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Aerospace equipages encounter potential radiation footprints through which soft errors occur in the memories onboard. Hence, robustness against radiation with reliability in memory cells is a crucial factor in aerospace electronic systems. This work proposes a novel Carbon nanotube field-effect transistor (CNTFET) in designing a robust memory cell to overcome these soft errors. Further, a petite driver circuit to test the SRAM cells which serve the purpose of precharge and sense amplifier, and has a reduction in threefold of transistor count is recommended. Additionally, analysis of robustness against radiation in varying memory cells is carried out using standard GPDK 90 nm, GPDK 45 nm, and 14 nm CNTFET. The reliability of memory cells depends on the critical charge of a device, and it is tested by striking an equivalent current charge of the cosmic ray’s linear energy transfer (LET) level. Also, the robustness of the memory cell is tested against the variation in process, voltage and temperature. Though CNTFET surges with high power consumption, it exhibits better noise margin and depleted access time. GPDK 45 nm has an average of 40% increase in SNM and 93% reduction of power compared to the 14 nm CNTFET with 96% of surge in write access time. Thus, the conventional MOSFET’s 45 nm node outperforms all the configurations in terms of static noise margin, power, and read delay which swaps with increased write access time.

Bioactive constituents from natural sources are of great interest as alternatives to synthetic compounds for the treatment of various diseases, including diabetes mellitus. In the present study, phytochemicals present in Leucaena leucocephala (Lam.) De Wit leaves were identified by gas chromatography-mass spectrometry and further examined by qualitative and quantitative methods. α-Amylase enzyme activity assays were performed and revealed that L. leucocephala (Lam.) De Wit leaf extract inhibited enzyme activity in a dose-dependent manner, with efficacy similar to that of the standard α-amylase inhibitor acarbose. To determine which phytochemicals were involved in α-amylase enzyme inhibition, in silico virtual screening of the absorption, distribution, metabolism, excretion, and toxicity properties was performed and pharmacophore dynamics were assessed. We identified hexadecenoic acid and oleic acid ((Z)-octadec-9-enoic acid) as α-amylase inhibitors. The binding stability of α-amylase to those two fatty acids was confirmed in silico by molecular docking and a molecular dynamics simulation performed for 100 ns. Together, our findings indicate that L. leucocephala (Lam.) De Wit-derived hexadecanoic acid and oleic acid are natural product-based antidiabetic compounds that can potentially be used to manage diabetes mellitus.

V-ATPases are membrane-embedded protein complexes that function as ATP hydrolysis-driven proton pumps. V-ATPases are the primary source of organellar acidification in all eukaryotes, making them essential for many fundamental cellular processes. Enzymatic activity can be modulated by regulated and reversible disassembly of the complex, and several subunits of mammalian V-ATPase have multiple isoforms that are differentially localized. Although the biochemical properties of the different isoforms are currently unknown, mutations in specific subunit isoforms have been associated with various diseases, making V-ATPases potential drug targets. V-ATPase structure and activity have been best characterized in Saccharomyces cerevisiae, where recent structures have revealed details about the dynamics of the enzyme, the proton translocation pathway, and conformational changes associated with regulated disassembly and autoinhibition.

Targeted drug delivery systems using natural polysaccharide/protein biopolymer for tumor cells are an attractive platform for enriching the therapeutic effects and reducing the side effects of the drug. Carboxymethyl cellulose (CMC) and casein (CA) nanogels (NGs) loaded with curcumin (CUR) were prepared by self-assembly method and fabricated with folic acid (FA) and casein using layer-by-layer (LbL) technique for skin cancer drug delivery. The prepared samples were characterized by techniques like zeta potential, FTIR, XRD, TGA and Cryo-SEM. Both the swelling and in vitro drug release was performed in acidic pH (4.5 and 6.8) and physiological pH 7.4. Hemolysis assays demonstrated that the drug carriers are hemocompatible. Confocal microscope studies indicate facilitated uptake of 2-FA/CA/CUR@CMC-CA NGs in MEL-39 melanoma cancer cell line, which in turn result in a higher potential for apoptosis. Compared to pure CUR and CUR@CMC-CA NGs, the 2-FA/CA/CUR@CMC-CA NGs has lower IC50 value and superior cytotoxicity in MEL-39 cells because of folate-receptor mediated endocytosis evaluated by the cellular viability quantification using MTT assay and optical microscope images. Finally from in vitro skin permeation experiments, 2-FA/CA/CUR@CMC-CA NGs showed 3.47 ± 0.03 to 4.15 ± 0.25 μg/ml CUR concentrations at the stratum corneum, epidermal and dermal layers. Overall, our results put forth 2-FA/CA/CUR@CMC-CA NGs as an aspiring candidate to achieve enhanced anticancer effects against melanoma skin cancer. Keywords: Carboxymethyl cellulose, Casein, Folic acid, Curcumin, Nanogels, Skin cancer

Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast Saccharomyces cerevisiae , isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that V O complexes containing Stv1p could be readily purified without attached V 1 regions. We used this effect to determine structures of the membrane-embedded V O region with Stv1p at 3.1-Å resolution, which we compare with a structure of the V O region with Vph1p that we determine to 3.2-Å resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.

Recently, several attempts have been made to use the phytopharmaceuticals from plant extracts as reducing, capping and stabilizing agents for the biomimetic synthesis of various metal nanoparticles conjugated to the phytopharmaceuticals. These biogenic metal nanoparticles are non-toxic and can be used as contrast agents, drug delivery vehicles and photothermal agents for cancer therapy. Herein, we report the synthesis of both silver and gold nanoparticles using the pollen extract of Phoenix dactylifera (Date Palm), characterization using UV-visible spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, quantitation of phytochemicals capping the nanoparticles using Folin - Ciocalteu's method, cytotoxicity studies on MCF-7 breast cancer cells, cancer cell death analysis using fluorescent microscopy, and modulation of expression of the pro-apoptotic p53 and anti-apoptotic Bcl-2 proteins. The biosynthesis resulted in stable and poly-dispersed silver nanoparticles and gold nanoparticles, exhibiting strong and broad surface plasmon absorption peaks. The elemental analysis confirmed the presence of gold and silver of high purity and also the organic moieties from the plant extract acting as capping and stabilizing agents. The biogenic nanoparticles also exhibited dose-dependent cytotoxicity on MCF-7 cells and showed signs of apoptotic cell death. Immunoassays revealed the upregulation of the pro-apoptotic protein p53 and down-regulation of the anti-apoptotic protein Bcl-2 after the nanoparticle treatment.

The biogenic synthesis of silver nanoparticles (AgNPs) has a wide range of applications in the pharmaceutical industry. Here, we synthesized AgNPs using the aqueous flower extract of Bauhinia tomentosa Linn. Formation of AgNPs was observed using ultraviolet-visible light spectrophotometry at different time intervals. Maximum absorption was observed after 4 h at 420 nm due to the reduction of Ag+ to Ag0. The stabilizing activity of functional groups was identified by Fourier-transform infrared spectroscopy. Size and surface morphology were also analyzed using scanning electron microscopy. The present study revealed the AgNPs were spherical in form with a diameter of 32 nm. The face-centered cubic structure of AgNPs was indexed using X-ray powder diffraction with peaks at 2θ = 37°, 49°, 63°, and 76° (corresponding to the planes of silver 111, 200, 220, 311), respectively. Energy-dispersive X-ray spectroscopy revealed that pure reduced silver (Ag0) was the major constituent (59.08%). Antimicrobial analyses showed that the biosynthesized AgNPs possess increased antibacterial activity (against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), with larger zone formation against S. aureus (9.25 mm) compared with that of E. coli (6.75 mm)) and antifungal activity (against Aspergillus flavus and Candida albican (with superior inhibition against A. flavus (zone of inhibition: 7 mm) compared with C. albicans (zone of inhibition: 5.75 mm)). Inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was found to be dose-dependent with half-maximal inhibitory concentration (IC50) values of 56.77 μg/mL and 43.03 μg/mL for AgNPs and ascorbic acid (control), respectively, thus confirming that silver nanoparticles have greater antioxidant activity than ascorbic acid. Molecular docking was used to determine the mode of antimicrobial interaction of our biosynthesized B. tomentosa Linn flower-powder extract-derived AgNPs. The biogenic AgNPs preferred hydrophobic contacts to inhibit bacterial and fungal sustainability with reducing antioxidant properties, suggesting that biogenic AgNPs can serve as effective medicinal agents.

