Effect of Environmental Factors on Nuclear Organization and Transformation of Human B Lymphocytes.

Chromosomal translocations have long been known for their association with malignant transformation, particularly in hematopoietic disorders such as B-cell lymphomas. In addition to the physiological process of maturation, which creates double strand breaks in immunoglobulin gene loci, environmental factors including the Epstein-Barr and human immunodeficiency viruses, malaria-causing parasites (Plasmodium falciparum), and plant components (Euphorbia tirucalli latex) can trigger a reorganization of the nuclear architecture and DNA damage that together will facilitate the occurrence of deleterious chromosomal rearrangements.

HIV Tat induces a prolonged MYC relocalization next to IGH in circulating B-cells.

With combined antiretroviral therapy (cART), the risk for HIV-infected individuals to develop a non-Hodgkin lymphoma is diminished. However, the incidence of Burkitt lymphoma (BL) remains strikingly elevated. Most BL present a t(8;14) chromosomal translocation which must take place at a time of spatial proximity between the translocation partners. The two partner genes, MYC and IGH, were found colocalized only very rarely in the nuclei of normal peripheral blood B-cells examined using 3D-FISH while circulating B-cells from HIV-infected individuals whose exhibited consistently elevated levels of MYC-IGH colocalization. In vitro, incubating normal B-cells from healthy donors with a transcriptionally active form of the HIV-encoded Tat protein rapidly activated transcription of the nuclease-encoding RAG1 gene. This created DNA damage, including in the MYC gene locus which then moved towards the center of the nucleus where it sustainably colocalized with IGH up to 10-fold more frequently than in controls. In vivo, this could be sufficient to account for the elevated risk of BL-specific chromosomal translocations which would occur following DNA double strand breaks triggered by AID in secondary lymph nodes at the final stage of immunoglobulin gene maturation. New therapeutic attitudes can be envisioned to prevent BL in this high risk group.

Changes in pro-inflammatory markers and leucine concentrations in response to Nordic Walking training combined with vitamin D supplementation in elderly women.

Mechanisms underpinning age-related decreases in muscle strength and muscle mass relate to chronic inflammation. Physical activity induces an anti-inflammatory effect, but it is modulated by additional factors. We hypothesized that vitamin D, which has also anti-inflammatory activity will modify adaptation to exercise and reduce inflammation in elderly women. Twenty-seven women aged 67 ± 8 years were included and divided into groups with baseline vitamin D concentration more than 20 ng mL-1 (MVD) and less than 20 ng mL-1 (LVD). Both groups performed 1 h Nordic Walking (NW) training combined with vitamin D supplementation for 12 weeks. Serum concentrations of inflammation markers, branched amino acids, vitamin D, muscle strength and balance were assessed at the baseline and three days after intervention. The training caused the significant decrease in concentration of pro-inflammatory proteins HMGB1 (30 ± 156%; 90% CI) and IL-6 (-10 ± 66%; 90% CI) in MVD group. This effects in group MVD were moderate, indicating vitamin D as one of the modifiers of these exercise-induced changes. Rise of myokine irisin induced by exercise correlated inversely with HMGB1 and the correlation was more pronounced at the baseline as well as after training among MVD participants. Although the intervention caused the leucine level to rise, a comparison of the recorded response between groups and the adjusted effect indicated that the effect was 20% lower in the LVD group. Overall the applied training program was effective in reducing HMGB1 concentration. This drop was accompanied by the rise of myokine irisin and better uptake of leucine among women with higher baseline vitamin D.

Metabolism of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in rodent tissues and in vivo.

Our previous studies identified 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) phosphates in human erythrocytes. We demonstrated formation of these nucleotides by phosphorylation of 4PYR and potential toxicity due to disruption of erythrocyte energy balance. This study aimed to evaluate the ability of the other cell types to phosphorylate 4PYR to characterize function and toxicity of these compounds. Homogenates of rat heart, kidneys, and liver were used to study the rate of 4PYR phosphorylation in the presence of ATP. In another experiment, 4PYR was administered into mouse as repeated subcutaneous injections and into rats as intraperitoneal infusion. After 7 days, heart, liver, kidney, lungs, and skeletal muscle were collected, and the concentration of 4PYR nucleotides was evaluated. HPLC was used to measure 4PYR and 4PYR nucleotides in homogenate and specimens from in vivo experiments. 4PYR was rapidly phosphorylated by the liver homogenate (390 ± 27 nmol/min/g wet wt). Significant rates were reported in the heart and kidneys' homogenates: 34.3 ± 4.3 nmol/min/g and 33.2 ± 9.2 nmol/min/g, respectively. Phosphorylation of 4PYR was almost completely inhibited by adenosine kinase inhibitor 5'-iodotubercidin. Administration of 4PYR in vivo resulted in accumulation of 4PYR monophosphate in the liver, heart, skeletal muscle, and lung (20-220 nmol/g dry wt) except kidney (<1 nmol/g). In contrast to erythrocytes, no 4PYR triphosphate formation (<1 nmol/g) was observed in any of the organs studied. We conclude that not only the erythrocytes but also other cell types are capable of phosphorylating 4PYR to form 4PYR monophosphate. Potential toxicity or physiological role of 4PYR in peripheral organs could be considered, but mechanisms will be different from that in erythrocytes.

