Effects of Novel Synthetic Nucleosides as Anti-Tumor Agents on Human Acute Promyelocytic Leukemia Cell Line (HL-60)

Nucleosides and their analogs are considered a clinically proven class of therapeutic agents possessing anticancer and antiviral activity. Several trifluoromethyl-substituted pyrazole N-nucleosides (coded NIA, NIIA and NIIA) and their nucleobases were synthesized and tested for the ability to induce apoptosis in acute human promyelocytic cell line (HL-60). The growth and proliferation of HL-60 was more effectively inhibited by NIA, NIIA and their nucleobases compared to NIIIA and its nucleobase. In addition, DNA fragmentation was detected in a concentration-dependent manner as a result of nucleosides treatment. A caspase-3-dependent apoptosis was observed based on the Western blot analysis of poly-ADP-ribose polymerase (PARP) and caspase-3. Nucleosides and nucleobases also trigger the release of cytochrome-c from the mitochondria by disruption of mitochondrial membrane potential and ROS formation. Furthermore, the use of zDEVD-fmk (caspase-3 inhibitor) and zLEHD-fmk (caspase-9 inhibitor) resulted in an inhibition of the activity of caspase-3 and 9, accompined with no change in the activity of caspase-8 after the use of zIETD-fmk (casapse-8 inhibitor). These findings implicate the involvement of the caspase-9-dependent mitochondrial pathway. Treatment with nucleosides also resulted in a concentration-dependent upregulation and translocation of the proapoptotic molecule Bax, increased expression of other proapoptotic proteins Bad, Bak, decreased expression of antiapoptotic proteins (i.e. Bcl-2 and Bcl-xL), and enhancement of p53 expression. Moreover, treatment with these agents resulted in accumulation of cells in the G0-G1 phase of the cell cycle, indicating the degradation of the cellular DNA. Interestingly, these nucleosides were found to posses anti-histone deacetyl transferase(s) (HDACs) potential, which is an exciting turn in cancer therapy. This conclusion was based on the observed decease in the expressed level of HDAC-1, -2 and -3. Our results suggest that these novel synthetic nucleosides can induce apoptosis via the mitochondrial pathway, and therefore they could be considered as candidate anti-tumor agents

Investigating the mechanisms of action of the SNF2-homolog protein Fun30

In eukaryotes, the manipulation of chromatin structure represents a means of regulating access to genetic information. One way in which this can be achieved is via the action of ATP-dependent chromatin remodelling complexes related to the S.cerevisiae Snf2 proteins. The Fun30 protein (Function unknown now 30) shares sequence homology with the Snf2 family of chromatin remodelling proteins. Here the activities and roles of Fun30 have been investigated further. In this study, Fun30 was found to exist predominantly as a homodimer with an apparent molecular weight of 250 KDa. Fun30 binds to DNA, mononucleosomes and nucleosomal arrays. Moreover, Fun30 was shown to cause ATP-dependent alterations to nucleosome structure. This can involve increasing the accessibility of DNA, octamer displacement by sliding in cis or transfer to separate DNA molecules.However, Fun30 was shown to be proficient in catalysing the exchange of histone dimers. Deletion of fun30 was observed to increases resistance to UV, IR and 6-azauracil raising the possibility that this protein has functions in DNA repair. Deletion of fun30 was also found to result in temperature sensitivity. At the non permissive temperature cells were found to accumulate in the S-phase of the cell cycle. Under these conditions Rad53 was found to be phosphorylated which is consistent with the activation of the S-phase checkpoint. In order to investigate the role of Fun30 in DNA replication samples were prepared to map the genome-wide locations of Fun30 at different stages in the cell cycle. Preliminary analysis of this data suggests that there is a transient enrichment of Fun30 protein within the vicinity of replication origins during S-phase.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in pathological cardiac hypertrophy, but the mechanisms by which it modulates gene activity in the nucleus to mediate hypertrophic signaling remain unclear. Here, we report that nuclear CaMKII activates cardiac transcription by directly binding to chromatin and regulating the phosphorylation of histone H3 at serine-10. These specific activities are demonstrated both in vitro and in primary neonatal rat cardiomyocytes. Activation of CaMKII signaling by hypertrophic agonists increases H3 phosphorylation in primary cardiac cells and is accompanied by concomitant cellular hypertrophy. Conversely, specific silencing of nuclear CaMKII using RNA interference reduces both H3 phosphorylation and cellular hypertrophy. The hyper-phosphorylation of H3 associated with increased chromatin binding of CaMKII occurs at specific gene loci reactivated during cardiac hypertrophy. Importantly, H3 Ser-10 phosphorylation and CaMKII recruitment are associated with increased chromatin accessibility and are required for chromatin-mediated transcription of the Mef2 transcription factor. Unlike phosphorylation of H3 by other kinases, which regulates cellular proliferation and immediate early gene activation, CaMKII-mediated signaling to H3 is associated with hypertrophic growth. These observations reveal a previously unrecognized function of CaMKII as a kinase signaling to histone H3 and remodeling chromatin. They suggest a new epigenetic mechanism controlling cardiac hypertrophy

Deletion of low molecular weight protein tyrosine phosphatase (Acp1) protects against stress-induced cardiomyopathy.

