Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment)

To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A.     

Composite B-cell and T-cell lymphoma arising 24 years after nodular lymphocyte predominant Hodgkin''s disease

Surfactant protein A (SP-A) is the major protein of pulmonary surfactant. This protein is implicated in regulating surfactant secretion, alveolar processing, recycling, and in non-serum-induced immune response. An increasing body of work indicates the importance of cations, particularly calcium, on SP-A function. However, little information exists on the effects of cations on SP-A quaternary structure. Here, the quaternary organisation of bovine surfactant protein A in the presence of cations has been quantitatively and systematically studied by transmission electron microscopy. The conformation of SP-A is altered by the presence of cations, especially calcium, then sodium, and to a small extent, magnesium. There is a transition concentration, unique for each cation, at which a conformational switch occurs. These transition concentrations are: 5 mM for CaCl2, 100 mM for NaCl and 1 mM for MgCl2. Below these concentrations, SP-A exists primarily in an opened form with a large head diameter of 20 nm; above it, SP-A is mostly in a closed form due to a compaction of the headgroups resulting in a head diameter of 11 nm. There is a corresponding increase in particle length from 17 nm for opened SP-A to 20 nm for closed SP-A. The fact that the transition concentrations are within physiological range suggests that cation-mediated conformational changes of SP-A could be operative in vivo.     

Evaluation of enterolithiasis in equids: 900 cases (1973-1996)

In this study we investigated the effect of lesioning the noradrenergic systems on the behavioral effects of (5R, 10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10-imine hydrogen maleate--MK-801, in rats. The noradrenergic system was lesioned with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride--DSP4 (60 mg/kg IP). MK-801 increased the locomotor activity and rearing. DSP4 significantly further increased the hyperlocomotor activity, circling (especially to the left side), sniffing, rolling, and falling that were induced by MK-801. These results showed that destruction of the noradrenergic system increased MK-801-hyperlocomotor activity, ataxia and stereotypy.     

Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants

OBJECTIVES: To evaluate the effectiveness and morbidity of salvage brachytherapy for locally recurrent or persistent prostate cancer after radiotherapy failure. METHODS: In this retrospective study, 49 patients of median age 73.3 years (range 52.9 to 86.9) with biopsy-proven localized prostate cancer underwent interactive transperineal fluoroscopic-guided and biplane ultrasound-guided brachytherapy with either iodine 125 or palladium 103 after prior radiotherapy failure. Post-treatment follow-up was conducted for a median of 64.1 months (range 26.6 to 96.8) and included clinical assessment of disease status, assays of serum prostate-specific antigen (PSA) levels, and documentation of treatment-related symptoms and complications. Determination of biochemical treatment failure was based on two successive rising PSA values above the post-treatment PSA nadir value. RESULTS: The actuarial rate of local prostate cancer control was 98% (95% confidence interval [CI] 94% to 99%). Actuarial disease-specific survival at 3 and 5 years was 89% (95% CI 73% to 96%) and 79% (95% CI 58% to 91%), respectively. At 3 and 5 years, actuarial biochemical disease-free survival was 48% (95% CI 32% to 63%) and 34% (95% CI 17% to 51%), respectively. Post-treatment PSA nadir was found to be a significant predictor of biochemical disease-free survival. Actuarial biochemical disease-free survival of patients who achieved a PSA nadir less than 0.5 ng/mL was 77% (95% CI 53% to 90%) and 56% (95% CI 25% to 78%) at 3 and 5 years, respectively. Of 49 patients, 23 (47%) achieved a post-treatment PSA nadir less than 0.5 ng/mL. The incidence of serious complications after salvage brachytherapy, such as incontinence and rectal complications, was lower than that reported after other types of salvage procedures. CONCLUSIONS: Interactive transperineal fluoroscopic-guided and biplane ultrasound-guided brachytherapy is a novel, potentially curative salvage modality for patients in whom prior radiotherapy failed. In a population of patients with poor prognosis, this modality was associated with a high rate of local prostate cancer control and a 34% actuarial rate of biochemical disease-free survival at 5 years. The incidence of major complications after salvage brachytherapy appears to be lower than that after other potentially curative salvage procedures, such as radical prostatectomy and cryoablation. Salvage brachytherapy warrants further investigation.     

Prevalence of anxiety and depressive illness and help seeking behaviour in African Caribbeans and white Europeans: two phase general population survey

OBJECTIVE: To determine the prevalence of common mental disorders (anxiety and depression) and help seeking behaviour in African Caribbeans and white Europeans. DESIGN: Two phase survey in a general population sample. The first phase comprised screening with the 12 item general health questionnaire; the second phase was standardised psychiatric assessment and interview about help seeking. SETTING: People registered with four general practices in central Manchester. PARTICIPANTS: Of 1467 people randomly selected from family health services authority lists, 864 were still resident. 337 African Caribbeans and 275 white Europeans completed the screening phase (response rate 71%); 127 African Caribbeans and 103 white Europeans were interviewed in the second phase. MAIN OUTCOME MEASURES: One month period prevalence of anxiety and depressive disorders in each ethnic group. RESULTS: 13% of African Caribbeans (95% confidence interval 10% to 16%) and 14% (10% to 18%) of white Europeans had one or more disorder. Anxiety disorders were significantly less common among African Caribbeans (3% (1% to 5%) v 9% (6% to 12%) in white Europeans). Depressive disorders were significantly more common among African Caribbean women than white women (difference 8% (1% to 15%)). Medical help seeking was similar in the two groups, but African Caribbeans with mental disorders were more likely to seek additional help from non-medical sources (12/29 v 5/29, P=0.082). CONCLUSIONS: In an inner city setting the prevalence of common mental disorders is similar in these two ethnic groups.     

Neurotrophins support the development of diverse sensory axon morphologies

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax-/- neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.     

Use of echocardiography in the management of congestive heart failure in the community

BACKGROUND: Inhibition of apoptosis, or programmed cell death, may be critical both in the development of cancer and in determining response to therapy. The authors examined the expression of two related apoptotic inhibitors, Bcl-2 and Bcl-xL, in pretreatment biopsies from a series of 42 patients with squamous cell carcinoma of the head and neck. The observed pattern of apoptotic inhibitor expression was compared with that of the p53 gene product, another factor implicated in carcinogenesis and therapeutic responsiveness. METHODS: Formalin fixed, paraffin embedded tumor biopsies from 42 patients with locally advanced squamous cell carcinoma of the head and neck were analyzed by immunohistochemistry using antibodies specific for Bcl-xL, Bcl-2, and p53. Measures of clinical outcome, including disease specific survival and overall survival, were compared among the groups. RESULTS: The majority of the tumors demonstrated enhanced expression of either Bcl-2 or Bcl-xL compared with surrounding normal epithelium. Fifty-two percent of the tumors had up-regulated Bcl-xL, and 17% had up-regulated Bcl-2. There was no overlap between these groups. Expression of Bcl-2, but not Bcl-xL, was correlated with improved disease specific survival. Immunohistochemically detectable p53 expression (48% of tumors) was not found to correlate with expression of either Bcl-xL or Bcl-2 and, in this series, was not a predictor of clinical outcome. CONCLUSIONS: These results suggest that disruption of apoptotic control pathways is an important event in the evolution of squamous cell carcinoma of the head and neck. A common mechanism for this disruption involves overexpression of Bcl-xL, Patients whose tumors demonstrate Bcl-2 positivity, even with locoregionally advanced disease, appear to have a high likelihood of cure with aggressive combined modality therapy and may be treated successfully with less toxic therapy.     

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.