contribute to further understanding the role of familial susceptibility in non-Hodgkin's lymphoma.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

How complete has skin cancer registration been in the UK? A study from Yorkshire.

The completeness of skin cancer registration in the Yorkshire region was evaluated for the year 1994 by the independent case ascertainment method. Patients diagnosed with skin cancer were identified from regional pathology laboratories, inpatient and outpatient hospital departments and general practices, and were matched against records held by the Northern and Yorkshire Cancer Registry and Information Services (NYCRIS). Out of 5987 skin cancer cases identified from 14 pathology laboratories, 123 general practices, 16 NHS Trusts inpatient databases and 7 dermatology outpatient departments, 83.5% had a matching record on the Cancer Register. The proportion of registered malignant melanoma (MM) and non-melanoma skin cancer (NMSC) cases were 87.5% (95% confidence interval (CI) 84.0-90.4) and 83.1% (95% CI 81.9-84.2) respectively. Skin cancers found in the pathology laboratories, the main notification sources of the registry, were under-ascertained by 15% (10% MM and 15% NMSC). Cases identified from general practices had a significantly lower proportion of matching registry records in comparison with other information sources. No record of histological confirmation could be found for 11% MM and 13% NMSC. Complete capture of pathology laboratory information, histological confirmation of all lesions suspected of skin cancer and routine receipt of hospital patient administration system information supplementary to that from pathology laboratories are measures that would provide the most substantial improvement to ascertainment of skin cancer data.

Antibody against CD20 in patients with B cell malignancy.

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogren's syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.

Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

Influence of age on the disposition kinetics of chloramphenicol in equine neonates.

The effect of age on the pharmacokinetics of chloramphenicol was determined after IV administration of chloramphenicol sodium succinate (25 mg/kg of body weight) to 6 foals at 1 day and 3, 7, 14, and 42 days of age. The disposition of chloramphenicol was best described, using a two-compartment open model in all foals at all ages evaluated. Significant age-related changes were observed in values for the major kinetic terms describing the disposition of chloramphenicol in foals; the greatest changes were observed between 1 day and 3 days of age. The mean +/- SD value for elimination rate constant (beta) for chloramphenicol in 1-day-old foals (0.131 +/- 0.06 h-1) was significantly (P less than 0.005) lower than the value in 3-day-old foals (0.514 +/- 0.156 h-1), and both values were significantly (P less than 0.05) lower than values for beta in 7-, 14-, and 42-day-old foals. With increasing age, the increase in the mean value for beta resulted in decrease in the harmonic mean elimination half-time (t1/2 beta) for chloramphenicol, from 5.29 hours in 1-day-old foals to: 1.35 hours in 3-day-old foals; 0.61 hour in 7-day-old foals; 0.51 hour in 14-day-old foals; and 0.34 hour in 42-day-old foals. At 1, 3, and 7 days of age, values for t1/2 beta of chloramphenicol in a premature foal born after parturition was induced with oxytocin, were considerably longer than comparable t1/2 beta values for term foals born naturally.(ABSTRACT TRUNCATED AT 250 WORDS)

Ormetoprim-sulfadimethoxine in cattle: pharmacokinetics, bioavailability, distribution to the tears, and in vitro activity against Moraxella bovis.

The pharmacokinetics, bioavailability, and distribution to the tears of ormetoprim (OMP; 5.5 mg/kg of body weight) and sulfadimethoxine (SDM; 27.5 mg/kg of body weight) were determined following IV or oral administration to 6 Holstein steers. After IV administration, the disposition kinetics of both drugs were best described by a 2-compartment open model. Sulfadimethoxine had a moderately rapid distribution phase, followed by a slower elimination phase, with a mean half-life (t 1/2) of 7.91 hours. The mean volume of distribution of SDM was 185 ml/kg, and the mean body clearance was 0.28 ml/min X kg. The concentration of SDM in tears was lower than the corresponding plasma concentration, and the elimination of SDM from tears (t 1/2 = 3.02 hours) was significantly faster than its elimination from plasma (t 1/2 = 7.91 hours). The disposition of OMP administered IV was characterized by a rapid distribution phase, followed by a rapid elimination phase (t 1/2 = 1.37 hours). The high values of the mean volume of distribution (1,450 ml/kg) and mean rate of body clearance (13.71 ml/min X kg) indicated that OMP was widely distributed in the body and was rapidly cleared from the body. Ormetoprim concentrations in tears exceeded corresponding plasma concentrations, and the elimination of OMP from tears was significantly slower (t 1/2 = 1.91 hours) than from plasma (t 1/2 = 1.37 hours). After oral administration of an OMP-SDM combination in bolus form, the absorption of SDM was slow (absorption t 1/2 = 3.32 hours), but complete.(ABSTRACT TRUNCATED AT 250 WORDS)

Cefadroxil in the horse: pharmacokinetics and in vitro antibacterial activity.

