The in silico identification and characterization of a bread wheat/Triticum militinae introgression line

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross‐bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.     

Cardiac shock-wave therapy in the treatment of coronary artery disease: systematic review and meta-analysis

Aim

To systematically review currently available cardiac shock-wave therapy (CSWT) studies in humans and perform meta-analysis regarding anti-anginal efficacy of CSWT.

Methods

The Cochrane Controlled Trials Register, Medline, Medscape, Research Gate, Science Direct, and Web of Science databases were explored. In total 39 studies evaluating the efficacy of CSWT in patients with stable angina were identified including single arm, non- and randomized trials. Information on study design, subject’s characteristics, clinical data and endpoints were obtained. Assessment of publication risk of bias was performed and heterogeneity across the studies was calculated by using random effects model.

Results

Totally, 1189 patients were included in 39 reviewed studies, with 1006 patients treated with CSWT. The largest patient sample of single arm study consisted of 111 patients. All selected studies demonstrated significant improvement in subjective measures of angina symptoms and/or quality of life, in the majority of studies left ventricular function and myocardial perfusion improved. In 12 controlled studies with 483 patients included (183 controls) angina class, Seattle Angina Questionnaire (SAQ) score, nitrates consumption were significantly improved after the treatment.In 593 participants across 22 studies the exercise capacity was significantly improved after CSWT, as compared with the baseline values (in meta-analysis standardized mean difference SMD?=??0.74; 95% CI, ?0.97 to ?0.5; p?<?0.001).

Conclusions

Systematic review of CSWT studies in stable coronary artery disease (CAD) demonstrated consistent improvement of clinical variables. Meta-analysis showed a moderate improvement of exercise capacity.Overall, CSWT is a promising non-invasive option for patients with end-stage CAD, but evidence is limited to small sample single-center studies. Multi-center adequately powered randomised double blind studies are warranted.

     

Improvement of potential therapeutic value of tumor necrosis-alpha (TNF-alpha) by charge modulation in the tip region

Analysis of published data reveals that the introduction of more basic amino acid residues in the flexible N-terminal region of the human tumour necrosis factor alpha (TNF) molecule indicates a weak but consistent trend towards increased in vitro cytotoxicity, especially when the effect of N-terminal length is taken into account. In our laboratory, a series of TNF analogues with a charge modification in the tip region of the molecule was prepared, and cytotoxicity measured. Similar trends in cytotoxicity with increasing basicity of the TNF analogue were found in this study for two mouse cell lines, L929 and WEHI-164 clone 13-1, as well as for the human line KYM-1D4. For the series of analogues as a whole, a general increase in in vitro cytotoxicity with increasing pI values was not apparent, but some analogues with charge reversal in the tip region, for example, the LK-805 analogue (E107K), exhibited significantly increased cytotoxicity in comparison to native TNF in a range of cell lines, including L929, KYM-1D4-K, WEHI-164 clone 13-1, HEPA 1-6 and EAhy926 cell lines. Experiments with heparinase-pre-treated cells demonstrated that the increased in vitro cytotoxicity of LK-805 is most probably due to interactions with cell surface heparan sulphates that effectively concentrate it before binding to TNF receptors occurs. Examination of structural models of TNF bound to soluble TNF receptor 1 (TNFR1) indicates that simple mutations in the tip region most probably cannot interact with receptor binding sites, and therefore do not directly modulate cytotoxicity.     

Some trends in the somatic development of children and adolescents under iodine-deficiency: materials from the Saratov region

2,106 girls and 2,169 boys from 7 to 17 were investigated in 2002-2004 in three urban settlements of the Saratov region (Povolzhje area): the town of Khvalynsk, population 15,000, with a low level of industrialization; the city of Balakovo, population 220,000, highly industrialized and with a nuclear power station; and the city of Saratov, population around 1,000,000 a regional capital, and also highly industrialized. The whole area, particularly the location of Khvalynsk, is also characterized by iodine deficiency (iodine concentration is 0.78 mkg/kg v. normal values of 5-7 mkg/kg).The program included about 30 anthropometric measurements, evaluation of developmental stages of secondary sexual characteristics, and information on menarcheal age by the status quo method. Information on parental occupation and education, as well as number of children per family was collected by questionnaire. For the analysis all the data were standardized, and further comparisons were made irrespective of age groups. The significance of differences was assessed by one-way ANOVA. For height, weight and chest circumference there are significant differences among the three populations (p < 0.001), with Khvalynsk children being the smallest in body height and weight. However, in chest circumference they are close to or even bigger (girls) than Balakovo children. The children from Khvalynsk are characterized by the lowest values for subcutaneous fat layer, both on the trunk and extremities. For the age of menarche, Khvalynsk girls have the highest values: 13 years 5 months (13.42). In Balakovo and Saratov, the corresponding figures are identical: 13 years 2 months (13.17). Secular changes in Khvalynsk and Saratov children are discussed as compared to the literature.     