Iron deficiency anemia and cardiac mortality in patients with left ventricular systolic dysfunction undergoing coronary stenting.

AIM: The aim of this study was to assess cardiac mortality in patients with reduced ejection fraction (EF< or =45%) and anemia (Hb< or =12 g/dL) undergoing coronary stenting and to investigate whether iron-deficiency anemia influenced outcome when compared to non-anemic patients or patients with other types of anemia. METHODS: One hundred twenty consecutive patients undergoing percutaneous coronary intervention (PCI) between April 2003 and December 2005 were identified and followed for a median of 30 months. Patients were divided into 2 groups, anemic (Hb< or =12 g/dL) and non-anemic. Anemic patients were then divided into 3 sub-groups based on laboratory analysis and anemia work-up: iron-deficiency, malignancy-associated, and anemia of chronic disease (including chronic kidney disease). Mortality rates and cause of death were retrieved using both the Social Security database and the hospital records. RESULTS: Thirty-one percent of patients had iron deficiency, 24% had a malignancy-associated anemia and 45% had anemia of chronic disease. Overall mortality was 12% of which 29% was cardiac death. All-cause and cardiac mortality were significantly higher in anemic vs. non-anemic patients, (31% vs. 6%, P<0.001, and 10% vs. 1%, P=0.016, respectively). Iron-deficiency anemia strongly predicted cardiac mortality (33% vs. 1% in non-anemic patients, P<0.001), while malignancy-associated anemia was the strongest predictor of non-cardiac death (57% vs. 4% in non-anemic patients, P<0.001). Anemia of chronic disease neither predicted cardiac nor non-cardiac death. CONCLUSIONS: To the authors' knowledge, this is the first study to show that iron-deficiency anemia is a strong predictor of cardiac death when compared to patients with other types of anemia or to non-anemic patients.

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes.

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.

Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma.

Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.

Strong and selective inhibitors of hepatitis B virus replication among novel N4-hydroxy- and 5-methyl-beta-L-deoxycytidine analogues.

Novel N(4)-hydroxy- and 5-methyl-modified beta-L-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. beta-L-2',3'-Didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (beta-L-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC(50)], 0.03 microM), followed by beta-L-2',3'-dideoxy-3'-thia-N(4)-hydroxycytidine (EC(50), 0.51 microM), beta-L-2',3'-dideoxy-N(4)-hydroxycytidine (EC(50), 0.55 microM), and beta-L-5-methyl-2'-deoxycytidine (EC(50), 0.9 microM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the beta-L-cytidine derivatives was also assessed. In accordance with the cell culture data, beta-L-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 microM. The cytotoxicities of some of the 4-NHOH-modified beta-L-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH(2) group. The 50% cytotoxic concentrations for beta-L-Hyd4C in HepG2 and HL-60 cells were 2,500 microM and 3,500 microM, respectively. In summary, our results demonstrate that at least beta-L-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

Comparison of the bladder tumour antigen test with photodynamic diagnosis in patients with pathologically confirmed recurrent superficial urinary bladder tumours.

OBJECTIVE: To verify the sensitivity of the bladder tumour antigen (BTAstat, Bard Urological, Covington, GA) test against the sensitive procedure of photodynamic diagnosis (PDD), in which 5-aminolaevulinic acid (5-ALA, a precursor of fluorescent porphyrins) is absorbed by the tumour and detected by ultraviolet cystoscopy, in the early diagnosis of urinary bladder tumours. PATIENTS AND METHODS: Forty-three patients (31 men and 12 women, age range 21-87 years) were assessed after transurethral resection of their bladder tumour using the BTAstat test and PDD. Sixty-nine biopsies from suspect areas of bladder mucosa were taken during cystoscopy under ultraviolet light and all suspect lesions electrocoagulated. RESULTS: Thirty-five patients (81%) had a positive BTAstat test; in these patients PDD detected malignant lesions (17 Ta1G1-2, two T1G2, two T1G3 and 14 Tis). In eight patients (19%) the BTAstat was negative but PDD detected three malignant lesions (two Tis and one TaG1). CONCLUSIONS: PDD is valuable for detecting bladder malignancy and can identify small lesions not detected by the BTAstat test.

Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system.

The retinoblastoma tumor suppressor (RB) plays an important role in the regulation of cell cycle progression and terminal differentiation of many cell types. Rb(-/-) mouse embryos die at midgestation with defects in cell cycle regulation, control of apoptosis and terminal differentiation. However, chimeric mice composed of wild-type and Rb-deficient cells are viable and show minor abnormalities. To determine the role of Rb in development more precisely, we analyzed chimeric embryos and adults made with marked Rb(-/-) cells. Like their germline Rb(-/-) counterparts, brains of midgestation chimeric embryos exhibited extensive ectopic S-phase entry. In Rb-mutants, this is accompanied by widespread apoptosis. However, in chimeras, the majority of Rb-deficient cells survived and differentiated into neuronal fates. Rescue of Rb(-/-) neurons in the presence of wild-type cells occurred after induction of the p53 pathway and led to accumulation of cells with 4n DNA content. Therefore, the role of Rb during development can be divided into a cell-autonomous function in exit from the cell cycle and a non-cell-autonomous role in the suppression of apoptosis and induction of differentiation.

HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression.

Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1). Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

Microchip vagal pacing reduces food intake and body mass.

BACKGROUND/AIMS: Morbid obesity is an increasing problem worldwide. In many patients pharmacotherapy is ineffective and these cases are treated by surgery. Different types of gastroplasty and gastric bypasses have been described. However, all of these ablative surgical methods are irreversible and often replace obesity by other disorders. Neuromodulation of vagal activity is a method of inducing significant changes in stomach motility. We developed a pre-programmed microchip able to pace vagal afferent activity by changing current parameters. The aim of our study was to evaluate long-term effects of vagal neuromodulation on food intake and body mass in rabbits. METHODOLOGY: Twenty-seven healthy male adult New Zealand white rabbits were included into the study and divided into three groups: A, B and C, 9 animals each. Microchips were implanted by laparotomy access. Anesthesia was obtained by continuous intravenous infusion of propofol. Microchips were fixed in the preperitoneal pocket and two electrodes were positioned on the posterior vagus in group A by forward, and in group B by backward pacing. Control group C was sham operated by laparotomy and only vagal nerves preparation was performed. The following parameters were estimated: daily solid food and liquids intake, amount of feces, body mass and heart rate. RESULTS: Within four weeks after operation body mass in group B had decreased up to 12% (P = 0.029), whereas in group A and C changed to -3% and +2%, respectively. An 87% solid food intake was observed in group A, 60% in group B (P < 0.01), and 143% in group C, compared to preoperative period. No significant differences were observed between groups A, B and C for liquids intake. Total feces weight changes corresponded to solid food intake. Heart rate decreased intraoperatively to 78% and 74% in groups A and B, respectively. CONCLUSIONS: Microchip mediated functional gastroplasty significantly reduces food intake and body mass. Obtained results encourage using similar treatment in morbid obesity human patients. However, further studies are required.

Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level.

Adenovirus (Ad) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts. There is currently no licensed chemotherapy effective in dealing with this virus infection. In this study the anti-adenoviral activity of a group of modified nucleoside analogs was investigated. The most efficient 3-fluorosubstituted nucleoside triphosphate inhibitors of Ad DNA polymerase were 3'-fluorothymidine triphosphate (IC50 0.63 microM), 2',3'-dideoxy-3'-fluoroguanosine triphosphate (IC50 0.71 microM) and 2',3'-dideoxy-3'-fluorouridine triphosphate (IC50 2.96 microM). The most efficient 2',3'-dideoxynucleoside triphosphates were 2',3'-dideoxycytidine triphosphate (ddCTP; IC50 1.0 microM), 2',3'-dideoxyadenosine triphosphate (IC50 1.6 microM) and 2',3'-dideoxythymidine triphosphate (IC50 1.82 microM). Kinetic studies indicate competitive inhibition of adenovirus DNA polymerase by ddCTP. These data confirm results previously obtained at the cellular level using a focus reduction assay involving Ad2-infected FL cells. Whereas the D-enantiomers 3'-fluorothymidine and 2',3'-dideoxycytidine are potent inhibitors of adenoviral replication, the corresponding L-enantiomers exhibited no inhibitory activity.