In annelid worms, the nerve cord serves as a crucial organ to control the sensory and behavioral physiology. The inadequate genome resource of earthworms has prioritized the comprehensive analysis of their transcriptome dataset to monitor the genes express in the nerve cord and predict their role in the neurotransmission and sensory perception of the species. The present study focuses on identifying the potential transcripts and predicting their functional features by annotating the transcriptome dataset of nerve cord tissues prepared by Gong et al., 2010 from the earthworm Eisenia fetida. Totally 9762 transcripts were successfully annotated against the NCBI nr database using the BLASTX algorithm and among them 7680 transcripts were assigned to a total of 44,354 GO terms. The conserve domain analysis indicated the over representation of P-loop NTPase domain and calcium binding EF-hand domain. The COG functional annotation classified 5860 transcript sequences into 25 functional categories. Further, 4502 contig sequences were found to map with 124 KEGG pathways. The annotated contig dataset exhibited 22 crucial neuropeptides having considerable matches to the marine annelid Platynereis dumerilii, suggesting their possible role in neurotransmission and neuromodulation. In addition, 108 human stem cell marker homologs were identified including the crucial epigenetic regulators, transcriptional repressors and cell cycle regulators, which may contribute to the neuronal and segmental regeneration. The complete functional annotation of this nerve cord transcriptome can be further utilized to interpret genetic and molecular mechanisms associated with neuronal development, nervous system regeneration and nerve cord function.

Synthesis of a series of half-sandwich Ru(II) complexes (1-5) containing acylthiourea ligand is reported herein. All the Ru(II) complexes were well characterized by analytical and spectroscopic (UV-Vis, FT-IR, NMR and mass spectrometry) methods. Molecular structures of two (2 and 3) of the complexes were confirmed by single crystal X-ray diffraction, and the complexes adopted pseudo-octahedral geometry around Ru. Catalytic ability of the Ru complexes was evaluated in the synthesis of quinoxaline compounds from various 2-nitroaniline and hydroxy ketone derivatives via transfer hydrogenation approach. Active homogeneous catalyst was heterogenized by supporting it on graphene oxide, and the heterogeneous equivalent was characterized by Raman, XPS, TEM, SEM and ICP-OES techniques. Activity of the heterogeneous catalyst was tested, and it can be reused up to five cycles without any loss in activity.

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation inStreptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced inEscherichia coliand purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space groupP3121, with unit-cell parametersa=b= 126.62,c= 95.63 Å.

The infected diabetic wound ulcer is a significant problem for the diabetic patients, which leads to removal of affected foot site due to its delayed/non-healing tissues. The poly-microbial infections and active matrix metalloproteinases (MMP) are the significant influencing factors to delayed healing in diabetic mice. The main purposes of present investigation are to evaluation the targeted inactivation of MMP and avoid polymicrobial infections by using a combined therapeutic effect of metal chelating dipeptide (L-carnosine) and curcumin with the biocompatible silk protein hydrogel (L-car@cur/SF) dressing in the infected diabetic wound ulcer. The in vitro biological assay methods, such as, cell viability, anti-oxidant activity, anti-inflammatory macrophage cells and inhibition collagenase exhibited that the designed hydrogel matrix to be human cell compatible and could be accelerate for significant diabetic healing potential. The activation of cur/SF matrix by the L-carnosine was persuading the inactivation of matrix metalloproteinase-9 (MMP-9) through its potent chelating effects of Zn2+ ions from the MMP-9 active center. The L-car@cur/SF hydrogel was demonstrated for the effective MMP-9 inactivation and bacterial inhibition via in vivo mice wound site, which indorsed the diabetic wound healing efficiency in streptozotocin-tempted diabetic mice.

The synthesis of a rigid 4,5-bis(triphenylphosphinimino)-2,7-di-tert-butyl-9,9-dimethylxanthene {(Ph3PN)2XT} ligand is outlined, along with a modified synthesis for previously reported 1,8-bis(triphenylphosphinimino)naphthalene {(Ph3PN)2NAP}. Reaction of neutral (Ph3PN)2XT with [Y(CH2SiMe3)3(THF)2] resulted in double cyclometallation, yielding the base-free monoalkyl complex, [({Ph2(C6H4)PN2XT)Y(CH2SiMe3)] (1). Layering a concentrated THF solution of 1 with hexanes at −28 °C afforded THF-coordinated [({Ph2(C6H4)PN}2XT)Y(CH2SiMe3)(THF)]·2THF (1-THF·2THF), with a distorted pentagonal bipyramidal geometry and approximately meridional coordination of the pentadentate {Ph2(C6H4)PN}2XT dianion. Similarly, (Ph3PN)2NAP reacted with [Y(CH2SiMe3)3(THF)2] to afford a THF-coordinated monoalkyl complex, [{(Ph2(C6H4)PN)2NAP}Y(CH2SiMe3)(THF)] (2-THF). Layering a DME solution of 2-THF with hexanes at −28 °C afforded X-ray quality crystals of [[{(Ph2(C6H4)PN)2NAP}Y(CH2SiMe3)(κ2-DME)]·hexane (2-DME·hexane), with a highly distorted pentagonal bipyramidal geometry and a facial coordination mode of the tetradentate {Ph2(C6H4)PN}2NAP dianion.