Sodium cefadroxil was administered as a single intravenous dose (25 mg/kg) to six healthy adult mares. Plasma samples were collected over a 24-h period and cefadroxil concentrations were measured by microbiological assay. The pharmacokinetic behavior of the drug was appropriately described in terms of a one-compartment open model. Values for the major pharmacokinetic terms were: extrapolated initial plasma concentration = 59.2 +/- 15.0 micrograms/ml; half-life = 46 +/- 20 min; apparent volume of distribution = 462 +/- 191 ml/kg; and body clearance = 7.0 +/- 0.6 ml/min.kg. In a subsequent study, a suspension of cefadroxil monohydrate was administered intragastrically (25 mg/kg) to the same six horses. Plasma concentrations of the drug peaked at 1-2 h but, in general, absorption was both poor and inconsistent. The data were unsuitable for determination of cefadroxil bioavailability from this oral dosage form. Ninety-nine isolates of eleven bacterial species obtained from clinically ill horses were tested for susceptibility to cefadroxil. All strains of Streptococcus equi, Streptococcus zooepidemicus, coagulase-positive staphylococci, Corynebacterium pseudotuberculosis and five out of six strains of Actinobacillus suis were highly susceptible to the drug (MIC less than 4 micrograms/ml). Escherichia coli, Klebsiella pneumoniae and Salmonella sp. showed intermediate susceptibility (MIC 4-16 micrograms/ml), while all isolates of Corynebacterium (Rhodococcus) equi, Enterobacter cloacae and Pseudomonas aeruginosa proved to be highly resistant to cefadroxil (MIC greater than 128 micrograms/ml).

Ulcerative keratitis associated with Salmonella arizonae infection in a horse.

Salmonella arizonae 61:1,5, was isolated in pure culture from the eye of a horse with unilateral ulcerative keratitis. The eye responded well to treatment with atropine sulfate and polymyxin B-bacitracin-neomycin ophthalmic ointments. In swab specimens taken after the lesion had healed, Salmonella was not found to be a constituent of the bacterial flora of the horse's eyes.

Susceptibility of equine bacterial isolates to antimicrobial agents.

In vitro antimicrobic susceptibility patterns of commonly isolated aerobic gram-positive and gram-negative bacterial pathogens of equine origin were determined, using the agar-plate dilution method. All organisms were recent clinical isolates and included Corynebacterium (Rhodococcus) equi, Corynebacterium pseudotuberculosis, (coagulase positive) Staphylococcus sp, Streptococcus equi, Streptococcus zooepidemicus, Actinobacillus sp, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella. In vitro susceptibility levels were outlined for 14 antimicrobics as follows: amikacin less than or equal to 4.0 micrograms/ml, ampicillin less than or equal to 1.0 microgram/ml, amoxicillin less than or equal to 1.0 microgram/ml, cefadroxil less than or equal to 8.0 micrograms/ml, chloramphenicol less than or equal to 8.0 micrograms/ml, erythromycin less than or equal to 1.0 microgram/ml, gentamicin less than or equal to 2.0 micrograms/ml, kanamycin less than or equal to 4.0 micrograms/ml, penicillin less than or equal to 1.0 microgram/ml, tetracycline less than or equal to 1.0 microgram/ml, sulfadimethoxine less than or equal to 10.0 micrograms/ml, ormetoprim/sulfadimethoxine less than or equal to 0.5/9.5 micrograms/ml, sulfadiazine less than or equal to 10.0 micrograms/ml, and trimethoprim/sulfadiazine less than or equal to 0.5/9.5 micrograms/ml.

Automated serum chemical analysis in the foal.

Blood was collected from healthy Quarter Horse and Appaloosa foals at birth and at intervals until 18 weeks of age and then was processed in an automated system for serum chemical analysis, grouping the results by age. The test values were markedly different from those normally expected in adult horses. The greatest differences were in alkaline phosphatase, lactate dehydrogenase, glucose, and total bilirubin measurements. It was concluded that serum biochemical test results be compared with age-specific normal values before diagnoses are made in cases of illness.