The overexpression of a Saccharomyces cerevisiae centromeric histone H3 variant mutant protein leads to a defect in kinetochore biorientation

Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA and mediate attachment to the mitotic spindle. Because centromeric sequences are not conserved, centromere identity is propagated by an epigenetic mechanism. All eukaryotes contain an essential histone H3 variant (CenH3) that localizes exclusively to centromeres. Because CenH3 is required for kinetochore assembly and is likely to be the epigenetic mark that specifies centromere identity, it is critical to elucidate the mechanisms that assemble and maintain CenH3 exclusively at centromeres. To learn more about the functions and regulation of CenH3, we isolated mutants in the budding yeast CenH3 that are lethal when overexpressed. These CenH3 mutants fall into three unique classes: (I) those that localize to euchromatin but do not alter kinetochore function, (II) those that localize to the centromere and disrupt kinetochore function, and (III) those that no longer target to the centromere but still disrupt chromosome segregation. We found that a class III mutant is specifically defective in the ability of sister kinetochores to biorient and attach to microtubules from opposite spindle poles, indicating that CenH3 mutants defective in kinetochore biorientation can be obtained.     

Successful bone marrow transplantation in a patient with DNA ligase IV deficiency and bone marrow failure

Cirrhotic cardiomyopathy is the term used to describe a constellation of features indicative of abnormal heart structure and function in patients with cirrhosis. These include systolic and diastolic dysfunction, electrophysiological changes, and macroscopic and microscopic structural changes. The prevalence of cirrhotic cardiomyopathy remains unknown at present, mostly because the disease is generally latent and shows itself when the patient is subjected to stress such as exercise, drugs, hemorrhage and surgery. The main clinical features of cirrhotic cardiomyopathy include baseline increased cardiac output, attenuated systolic contraction or diastolic relaxation in response to physiologic, pharmacologic and surgical stress, and electrical conductance abnormalities (prolonged QT interval). In the majority of cases, diastolic dysfunction precedes systolic dysfunction, which tends to manifest only under conditions of stress. Generally, cirrhotic cardiomyopathy with overt severe heart failure is rare. Major stresses on the cardiovascular system such as liver transplantation, infections and insertion of transjugular intrahepatic portosystemic stent-shunts (TIPS) can unmask the presence of cirrhotic cardiomyopathy and thereby convert latent to overt heart failure. Cirrhotic cardiomyopathy may also contribute to the pathogenesis of hepatorenal syndrome. Pathogenic mechanisms of cirrhotic cardiomyopathy are multiple and include abnormal membrane biophysical characteristics, impaired β-adrenergic receptor signal transduction and increased activity of negative-inotropic pathways mediated by cGMP. Diagnosis and differential diagnosis require a careful assessment of patient history probing for excessive alcohol, physical examination for signs of hypertension such as retinal vascular changes, and appropriate diagnostic tests such as exercise stress electrocardiography, nuclear heart scans and coronary angiography. Current management recommendations include empirical, nonspecific and mainly supportive measures. The exact prognosis remains unclear. The extent of cirrhotic cardiomyopathy generally correlates to the degree of liver insufficiency. Reversibility is possible (either pharmacological or after liver transplantation), but further studies are needed.     

Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.     

The functional cycle and regulation of the Thermus thermophilus DnaK chaperone system