The purine nucleotide content in human leukemia cell lines

The HPLC method was used to determine the purine nucleotide (ATP, ADP, AMP, GTP, GDP, GMP, NAD(+)) contents and the values of the adenylate energy charge (AEC) and guanylate energy charge (GEC) for three human acute myelogenous leukemia (AML) cell lines: HL60 (M3 subtype of AML), THP1 (M5 subtype of AML), and HEL (M6 subtype of AML) in French-American-British classification (FAB) and for one chronic myelogenous leukemia (CML) cell line: K562. The results showed that the examined leukemic cells had some significant changes in their purine nucleotide concentrations relative to healthy cells. On the basis of the obtained results, it seems that two of the tested acute myelogenous leukemia cell lines, HL60 and HEL, have similar purine nucleotide metabolisms, while the third AML cell line, THP1, has a purine nucleotide metabolism like that of the chronic myelogenous leukemia cell line, K562.     

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N-terminal polypeptide chain, as observed experimentally in a model system-immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N-terminal fragment and self-assembled ligand in coupling between V and C domains. Root-mean-square distance (RMSD) time profiles show that removal of N-terminal fragment leads to destabilization of V domain. A micelle composed of four self-assembled dye molecules stabilizes and fixes the domain. Analysis of root-mean-square fluctuation (RMSF) values and dynamic cross-correlation matrices (DCCM) reveals that removal of N-terminal fragment results in complete decoupling between V and C domains. Binding of self-assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small beta-sheet composed of N- and C-terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self-assembled ligand, bound in the place originally occupied by N-terminal fragment, is not able to take over the function of the beta-sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain-Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction.     

Cataract surgery and intraocular lens implantation in children with juvenile rheumatoid arthritis associated uveitis

Clinical records of 6 children (7 eyes) with juvenile rheumatoid arthritis (JRA) who underwent cataract surgery with IOL implantation between January 1998 and December 2002 were reviewed. The median age at the time of cataract surgery was 8 years (range 5-14 years). The median follow up was 48 months (range 26 to 60 months). Five of six children (6 eyes) were on systemic immunosuppressive or anti-inflammatory therapy. Glaucoma was present in three eyes before surgery, and all three eyes underwent combined cataract surgery and trabeculectomy with mitomycin C. A final best corrected visual acuity of 0.5 or better was achieved in all eyes Postoperative complications included posterior capsule opacification (n = 5), glaucoma (n = 1), and cystoid macular edema (n = 1). Intraocular lens implantation in children with control of preoperative and postoperative ocular inflammation could lead to favorable visual results.     

B cell receptor-mediated nuclear fragmentation proceeds in WEHI 231 cells in the absence of detectable DEVDase and FRase activity

Crosslinking of the WEHI 231 lymphoma B cell receptor (BCR) leads to growth arrest followed by apoptosis. In a study of the role of lysosomal cysteine proteinases in BCR-mediated apoptosis we provide evidence that commitment to apoptosis correlates with a time-dependent increase in caspase and cathepsin activities. We also show that activation of cathepsins is a caspase-independent process, and caspase cascade activation is independent of lysosomal endopeptidases. BCR-induced nuclear fragmentation was not prevented, but rather delayed in the absence of detectable caspase and cathepsin activities, suggesting that BCR-driven apoptosis of these cells may use an alternative proteolytic mechanism independent of caspases and cathepsins.     

Purification,cloning, expression,and properties of mycobacterial trehalose-phosphate phosphatase

The trehalose-phosphate phosphatase (TPP) was purified from the cytosol of Mycobacterium smegmatis to near homogeneity using a variety of conventional steps to achieve a purification of about 1600-fold with a yield of active enzyme of about 1%. Based on gel filtration, the active enzyme had a molecular weight of about 27,000, and the most purified fraction also gave a major band on SDS-PAGE corresponding to a molecular weight of about 27,000. A number of peptides from the 27-kDa protein were sequenced and these sequences showed considerable homology to the trehalose-P phosphatase (otsB) of Escherichia coli. Based on these peptides, the M. smegmatis gene for TPP was cloned and expressed in E. coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. Most of the TPP activity in the crude E. coli sonicate was initially found in the membrane fraction, but it became solubilized in the presence of 0.2% Sarkosyl. The solubilized protein was purified to apparent homogeneity on a metal ion column and this fraction had high phosphatase activity that was completely specific for trehalose-P. The purified enzyme, either isolated from M. smegmatis, or expressed in E. coli, rapidly dephosphorylated trehalose-6-P, but had essentially no activity on any other sugar phosphates, or on p-nitrophenyl phosphate. The K(m) for trehalose-6-P was about 1.6 mm, and the pH optimum was about 7.5. The native enzyme showed an almost absolute requirement for Mg(2+) and was not very active with Mn(2+), whereas both of these cations were equally effective with the recombinant TPP. The enzyme activity was inhibited by the antibiotics, diumycin and moenomycin, but not by a number of other antibiotics or trehalose analogs. TPP activity was strongly inhibited by the detergents, Sarkosyl and deoxycholate, even at 0.025%, but it was not inhibited by Nonidet P-40, Triton X-100, or octyl glucoside, even at concentrations up to 0.3%. The purified enzyme was stable to heating at 60 degrees C for up to 6 min, but was slowly inactivated at 70 degrees C. Circular dichroism studies on recombinant TPP indicate that the secondary structure of this protein has considerable beta-pleated sheet and is very compact. TPP may play a key role in the biosynthesis of trehalose compounds, such as trehalose mycolates, and therefore may represent an excellent target site for chemotherapy against tuberculosis and other mycobacterial diseases.