Synthesis and biological evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives and their precursors as antileukemic agents

We report here the synthesis and preliminary evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives 6(a–k) and their precursors 5(a–k) as potential chemotherapeutic agents. In each case, the structures of the compounds were determined by FTIR, 1H NMR and mass spectroscopy. Among the synthesized molecules, methyl 1-(4-methoxyphenethyl)-2-(4-fluoro-3-nitrophenyl)-1H-benzimidazole-5-carboxylate (5a) induced maximum cell death in leukemic cells with an IC50 value of 3 μM. Using FACS analysis we show that the compound 5a induces S/G2 cell cycle arrest, which was further supported by the observed down regulation of CDK2, Cyclin B1 and PCNA. The observed downregulation of proapoptotic proteins, upregulation of antiapoptotic proteins, cleavage of PARP and elevated levels of DNA strand breaks indicated the activation of apoptosis by 5a. These results suggest that 5a could be a potent anti-leukemic agent.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Complexes of Rare-Earth Metals with Meconic Acid

Two series of complexes of meconic acid (H3 Mec) with rare-earths have been prepared by varying the preparative procedure. The compounds have the general formulae, [Ln(Mec) (H2O)2]·3 H2O (whereLn=La, Ce, Pr, Nd, Sm, Ho and Y) and [Ln(HMec) (H2 Mec) (H2O)2]·4 H2O (whereLn=La, Pr, Nd and Sm). The infrared spectral data indicate that the carboxylate groups are bound to the rare-earth metal in a bidentate fashion. Thermal studies indicate that two water molecules are coordinated in each case. The complexes are probably polymeric.

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.

Photoelastic determination of mode i stress intensity factor in tensile strips—effect of crack length

This paper reports an experimental investigation carried out, using the photoelastic technique, to determine the Mode I stress intensity factor in case of cracks of varying a/w ratio in single edge-notch specimens. The photoelastic information was analysed using the several methods proposed by earlier workers. The experimental results are compared with the analytical expressions.

Reinvestigation of the structure of Feist's acid 3-methylene-trans-1,2-cyclopropanedicarboxylic acid

C6H604, Mr = 142, triclinic, P[, a = 4.842(1), b = 7.607(1), c = 9.168 (3) A, ~ = 98.41(2), fl = 99.89(2), y = 77.74(1) ° , V = 320.9/k 3, Z = 2, Dm= 1.45 (flotation), D x = 1.470 g cm -3, p(Mo Ktt, 2 = 0.7107 A) = 0.63 cm -~, F(000) = 148. The structure was solved by direct methods and refined to an R value of 0.038 for 723 intensity measurements. The geometrical changes in the cyclopropane ring are discussed in the light of substituent effects. In the crystal structure the carboxylic groups are disordered.

Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2x10(-201)), ABCG2 (p = 3.1x10(-26)), SLC17A1 (p = 3.0x10(-14)), SLC22A11 (p = 6.7x10(-14)), SLC22A12 (p = 2.0x10(-9)), SLC16A9 (p = 1.1x10(-8)), GCKR (p = 1.4x10(-9)), LRRC16A (p = 8.5x10(-9)), and near PDZK1 (p = 2.7x10(-9)). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0x10(-26)) and propionyl-L-carnitine (p = 5.0x10(-8)) concentrations, which in turn were associated with serum UA levels (p = 1.4x10(-57) and p = 8.1x10(-54), respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Rain Damage to Strawberries Grown in Southeast Queensland: Evaluation and Genetic Control.

In Queensland, Australia, strawberries (Fragaria xananassa Duchesne) are grown in open fields and rainfall events can damage fruit. Cultivars that are resistant to rain damage may reduce losses and lower risk for the growers. However, little is known about the genetic control of resistance and in a subtropical climate, unpredictable rainfall events hamper evaluation. Rain damage was evaluated on seedling and clonal trials of one breeding population comprising 645 seedling genotypes and 94 clones and on a second clonal population comprising 46 clones from an earlier crossing to make preliminary estimates of heritability. The incidence of field damage from rainfall and damage after laboratory soaking was evaluated to determine if this soaking method could be used to evaluate resistance to rain damage. Narrow-sense heritability of resistance to rain damage calculated for seedlings was low (0.21 +/- 0.15) and not significantly different from zero; however, broad-sense heritability estimates were moderate in both seedlings (0.49 +/- 0.16) and clones (0.45 +/- 0.08) from the first population and similar in clones (0.56 +/- 0.21) from the second population. Immersion of fruit in deionized water produced symptoms consistent with rain damage in the field. Lengthening the duration of soaking of 'Festival' fruit in deionized water exponentially increased the proportion of damage to fruit ranging in ripeness from immature to ripe during the first 6-h period of soaking. When eight genotypes were evaluated, the proportion of sound fruit after soaking in deionized water in the laboratory for up to 5 h was linearly related (r(2) = 0.90) to the proportion of sound fruit in the field after 89 mm of rain. The proportion of sound fruit of the breeding genotype '2008-208' and 'Festival' under soaking (0.67, 0.60) and field (0.52, 0.43) evaluations, respectively, is about the same and these genotypes may be useful sources of resistance to rain damage.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.

Texture dependent stress corrosion failure of commercial titanium sheets in bromine-methanol solution

The stress corrosion cracking (SCC) characteristics of agr-titanium sheets in a bromine-methanol solution have been studied in the annealed and cold-rolled conditions using longitudinal and transverse specimens. The times to failure for annealed longitudinal specimens were longer than those for similarly tested transverse specimens. The cold-rolled specimens developed resistance to SCC, but failed by cleavage when notched, unlike the intergranular separation in annealed titanium. The apparent activation energy was found to be texture dependent and was in the range 30 to 51 kJ mol–1 for annealed titanium, and 15kJ mol–1 for cold-rolled titanium. The dependence of SCC behaviour on the texture is related to the changes in the crack initiation times. These are caused by changes in the passivation and repassivation characteristics of the particular thickness plane. The thickness planes are identified with the help of X-ray pole figures obtained on annealed and cold-rolled material. On the basis of the activation energy and the electrochemical measurements, the mechanism of SCC in annealed titanium is identified to be the one involving stress-aided anodic dissolution. On the other hand, the results on the cold-rolled titanium are in support of the hydrogen embrittlement mechanism consisting of hydride precipitation. The cleavage planes identified from the texture data match with the reported habit planes for hydride formation.

Structure of a new crystal form of 2- ([3-(trifluoromethyl)phenyl] amino)benzoic acid (flufenamic acid)

C14Ht0F3NO2, P2.Jc, a = 12.523 (4), b = 7.868(6), c = 12.874 (3)A, fl = 95.2 (2) ° , O,,, = 1.47 (4), D e = 1.47 Mg m -3, Z = 4. Final R = 0.074 for 2255 observed reflections. The carboxyl group and the phenyl ring bearing the carboxyl group are nearly coplanar whereas the two phenyl rings are inclined with respect to each other at 52.8 ° . The difference between the two polymorphs of flufenamic acid lies in the geometrical disposition of the [3-(trifluoromethyl)- phenyl]amino moiety with respect to the benzoic acid moiety. As in other fenamate structures, the carboxyl group and the imino N atom are connected through an intramolecular hydrogen bond; also, pairs of centrosymmetrically related molecules are connected through hydrogen bonds involving carboxyl groups.