Possible involvement of LKB1-AMPK signaling in non-homologous end joining.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.

Determinants of left ventricular untwisting behaviour in patients with dilated cardiomyopathy: analysis by two-dimensional speckle tracking.

BACKGROUND/OBJECTIVE: Left ventricular (LV) untwisting velocity has emerged as a novel index of LV diastolic function since it is thought to be related to LV diastolic suction. However, the pathophysiology of LV untwisting behavior has not been fully investigated. The aim of this study was to investigate the determinants of LV peak untwisting velocity in patients with dilated cardiomyopathy (DCM). METHODS: 101 patients with DCM (mean age 60 (SD 13) years) and 50 control subjects were evaluated. After a standard echocardiographic examination, peak torsion and peak untwisting velocity were measured using two-dimensional speckle-tracking imaging. Radial dyssynchrony was assessed by speckle-tracking radial strain analysis. Tissue Doppler derived systolic (Ts-SD) and diastolic (Te-SD) dyssynchrony indices were also assessed. RESULTS: The patients with DCM had significantly smaller peak torsion (p<0.001) and peak untwisting velocity (p<0.001) and greater radial dyssynchrony (p<0.001) and Ts-SD (p<0.001) and Te-SD (p = 0.001) compared with the control subjects. The peak untwisting velocity was correlated with end-systolic volume index (r = 0.524, p<0.001), E/e' (r = 0.365, p<0.001), radial dyssynchrony (r = 0.578, p<0.001), Ts-SD (p<0.001), Te-SD (p<0.001) and peak torsion (r = -0.635, p<0.001) in patients with DCM(. )Multivariate analysis revealed that peak torsion, radial dyssynchrony and E/e' were independent predictors of peak untwisting velocity in patients with DCM (standard coefficient -0.483, p<0.001, 0.330, p<0.001 and 0.241, p = 0.001, respectively). CONCLUSION: These results suggest that strain-based LV radial dyssynchrony and E/e' as well as LV torsion are related to diastolic untwisting behaviour in patients with DCM.

Possible link between large artery stiffness and coronary flow velocity reserve.

BACKGROUND: Population studies have shown that increased large artery stiffness is an independent predictor of cardiovascular events. Experimental studies have shown that a stiff aorta is associated with decreased coronary blood flow. However, a link between large artery stiffness and coronary microvascular function in the clinical setting has not been demonstrated previously. OBJECTIVE: To evaluate the relationship between large artery stiffness and coronary flow velocity reserve (CFVR). PATIENTS AND METHODS: 102 consecutive subjects (mean (SD) age 62 (10) years) without coronary and peripheral arterial disease were enrolled in the study. After 15 minutes' rest, measurements were obtained of brachial-ankle pulse wave velocity (baPWV), augmentation index (AIx) from a carotid pulse tracing, and transthoracic echocardiographic measures, including coronary flow velocity in the left anterior descending coronary artery. In addition, coronary flow velocity during hyperaemia was measured during an intravenous infusion of adenosine triphosphate. CFVR was defined as the ratio of hyperaemic to basal coronary velocity. RESULTS: Subjects with decreased CFVR (< 2.5; n = 40) had significantly higher baPWV (1848 (369) cm/s vs 1548 (333) cm/s; p<0.001), greater AIx (25.3 (11.0)% vs 16.3 (20.0)%; p = 0.01) and greater pulse pressure (PP) (64 (13) mm Hg vs 54 (13) mm Hg; p<0.001) than those with normal CFVR (> or = 2.5; n = 62). Multivariate analysis showed that AIx and PP were independent predictors of CFVR (r = -0.32, p<0.001 and -0.25, p = 0.02, respectively). CONCLUSIONS: The data suggest that large artery stiffening is linked to a reduction of CFVR, which may partially explain the higher cardiac event rate in patients with increased large artery stiffness.

Serotype-specific polysaccharide of Streptococcus mutans contributes to infectivity in endocarditis.

