Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer.

Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPalphaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.     

On the role of Hsp27 in regulating apoptosis

Heat shock proteins (Hsps) comprise several different families of proteins that are induced in response to a wide variety of physiological and environmental insults. One such protein which is highly induced during the stress response is a 27-kDa protein, termed Hsp27 whose expression is seen to correlate with increased survival in response to cytotoxic stimuli. It has been shown to prevent cell death by a wide variety of agents that cause apoptosis. Hsp27 is a molecular chaperone with an ability to interact with a large number of proteins. Recent evidence has shown that Hsp27 regulates apoptosis through an ability to interact with key components of the apoptotic signalling pathway, in particular, those involved in caspase activation and apoptosis. This article will review recent advances in the field and will address some of the potential mechanisms by which Hsp27 functions as an anti-apoptotic molecule.     

The switch from survival responses to apoptosis after chromosomal breaks

Eukaryotic cells have evolved highly sophisticated cellular responses to DNA double strand breaks (DSBs) that increase the likelihood of survival. However, cells can also respond to DSBs by undergoing programmed cell death. The mechanisms underlying the cellular decision on whether to repair and survive or to die are not well understood but may be related to the efficiency of repair or the extent of the damage. Presumably, a few easily reparable DSBs will not result in cell death in most cell types. However, abundant complex DSBs will present a severe challenge to the repair machineries with repeated attempts at repair likely to result in genome instability. For multicellular eukaryotes at least, struggling to complete repair is folly, whereas removal of severely damaged cells is a more sensible strategy. Here we discuss recent evidence linking DSBs to a highly regulated form of cell death termed, apoptosis. In particular, we focus on the roles of the tumour suppressor, p53 and a recently discovered role for an isotype of the linker histone H1. We present a hypothesis that the elevated levels of ssDNA produced during ongoing attempts at DSB repair may be involved in the switch from repair to apoptosis.     

Sulfonamido, azidosulfonyl and N-acetylsulfonamido analogues of rofecoxib: 4-[4-(N-acetylsulfonamido)phenyl]-3-(4-methanesulfonylphenyl)-2(5H)furanone is a potent and selective cyclooxygenase-2 inhibitor

4-[4-(N-Acetylsulfonamido)phenyl]-3-(4-methanesulfonylphenyl)-2(5H)furanone, possessing a N-acetylsulfonamido pharmacophore, has been identified as a potent and selective COX-2 inhibitor that has the potential to acetylate the COX-2 isozyme.     

Synthesis of carotenoid-cysteine conjugates

Isozeaxanthin under acidic conditions forms an allylic cation which reacts readily with thiol nucleophiles. With N-acetylcysteine as a nucleophile the products obtained are carotenoid-cysteine conjugates in which the amino acid moiety is attached to the carotenoid via sulphur in position 4. The water solubility of the products can be increased by deprotection of the amino group. The antioxidant activity of the products were examined on human liver cells under conditions of hydrogen-peroxide induced oxidative stress.     

Health-risk assessment related to the fluoride,nitrate, and nitrite in the drinking water in the Sanandaj,Kurdistan County,Iran

The concentration of fluoride and nitrate in groundwater is usually higher than that of surface water. The main objective of this study was to estimate the health-risk assessment associated with fluoride, nitrate, and nitrite in drinking water in Sanandaj and its villages. The number of samples in the Sanandaj and its rural was 106. The average concentration of fluoride in urban and rural drinking water was 0.22 mg/l and 0.27 mg/l, respectively. Fluoride concentration was also close to urban and rural drinking water. The concentration of nitrate in urban and rural drinking water was in the range between 0.28–27.77 mg/l and 1.28–80 mg/l, respectively. The concentration of nitrate reported in rural samples was higher than that of urban samples. The maximum concentration of nitrate reported in this study was 80 mg/l. The mean CDI for nitrate in the men, women, and children was 0.4258, 0.5110, and 1.1454, respectively. The findings of this study indicated that all three groups studied were exposed to nitrate contact hazards (HQ > 1). Therefore, the HQ in each of the three groups was higher than 1, which should be carefully monitored and necessary measures should be performed.     

Crystal Structures of PRK1 in Complex with the Clinical Compounds Lestaurtinib and Tofacitinib Reveal Ligand Induced Conformational Changes

Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain – the ‘C-tail’. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.     

Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.