Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle.

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.     

Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies.

Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.     

Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms

Background

We conducted a tuberculosis (TB) prevalence survey and evaluated the screening methods used in our survey, to assess if screening in TB prevalence surveys could be simplified, and to assess the accuracy of screening algorithms that may be applicable for active case finding.

Methods

All participants with a positive screen on either a symptom questionnaire, chest radiography (CXR) and/or sputum smear microscopy submitted sputum for culture. HIV status was obtained from prevalent cases. We estimated the accuracy of modified screening strategies with bacteriologically confirmed TB as the gold standard, and compared these with other survey reports. We also assessed whether sequential rather than parallel application of symptom, CXR and HIV screening would substantially reduce the number of participants requiring CXR and/or sputum culture.

Results

Presence of any abnormality on CXR had 94% (95%CI 88–98) sensitivity (92% in HIV-infected and 100% in HIV-uninfected) and 73% (95%CI 68–77) specificity. Symptom screening combinations had significantly lower sensitivity than CXR except for ‘any TB symptom’ which had 90% (95%CI 84–95) sensitivity (96% in HIV-infected and 82% in HIV-uninfected) and 32% (95%CI 30–34) specificity. Smear microscopy did not yield additional suspects, thus the combined symptom/CXR screen applied in the survey had 100% (95%CI 97–100) sensitivity. Specificity was 65% (95%CI 61–68). Sequential application of first a symptom screen for ‘any symptom’, followed by CXR-evaluation and different suspect criteria depending on HIV status would result in the largest reduction of the need for CXR and sputum culture, approximately 36%, but would underestimate prevalence by 11%.

Conclusion

CXR screening alone had higher accuracy compared to symptom screening alone. Combined CXR and symptom screening had the highest sensitivity and remains important for suspect identification in TB prevalence surveys in settings where bacteriological sputum examination of all participants is not feasible.     

The determination of huperzine A in European Lycopodiaceae species by HPLC-UV-MS

A rapid qualitative HPLC-UV-MS method was developed for the detection of huperzine A in Lycopodiaceae species. HPLC coupled with mass spectrometry experiments allowed the identification of the alkaloid and enabled targeted analysis of complex matrices such as herbal extracts. Huperzine A was detected by single ion monitoring of the pseudomolecular ion [M + H]+ at m/z 243.2. The alkaloid was also detected on TLC in a bioautographic enzyme assay with acetylcholinesterase to show the activity of the compound. Four Lycopodiaceae species collected in Switzerland were tested by these methods and Huperzia selago (L.) Bernh. ex Schrank et Martius was the only species found to contain huperzine A.     

Urban agricultural economy of the Early Islamic southern Levant: a case study of Ashkelon

Vegetation History and Archaeobotany - Archaeobotanical research in the southern Levant has focused on the development of agriculture and urban societies, but less so on how agriculture sustained...     

Interaction between the Product of the Breast Cancer Susceptibility Gene BRCA2 and DSS1, a Protein Functionally Conserved from Yeast to Mammals

Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.     

The role of shugoshin in meiotic chromosome segregation

During meiosis, DNA replication is followed by 2 successive chromosome segregation events, resulting in the production of gametes with a haploid number of chromosomes from a diploid precursor cell. Faithful chromosome segregation in meiosis requires that sister chromatid cohesion is lost from chromosome arms during meiosis I, but retained at centromeric regions until meiosis II. Recent studies have begun to uncover the mechanisms underlying this stepwise loss of cohesion in meiosis and the role of a conserved protein, shugoshin, in regulating this process.     

The triphenylphosphine-sulfur trioxide adduct as a coupling reagent in peptide synthesis

Several adducts of sulfur trioxide with electron-donor molecules have been prepared. Of these the air-stable triphenylphosphine-SO₃ complex has been shown to be the most useful in the synthesis of peptides. It has been demonstrated that a variety of solvents may be used for couplings, but that in order for reaction to occur the solvent must itself be able to form an adduct. This behaviour is due to the fact that SO₃ is transferred from the triphenylphosphine-SO₃ complex to the solvent, thus forming an intermediate complex which is responsible for activation of the carboxylate unit. The carboxyl component is generally present as its tetramethylguanidinium salt. The method has been used to synthesize several model peptides and to prepare a fully biologically active sample of methionine-enkephalin.