Streptococcus mutans and other viridans streptococci have been implicated as major etiological agents of infective endocarditis. The serotype-specific rhamnose-glucose polysaccharide (RGP) of S. mutans has several biological functions that appear to be essential for the induction of infective endocarditis. The aim of this study was to examine the contribution of RGP to the infectivity of S. mutans in infective endocarditis using a rat model. The RGP-defective mutant of S. mutans showed reduced ability to induce infective endocarditis compared to the parental strain. The ability of S. mutans to induce infective endocarditis was not consistent with the binding capacity of the organism to extracellular matrix proteins. The results suggest that S. mutans containing whole RGP is more virulent than the RGP-defective mutant, and the RGP has an important role for the induction of infective endocarditis by S. mutans.

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

SETTING: The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. OBJECTIVE: To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. DESIGN: A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. RESULTS: We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. CONCLUSION: The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.

Anticancer agent E7070 inhibits amino acid and uracil transport in fission yeast.

E7070 is a novel sulfonamide anticancer agent that inhibits cell cycle progression in G1 in mammalian cells, but its action targets are not known. We recently employed the genetically amenable fission yeast Schizosaccharomyces pombe as a model organism to search for its targets. Here, we show that E7070 inhibits imports of amino acid and uracil into S. pombe cells. Unlike their prototrophic counterparts, leucine- and uracil-auxotrophic strains are sensitive to E7070 and are unable to proliferate with a delayed G1-S transition in low-glucose yeast extract-polypeptone medium containing this drug because this chemical markedly inhibits the uptake of leucine and uracil in low glucose medium. Furthermore, addition of leucine or uracil to the culture medium or overexpression of genes encoding an amino acid or uracil transporter suppresses the E7070-imposed growth inhibition of these auxotrophic strains. Thus, some of the molecular targets for E7070 action in S. pombe are likely to be leucine and uracil transporters.

Hematologic abnormalities and outcome of 16 cats with myelodysplastic syndromes.

We investigated the hematologic abnormalities and prognoses in 16 cats with myelodysplastic syndromes (MDS). Nonregenerative anemia, thrombocytopenia, and neutropenia were observed in 15, 13, and 4, respectively, of the 16 cats with MDS. Morphologic abnormalities characteristic of MDS included megaloblastoid rubricytes (9 cats), hyposegmentation of neutrophils (7 cats), nuclear abnormality of rubricytes (10 cats) and neutrophils (13 cats), and micromegakaryocytes (10 cats). Disease in these 16 cats was subclassified into refractory anemia (RA; 8 cats), RA with excess of blasts (RAEB; 5 cats), RAEB in transformation (RAEB in T; 1 cat), and chronic myelomonocytic leukemia (CMMoL; 2 cats), according to the human French-American-British (FAB) classification. In the cats in which the clinical outcome was known, 3 of 6 cats with high blast cell count MDS, including RAEB, RAEB in T, and CMMoL, developed acute myeloid leukemia, but only 1 of 8 cats with low blast cell count MDS (RA) developed acute myeloid leukemia. Based on the Dusseldorf scoring system for the prognosis of human MDS, the survival times of the cats showing high scores (> or =3 points) were significantly shorter than those of the cats with low scores (<3 points). The FAB classification and Dusseldorf scoring system were considered to be useful for predicting the prognosis of feline MDS. Furthermore, 15 of the 16 cats with MDS in this study were infected with feline leukemia virus, indicating its possible etiologic role in the pathogenesis of feline MDS.

Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism.

During S phase, chromatid cohesion is established only between nascent sisters and with faithful pairing along their entire region, but how this is ensured is unknown. Here we report that sister chromatid cohesion is formed and maintained by a unique mechanism. In fission yeast, Eso1p, functioning in close coupling to DNA replication, establishes sister chromatid cohesion whereas the newly identified Cohesin-associated protein Pds5p hinders the establishment of cohesion until counteracted by Eso1p, yet stabilizes cohesion once it is established. Eso1p interacts physically with Pds5p via its Ctf7p/Eco1p-homologous domain.

Usefulness of selective myocardial contrast echocardiography in percutaneous transluminal septal myocardial ablation: a case report.