The Escherichia coli DnaK (DnaKEco) chaperone cycle is tightly regulated by the cochaperones DnaJ, which stimulates ATP hydrolysis, and GrpE, which acts as a nucleotide exchange factor. The Thermus thermophilus DnaK (DnaKTth) system additionally comprises the DnaK-DnaJ assembly factor (DafATth) that is mediating formation of a 300 kDa DnaKTth. DnaJTth.DafATth complex.A model peptide derived from the tumor suppressor protein p53 was used to dissect the regulation of the individual kinetic key steps of the DnaKTth nucleotide/chaperone cycle. As with DnaKEco the DnaKTth.ATP complex binds substrates with reduced affinity and large exchange rates compared to the DnaKTth.ADP.Pi state. In contrast to DnaKEco, ADP-Pi release is slow compared to the rate of hydrolysis, reversing the balance of the two functional nucleotide states. Whereas GrpETth stimulates nucleotide release from DnaKTth, DnaJTth does not accelerate ATP hydrolysis under various experimental conditions. However, it exerts influence on the interaction of DnaKTth with substrates: in the presence of DafATth, DnaJTth inhibits substrate binding, and substrate already bound to DnaKTth is displaced by DnaJTth and DafATth, indicating competitive binding of DnaJTth/DafATth and substrate. It thus appears that the DnaKTth. DnaJTth.DafATth complex as isolated from T. thermophilus does not represent the active species in the DnaKTth chaperone cycle. Isothermal titration calorimetry showed that the ternary complex of DnaKTth, DnaJTth and DafATth is assembling with high affinity, whereas binary complexes of DnaKTth and DnaJTth or DafATth were not detectable, indicating highly synergistic formation of the 300 kDa DnaKTth. DnaJTth.DafATth complex.Based on these results, a model describing the DnaKTth chaperone cycle and its regulation by cochaperones is proposed where DnaKTth. DnaJTth.DafATth constitutes the resting state, and a DnaKTth. substrate.DnaJTth complex is the active chaperone species. The novel factor DafATth that mediates interaction of DnaKTth with DnaJTth would thus serve as a "template" to stabilise the ternary DnaKTth.DafATth.DnaJTth complex until it is replaced by substrate proteins under heat shock conditions.     

Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.     

Subcellular distribution of the V-ATPase complex in plant cells,and <Emphasis Type="Italic">in vivo</Emphasis> localisation of the 100 kDa subunit VHA-a within the complex

Background  

Vacuolar H⁺-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.     

A data management system for structural genomics

Background  

Structural genomics (SG) projects aim to determine thousands of protein structures by the development of high-throughput techniques for all steps of the experimental structure determination pipeline. Crucial to the success of such endeavours is the careful tracking and archiving of experimental and external data on protein targets.     

Effects of gonadotropins on bovine oocytes matured in TCM-199

The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during in vitro maturation of bovine oocytes in TCM-199 without serum were evaluated. Bovine oocytes with compact cumulus cells were collected from slaughterhouse-derived ovaries and cultured in Hepes-buffered TCM-199 supplemented with 5 mg/mL BSA, 1 microg/mL estradiol-17beta, FSH (0, 0.015, 0.05, 0.15, 1.5 or 15 ng/mL; Experiment 1), LH (0, 0.14, 1, 7 or 49 microg/mL; Experiment 2) and combinations of 1 or 10 ng/mL FSH and 1 or 10 microg/mL LH (Experiment 3) at 39 degrees C in 5% CO2 in air. After 22 h of maturation, cumulus expansion was estimated by scoring from 0 (no expansion) to 4 (full expansion of cumulus mass). In vitro fertilization was done with Percoll (45/90%) separated bull sperm at 1 x 10(6) sperm/mL in fert-TALP with 5 U/mL heparin. At 18 to 20 h post-insemination, presumptive zygotes were transferred to a chemically defined medium (CDM-1) supplemented with 0.5% BSA and nonessential amino acids for 72 h and then moved to CDM-2, additionally supplemented with essential amino acids. Zygotes were cultured at 39 degrees C in 5% CO2, 5% O2 and 90% N2 for 8 days. During Experiments 1 and 2, cumulus expansion increased in proportion to concentrations of FSH and LH. Cleavage rates and development to blastocysts were not significantly different among FSH and LH treatments. In Experiment 3, cumulus expansion of bovine oocytes was maximal when 1 ng/mL FSH and 1 microg/mL LH were added to IVM medium, but cumulus expansion again was not related to developmental ability, although cleavage rates were improved slightly (P<0.05) by the combination of LH and FSH. Blastocyst quality, estimated by the size of inner cell mass, was not different between combinations of FSH and LH, and the numbers of nuclei were not different. Although expansion of cumulus cells surrounding bovine oocytes was altered in response to FSH and/or LH in semi-defined medium, cumulus expansion was not related to rates of cleavage or subsequent embryonic development in vitro. The effects of LH on cumulus expansion can be explained by as little as 1 part per 10, 000 contamination with FSH.