Both the evaluation and the determination of toxicity of chemical substances present in the environment have implications in human health. In this present study, the natural phenomenon named autotomy, a self-defense mechanism employed by several animals against the toxic chemical contaminants, was considered to assess the toxicity of different chemical substances. We investigated the effects of glucose, sodium chloride, kanamycin, mercuric chloride, arsenic trioxide, and lead oxide on the phenotypes of earthworm Eudrilus eugeniae. Depending on the concentration of different chemicals, worms exhibit unique phenotypes. These phenotypes can be used to identify the toxicity as well as the toxic concentration of the chemicals. Upon exposure to toxic chemicals, worms use different mechanical forces at the site of cleavage furrow to detach its segments. During the detachment, there is no apparent blood loss at both the ends of the worm. Our results show that the mercuric chloride is toxic at the concentration above 5 μg when compared to other chemicals. Based on our findings, the toxic effects of a chemical and the toxic concentration of a chemical can be evaluated in both cost and time-efficient manner; in addition, these chemicals can be classified into the following categories: (1) mercuric chloride is extreme-toxic, (2) arsenic trioxide and lead oxide is toxic, (3) kanamycin and sodium chloride is low-toxic, and (4) glucose is non-toxic.

Background Genetic variants of inosine triphosphatase (ITPA) that confer reduced ITPase activity are associated with protection against ribavirin(RBV)-induced hemolytic anemia in peginterferon(IFN)/RBV-based treatment of hepatitis C virus (HCV). Patients with reduced ITPase activity showed improved treatment efficacy when treated with IFN/RBV. In addition, a genetic polymorphism near the IL28B gene is associated with an improved response to IFN/RBV treatment. RBV has been an important component of IFN-containing regimens, and is currently recommended in combination with several IFN-free regimens for treatment of harder to cure HCV infections. Aim To evaluate whether genetic variations that reduce ITPase activity impact RBV-induced anemia in IFN-free/RBV regimens Methods In this study, genetic analyses were conducted in the PEARL-IV trial to investigate the effect of activity-reducing ITPA variants as well as IL28B polymorphism on anemia, platelet (PLT) counts, and virologic response in HCV genotype1a-infected patients treated with the direct-acting antiviral (DAA) regimen of ombitasvir/paritaprevir/ritonavir and dasabuvir±RBV. Results Reduction in ITPase activity and homozygosity for the IL28Brs12979860 CC genotype protected against RBV-induced anemia. In patients receiving RBV, reduced ITPase activity was associated with reduced plasma RBV concentration and higher PLT counts. ITPase activity had no impact on response to DAA treatment, viral kinetics, or baseline IP-10 levels. Conclusions Our study demonstrates that genetics of ITPA and IL28B may help identify patients protected from RBV-induced anemia when treated with IFN-free regimens. Our work demonstrates for the first time that IL28B genetics may also have an impact on RBV-induced anemia. This may be of particular significance in patients with difficult-to-cure HCV infections, such as patients with decompensated cirrhosis where RBV-containing regimens likely will continue to be recommended.

The role of inflammation in both diabetes and diabetic kidney disease (DKD) is becoming more widely accepted. However, the role of recently characterized T cell cytokines interleukin (IL)-9 and IL-17 in diabetes and especially DKD is less well studied. Transforming growth factor beta (TGF-β) controls the secretion of both of these cytokines. In this study, we estimated the levels of IL-9, IL-17, and TGF-β in the serum of subjects with normal glucose tolerance (NGT = 88) and subjects with type 2 diabetes without (diabetes mellitus (DM) = 65) and with DKD (DKD = 97) using enzyme-linked immunosorbent assay (ELISA), and we correlated these levels with the clinical risk factors of diabetes and DKD. IL-17 levels showed a serial decline and TGF-β levels showed a serial increase from NGT to DM to DKD (p < 0.001). However, the IL-9 levels were significantly reduced in the DM group compared to the NGT and DKD group (p < 0.001). While TGF-β and IL-17 showed a positive and negative correlation, respectively, with fasting and postprandial glucose levels and glycated hemoglobin (HbA1c), IL-9 showed positive correlation with urea and microalbuminuria. Apart from pro-inflammatory cytokines, T helper (Th) cytokines might play an important role in insulin resistance and DKD.