The retinoblastoma gene family in differentiation and development.

The retinoblastoma (Rb) tumor suppressor gene and its close relatives p107 and p130 are best known for their function in the control of cell cycle progression. In recent years, however, a new role for these proteins has been emerging as they have been linked with regulation of terminal differentiation of many tissues and cell types. In fact, Rb and its family members have been shown to be involved in multiple stages of the differentiation process including irreversible exit from the cell cycle, protection from apoptosis, induction of cell type specific gene expression and maintenance of the post-mitotic state. They also play a critical role in assuring the orderly progression through all these stages of differentiation.

[Methods of treatment of ureteral stenosis after radiotherapy in patients with gynecological tumors]. / Postepowanie w popromiennych zwezeniach moczowodów u chorych na nowotwory narzadu rodnego.

Authors present methods of treatments at the Clinic of Urology Medical University of Lódz in patients after gynecological tumors with ureteral strictures caused by radiotherapy or progression of cancer.

Noncovalent RNA-peptide complexes detected by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to explore noncovalent interactions between different peptides and ribose nucleic acids (RNAs). One RNA was mixed together with two or more peptides or vice versa to compare the different effects of the molecules for noncovalent complex formation. The matrix 2,4,6-trihydroxyacetophenone was considered optimal for these studies due to the fact that peptides and RNA showed roughly the same peak intensities, in negative ion mode, as well as RNA-peptide complexes being detected. The formation of the noncovalent RNA-peptide complexes showed a correlation with the number of the basic amino acids arginine, lysine and histidine. The strongest influence of these amino acids for complex formation was obtained with arginine. Although different RNA molecules were used with different compositions and secondary structures, no specific effects to complex formation was observed. The comparison of noncovalent complexes with covalent RNA-peptide complexes, which were obtained from ribosomal subunits after cross-linking and enzymatic cleavages, showed that the specific RNA-protein interactions are dependent on the three-dimensional structure of the ribosome and its components. The results of this report indicate that MALDI-MS may be useful for the study of noncovalent interactions, in particular for peptides and RNA.

Core histones and HIRIP3, a novel histone-binding protein, directly interact with WD repeat protein HIRA.

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second alpha helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development.

Chemo-enzymatic synthesis of a new type of enantiomerically pure carbocyclic nucleoside analogues with strong inhibitory effects on terminal deoxynucleotidyl transferase.

The synthesis of enantiomerically pure carbocyclic adenosine derivatives which have been prepared based on the kinetic resolution of a trans-2-(hydroxymethyl)cyclopentanol derivative is described. Their corresponding triphosphates were evaluated as inhibitors of DNA polymerase beta, terminal deoxynucleotidyl transferase (TdT), telomerase, Escherichia coli DNA polymerase I and reverse transcriptase of human immunodeficiency virus. Surprisingly, the triphosphate of (1S,2R)-1-(6-aminopurin-9-yl)-2-(hydroxymethyl)cyclopentane [(1S,2R)-6] and its enantiomer (1R,2S)-6 emerged as strong inhibitors of TdT (Ki = 0.5 and 1.9 mM, Kmapp dATP = 40 mM), whereas the activities of all other enzymes tested proved to be unaffected.

Biochemical classification of kidney carcinoma biopsy samples using magic-angle-spinning 1H nuclear magnetic resonance spectroscopy.

High resolution 1H nuclear magnetic resonance (NMR) spectra using spinning at the magic angle (1H MAS NMR) have been obtained on intact normal and pathological kidney tissue samples from patients undergoing surgery for renal cell carcinoma (RCC). The spectra were measured on ca. 80 mg samples and provided high resolution 1H NMR spectra in which effects of dipolar couplings, chemical shift anisotropy and magnetic susceptibility differences are minimised thus yielding high spectral resolution. Conventional one-dimensional and spin-echo spectra and two-dimensional J-resolved, TOCSY and 1H-13C HMQC spectra were also measured on selected samples and these allowed the assignment of resonances of endogenous substances comprising both cytosolic and membrane components. The tumour tissues were characterised principally by an increased lipid content. These are the first reported results on human tumour tissues using this technique and the approach offers potential for the rapid classification of different types of tumour tissue.