A 67-year-old woman with hypertrophic obstructive cardiomyopathy that was refractory to medical treatment underwent percutaneous transluminal septal myocardial ablation (PTSMA). The septal branch supplying the myocardium involved in the left ventricular outflow tract (LVOT) obstruction was identified by selective myocardial contrast echocardiography (MCE). MCE for the third and largest septal branch opacified the right side of the mid-septal myocardium and MCE for the second septal branch opacified the right side of the basal portion of the septal myocardium. Finally, contrast agent was injected into the first, small branch, which opacificied the myocardium protruding into the LVOT. Subsequently, septal myocardial ablation for this vessel with intracoronary alcohol was performed, followed by a reduction of the LVOT gradient and successful, dramatic improvement in the patient's clinical condition. Selective MCE was very useful to identify the appropriate septal branch for PTSMA and enabled maximal effect of this treatment with minimal myocardial damage.

Structure and function of the left ventricle and carotid artery in hemodialysis patients.

Hemodialysis patients frequently show associated hypertension, which can lead to a number of cardiovascular complications. The aim of this study was to assess the effects of hypertension on the structure and function of the carotid artery and left ventricle (LV) in hemodialysis patients. In addition, we investigated the contribution of hemodialysis and other risk factors. Fifty-two hemodialysis patients, 71 hypertensive patients (HT group) and 30 normotensive subjects (NT group) were included in this study. Hemodialysis patients were divided into two groups: 35 patients with hypertension (HDHT group), and 17 patients without hypertension (HDNT group). We measured intima-media thickness (IMT), plaque score, end-diastolic diameter, and stiffness index beta of the carotid artery by ultrasonography, and LV mass index (LVMi), endocardial fractional shortening (FS), and midwall FS (MWS) by echocardiography. A multiple stepwise regression analysis including hemodialysis, hypertension, diabetes mellitus, and other risk factors was also performed. IMT was significantly higher in the HT and HDHT groups than in the NT group. Plaque score and diameter of the carotid artery were higher in the HDHT group than in the other three groups. The stiffness index beta was higher in the HDHT group than in the non-hemodialysis groups. In multivariate analysis, IMT was independently correlated with age and hypertension. Plaque score and stiffness index beta were independently associated with age, hypertension, and hemodialysis. LVMi was higher in HT and hemodialysis-patients groups than in the NT group. Hypertension and hemodialysis were strong and independent predictors of LVMi. FS showed no significant differences among the four groups, but MWS was significantly lower among the hemodialysis patients than in the NT group. MWS was independently correlated with hemodialysis and diabetes. In conclusion, hemodialysis per se advanced both atherosclerosis and arteriosclerosis of the carotid artery. Moreover, it increased LVMi and caused cardiac dysfunction. Associated hypertension might thus accelerate the progression of atherosclerosis and arteriosclerosis of the carotid artery and the increase of LVMi.

Oncogenic cell cycle start control.

The ability to proliferate in the absence of anchorage is a fundamental attribute of cancer cells, yet how it is acquired is one central problem in cancer biology. By utilizing growth factor-transformable NRK cells and its insensitive mutants, we recently found that oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start, but not via the regulation of its catalytic activity and that Cdk6 participation closely correlates with the anchorage-independent growth ability. Since many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, this finding raises the possibility that the mechanism by which oncogenic stimulation invokes anchorage-independent growth of NRK cells is similar to the one used for hematopoietic cell proliferation. We discuss this novel mechanism and its implication.

Cdk6-cyclin D3 complex evades inhibition by inhibitor proteins and uniquely controls cell's proliferation competence.

Mammalian cells require a cyclin D-dependent kinase for the cell cycle start, yet many mesenchymal cells express three seemingly redundant D cyclins and similarly, seemingly redundant Cdk4 and Cdk6 as their kinase partners. We have found that the Cdk6-cyclin D3 complex is unique among the D cyclin and kinase combinations in the ability to promote the cell cycle start. In an anchorage-minus G(1)-arrested rat fibroblast, only Cdk6-D3 retains kinase activity due mainly to its ability to evade inhibition by p27(KIP1) and p21(CIP1) with a resemblance to viral cyclin-bound Cdk6. Rodent fibroblasts engineered to overexpress both Cdk6 and cyclin D3 highly resist serum starvation- or cell-cell contact-imposed G(1)-arrest. In BALB/c 3T3 cells, D3 is constitutively expressed, but Cdk6 is markedly induced with concomitant activation upon stimulation with a growth-promoting factor. These results suggest a role for the Cdk6-D3 complex in regulating cell's proliferation ability in response to external stimuli.