The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function in the heart and in cardiac diseases remains unknown. To investigate the in vivo role of LMPTP in cardiac function, we generated mice with genetic inactivation of the Acp1 locus and studied their response to long-term pressure overload. Acp1(-/-) mice develop normally and ageing mice do not show pathology in major tissues under basal conditions. However, Acp1(-/-) mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the postnatal stage, and increased in the adult mouse failing heart. We also show that LMPTP expression increases in end-stage heart failure in humans. Consistent with their protected phenotype, Acp1(-/-) mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of fibrotic genes. Transcriptional profiling and analysis of molecular signalling show that the resistance of Acp1(-/-) mice to pathological cardiac stress correlates with marginal re-expression of fetal cardiac genes, increased insulin receptor beta phosphorylation, as well as PKA and ephrin receptor expression, and inactivation of the CaMKIIδ pathway. Our data show that ablation of Lmptp inhibits pathological cardiac remodelling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of human heart failure

The Snf2 Homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones

FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Ubiquitination defect in cells expressing mutant FBXO32. a Co-immunopricipitation analysis. HEK293 cells were transfected with the indicated plasmids and immunoblot analysis was performed from total cell lysates using a specific anti-ubiquitin antibody. FBXO32 expression is shown as well as GAPDH. The blot is representative of three independent experiments. b Immunoblot analysis of the ubiquitination in cardiomyocytes. Cells were transfected with the Flag-FBXO32-WT or Flag-FBXO32-Mutant and whole cell extracts were analyzed by immunoblotting using the indicated antibodies. (TIF 1928 kb

Out-of-Wedlock Pregnancy Among Single Mothers in Khartoum, Sudan: Sociodemographic Characteristics, Causes, and Consequences

Background: Out-of-wedlock childbearing is a global phenomenon that has lifelong consequences on the lives of both mothers and their children. The aim of this study is to identify the sociodemographic characteristics, causes, and consequences of outof- wedlock pregnancy among single mothers in Khartoum, Sudan.Methods: This descriptive, cross-sectional study was conducted at the Mygoma Orphanage Center (MOC) and Shamaa Rehabilitation Center (SRC) using convenience sampling among 200 participants. A validated questionnaire with 25 items was used to collect data. The data were entered into Epi-Data Manager and analyzed using the SPSS. Results: The study found that most of the single mothers in Khartoum who gave birth out of wedlock were young and had just completed their university education. Most of them discovered their pregnancy during the second or third trimester, and nearly half of them did not receive any antenatal care. The majority of the children born to these mothers were preterm and had a low birth weight. Additionally, many mothers reported experiencing social stigma and rejection from their families due to their out-of-wedlock pregnancy. The study also highlighted loneliness, stress, and romantic relations as the main causes of out-of-wedlock pregnancy among single mothers in Khartoum, Sudan.Conclusion: The study provides useful insights into the sociodemographic characteristics, causes, and consequences of out-of-wedlock pregnancy among single mothers in Khartoum, Sudan. Social stigma and lack of support were identified as significant barriers to the reintegration of single mothers and their children into society. Future research should focus on investigating the long-term effects of outof- wedlock pregnancy on mothers and their children

Evaluating the Feasibility of Pro-Neurotensin and 25-Hydroxyvitamin D3 as Possible Indicators for Type 2 Diabetes Mellitus and Its Complications

(1) Background: Type 2 diabetes mellitus (T2DM) and metabolic syndrome are associated with decreased vitamin D. In contrast, high pro-neurotensin (pro-NT) levels are linked with an increased risk of T2DM and cardiovascular disease. We aimed to determine the validity of pro-NT and 25-dihydroxy vitamin D3 levels as predictors for T2DM complications; (2) Methods: One hundred T2DM, and one hundred healthy volunteers participated in this case-control study. Their Pro-NT and 25-hydroxyvitamin D3 levels were evaluated using the ELISA technique; (3) Results: Pro-NT and 25 (OH) vitamin D3 have significant validity and accuracy in T2DM prediction, 84.5%, and 90.5%, respectively (p = 0.001). At a value of \u3c29.5, 25-Hydroxy vitamin D3 showed 88% sensitivity and 93% specificity in predicting T2DM. At a value of \u3e124 Pmol/L, Pro-NT showed 81% sensitivity and 88% specificity in predicting T2DM. At a value of 16.5, 25-Hydroxy vitamin D3 had 78.4% sensitivity and 68.3% specificity in predicting T2DM complications. At a value of \u3e158 pmol/L, Pro-NT predicted T2DM complications with 67.6% sensitivity and 56.0% specificity; (4) Conclusions: 25 (OH) Vit D3 and Pro-NT could identify T2DM patients and predict T2DM complications. More extensive research is required to adequately validate this novel perspective with a large population study

Prevalence of Comorbidities Associated With Type 2 Diabetes Mellitus In Ksa: A Cross-Sectional Study

Objective: To determine the prevalence of comorbidities associated with type 2 diabetes mellitus among the Saudi adult population. Methods: A cross-sectional study design will be employed to investigate the prevalence of comorbidities associated with Type 2 Diabetes Mellitus (T2DM) within the Kingdom of Saudi Arabia (KSA) population. This design allows for the collection of data at a single point in time, providing insights into the existing relationship between T2DM and comorbidities.Results: The study included 601 participants. The most frequent body mass index BMI value among study participants was overweight 25-29.9 kg/m2 (n= 196, 33%), followed by Normal 18.5-24.9 kg/m2 (n= 177, 29%). The frequent gender among study participants was male years (n= 356, 59%) and female (n= 245, 41%). The most frequent age among study participants was 40-50 years (n= 145, 24%), followed by 18-28 years (n= 140, 23%). The most frequent marital status among study participants was married (n= 360, 60%), followed by single (n= 160, 27%). The perceived physical activity per week among study participants, most of whom did not do any activity (n=231,38%) followed one-time activity (n=98,16%). Conclusion: The results showed that most of the study participants were overweight according to their BMI. The majority of patients had normal blood pressure. Most of the participants suffer from diabetes. In addition, most of the participants do not do any activity, and the majority of participants are non-smokers