Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.

The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.     

Desialylation of transferrin by liver endothelium is selective for its triantennary chain.

Liver endothelium can remove and transport the glycoprotein transferrin (TF). During this process the molecules are desialylated; however, in contrast with other such glycoproteins, for example caeruloplasmin, only half of transported TF is desialylated. To explore which component of TF is desialylated, we double-labelled fully sialylated TF with [3H]sialic acid residues and a 125I-protein moiety. This was then 'chased' through purified liver endothelium in pulse-chase experiments. Endothelium-conditioned TF was fractionated on an RCA120 affinity column into sialylated and desialylated components. Each component was then re-fractionated on a concanavalin A affinity column, which separates the glycoprotein according to the branching pattern of its glycan chain. The desialylated fraction was eluted only as a triantennary component, whereas the non-desialylated fraction consisted only of bi- and tetra-antennary chains. The significance of this selective desialylation of triantennary chain of TF in the subsequent metabolism of its iron content and its possible role in the pathogenesis of alcohol-induced hepatic siderosis are discussed.     

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.     

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).     

Scaffolds derived from cancellous bovine bone support mesenchymal stem cells' maintenance and growth

Since bone defects can lead to various disabilities, in recent years, many increasing attempts have been made in bone tissue engineering. In this regard, scaffolds have attracted a lot of attention as three dimensional substrates for cell attachment which improve successful tissue engineering. The aim of the present study was to provide an interconnected porous scaffold to facilitate cell infiltration. To do so, cancellous bone from bovine femur was dissected in fragments and decellularized by physicochemical methods, including snap freeze/thaw, rinsing in hot water and treatment with different solutions of sodium dodecyl sulfate (SDS). Histological analysis and 4′,6-diamidino-2-phenylindole staining revealed that the best results were obtained after treatment with 2.5%, 5%, and 8% SDS for 8, 3, or 1 h respectively, which significantly removed bone cells with intact trabeculae geometry. Further characterization of decellularized scaffolds by the compression tests also revealed no significant difference between elastic modulus values of the three different SDS treatments. Moreover, studying the ratio of bone trabeculae to bone surfaces (BT/BS) as assessed by Clemex vision software 3.5 showed that treatment with 2.5% SDS for 8 h resulted in a BT/BS score in the range of native bone and therefore this treatment was used for further experiments. Histological studies and scanning electron microscopy revealed rat mesenchymal stem cells integration, adhesion, and maintenance during the 2 and 7 d of culture in vitro. In conclusion, the present results support the effective role of SDS in cancellous bovine bone decellularization and also propensity of treated samples in providing a suitable three-dimentional environment to support the maintenance and growth of mesenchymal stem cells.     

Clinical update on cancer: molecular oncology of head and neck cancer

Head and neck cancers encompass a heterogeneous group of tumours that, in general, are biologically aggressive in nature. These cancers remain difficult to treat and treatment can cause severe, long-term side effects. For patients who are not cured by surgery and/or (chemo)radiotherapy, there are few effective treatment options. Targeted therapies and predictive biomarkers are urgently needed in order to improve the management and minimise the treatment toxicity, and to allow selection of patients who are likely to benefit from both nonselective and targeted therapies. This clinical update aims to provide an insight into the current understanding of the molecular pathogenesis of the disease, and explores the novel therapies under development and in clinical trials.     

The role of bla(OXA-like carbapenemase) and their insertion sequences (ISS) in the induction of resistance against carbapenem antibiotics among Acinetobacter baumannii isolates in Tehran hospitals

This study aimed to evaluate the occurrence and dissemination of bla(OXA-like) carbapenemase genes and their insertion sequences among Acinetobacter baumannii isolates, taken from different hospitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs. A total number of 100 non duplicate Acinetobacter baumannii with different origins, were isolated from patients with proved nosocomial infections at eight university hospital in Tehran city. Antimicrobial susceptibility of these strains was done by E-test against 7 antimicrobial agents according to CLSI guideline. PCR of bla(OXA-51-like), bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), IS(ABA-1), IS(1133) was carried out by specialized primers and then these strains were typed by REP-fingerprinting. Colistin, imipenem and meropenem were the most sensitive antibiotics against Acinetobacter baumannii isolates with 96%, 51% and 51% sensitivity respectively. All the isolates had a bla(OXA-51-like) intrinsic to these species. The rates of bla(OXA-23), 23 and 58-like were 38%, 32% and 1% respectively. Coexistence of bla(OXA-51/23/24-like) was observed among 16% of these isolates. All bla(OXA-23-like) carbapenemase genes had only one IS(ABA1). REP fingerprinting showed 5 genotypes among carbapenem resistant isolates, 16 of them being genotype A. This study emphasized on the major role of bla(OXA-like) carbapenemase, particularly bla(OXA-23-like) carbapenemase and their IS(ABA1), in the dissemination of carbapenem resistant Acinetobacter baumannii. This study confirmed a presumptive role of IS element neighboring the carbapenemase gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates for the first time in Iran.