Calcium phosphate transfection.

This unit presents two methods of calcium phosphate-based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES-buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.

Calcium phosphate transfection.

This unit contains two methods of calcium phosphate-based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The Basic Protocol uses a HEPES-buffered solution to form a calcium phosphate precipitate that is directly layered onto the cells. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and is then dropped onto the cells. The alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, and may be helpful with linear or genomic DNA. Both methods of transfection require very high-quality plasmid DNA, which can be prepared as described in the second Support Protocol. Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first Support Protocol.

Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk-inhibitory kinases.

BACKGROUND: In eukaryotic cells, the kinase activity of the mitosis-promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain. RESULTS: We identified human Wee1B by searching a sequence database. The predicted human Wee1B protein comprises 561 amino acids. Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis. Interestingly, RT-PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression. GFP-Wee1B showed a predominantly nuclear localization in HeLa cells. Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1-50Deltamik1 mutant, and over-expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2-M transition. Recombinant Wee1B effectively phosphorylated cyclin B-associated Cdk1 on tyrosine-15, resulting in an inactivation of the kinase activity of Cdk1. CONCLUSION: We identified human Wee1B as a novel Cdk1-inhibitory kinase. The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals.

A pcl-like cyclin activates the Res2p-Cdc10p cell cycle "start" transcriptional factor complex in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the "start" of the cell cycle is controlled by the two functionally redundant transcriptional regulator complexes, Res1p-Cdc10p and Res2p-Cdc10p, that activate genes essential for the onset and progression of S phase. The activity of the Res2p-Cdc10p complex is regulated at least by the availability of the Rep2 trans-activator subunit in the mitotic cell cycle. We have recently isolated the pas1(+) gene as a multicopy suppressor of the res1 null mutant. This gene encodes a novel cyclin that shares homology with the Pho85 kinase-associated cyclins of the budding yeast Saccharomyces cerevisiae. Genetic analysis reveals that Pas1 cyclin is unrelated to phosphate metabolism and stimulates the G(1)-S transition by specifically activating the Res2p-Cdc10p complex independently of Rep2p. Pas1 cyclin also controls mating pheromone signaling. Cells lacking pas1(+) are highly sensitive to mating pheromone, responding with facilitated G(1) arrest and premature commitment to conjugation. Pas1 cyclin associates in vivo with both Cdc2 and Pef1 kinases, the latter of which is a fission yeast counterpart of the budding yeast Pho85 kinase, but genetic analysis indicates that the Pef1p-associated Pas1p is responsible for the activation of Res2p-Cdc10p during the G(1)-S transition.

Fission yeast Fizzy-related protein srw1p is a G(1)-specific promoter of mitotic cyclin B degradation.

Downregulation of cyclin-dependent kinase (Cdk)-mitotic cyclin complexes is important during cell cycle progression and in G(1) arrested cells undergoing differentiation. srw1p, a member of the Fizzy-related protein family in fission yeast, is required for the degradation of cdc13p mitotic cyclin B during G(1) arrest. Here we show that srw1p is not required for the degradation of cdc13p during mitotic exit demonstrating that there are two systems operative at different stages of the cell cycle for cdc13p degradation, and that srw1p is phosphorylated by Cdk-cdc13p only becoming dephosphorylated during G(1) arrest. We propose that this phosphorylation targets srw1p for proteolysis and inhibits its activity to promote cdc13p turnover.

Loop-mediated isothermal amplification of DNA.

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

Infection of left atrial thrombus associated with mitral stenosis: A case report.

We describe the first reported case of an infected left atrial thrombus. The case of the 65-year-old male patient in this report was associated with mitral stenosis and involved Escherichia coli, and was treated successfully with surgical resection of the infected thrombus. This case suggests that such infection should be considered as a possible complication of intracardiac thrombus when bacteremia is present.