Amidated kyotorphin (l-Tyr-l-Arg-NH2; KTP-NH2) causes analgesia when systemically administered. The lipophilic ibuprofen-conjugated derivative of KTP-NH2 has improved analgesic efficacy. However, fast degradation by peptidases impacts negatively in the pharmacodynamics of these drugs. In this work, selected derivatives of KTP and KTP-NH2 were synthesized to combine lipophilicity and resistance to enzymatic degradation. Eight novel structural modifications were tested for the potential to transverse lipid membranes and to evaluate their efficacy in vivo. The rationale behind the design of the pool of the eight selected molecules consisted in the addition of individual group at the N-terminus, namely the tert-butyloxycarbonyl (Boc), γ-aminobutyric acid (GABA), acetyl, butanoyl, and propanoyl or in the substitution of the tyrosine residue by an indole moiety and in the replacement of the peptidic bond by a urea-like bond in some cases. All the drugs used in the study are intrinsically fluorescent, which enables the use of spectrofluorimetry to sample the drugs in the permeation assays. The results show that the BOC and indolyl derivatives of KTP-NH2 have maximal ability to permeate membranes with concomitant maximal analgesic power. Overall, the results demonstrate that membrane permeation is correlated with analgesic efficacy. However, this is not the only factor accounting for analgesia. KTP-NH2 for instance has low passive permeation but is known to have central action. In this case, hypothetical transcytosis over the blood-brain barrier seems to depend on dipeptide transporters. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 1–10, 2015.

Enhancer of zeste 2 (Ezh2) mainly methylates lysine 27 of histone-H3 (H3K27me3) as part of the polycomb repressive complex 2 (PRC2) together with Suz12 and Eed. However, Ezh2 can also modify non-histone substrates, although it is unclear whether this mechanism has a role during development. Here, we present evidence for a chromatin-independent role of Ezh2 during T-cell development and immune homeostasis. T-cell-specific depletion of Ezh2 induces a pronounced expansion of natural killer T (NKT) cells, although Ezh2-deficient T cells maintain normal levels of H3K27me3. In contrast, removal of Suz12 or Eed destabilizes canonical PRC2 function and ablates NKT cell development completely. We further show that Ezh2 directly methylates the NKT cell lineage defining transcription factor PLZF, leading to its ubiquitination and subsequent degradation. Sustained PLZF expression in Ezh2-deficient mice is associated with the expansion of a subset of NKT cells that cause immune perturbation. Taken together, we have identified a chromatin-independent function of Ezh2 that impacts on the development of the immune system.

This article explores the application of hyperthermia mediated by alpha human folate receptor (αHFR) targeted gold nanoparticles (GNPs) for potentiating the cytotoxicity of cyclophosphamide (CPA) in αHFR positive breast cancer cells. Folate functionalized GNPs were delivered to highly αHFR positive breast cancer cells MDA-MB-231 and to MCF-7 breast cancer cells that does not express detectable levels of αHFR followed by hyperthermia. We have shown that hyperthermia induced by folate functionalized GNPs sensitized MDA-MB-231 cells by ten-fold to CPA treatment, whereas MCF-7 cells exhibited only onefold chemosensitization. Collectively, the study suggests the feasibility of using αHFR targeted GNPs for facilitating increased cellula r uptake of CPA in cancer cells expressing elevated αHFR, allowing reduction in drug dosage.

While the role of mitochondrial metabolism in controlling T-lymphocyte activation and function is becoming more clear, the specifics of how mitochondrial redox signaling contributes to T-lymphocyte regulation remains elusive. Here, we examined the global effects of elevated mitochondrial superoxide (O2•-) on T-lymphocyte activation using a novel model of inducible manganese superoxide dismutase (MnSOD) knock-out. Loss of MnSOD led to specific increases in mitochondrial O2•- with no evident changes in hydrogen peroxide (H2O2), peroxynitrite (ONOO−), or copper/zinc superoxide dismutase (CuZnSOD) levels. Unexpectedly, both mitochondrial and glycolytic metabolism showed significant reductions in baseline, maximal capacities, and ATP production with increased mitochondrial O2•- levels. MnSOD knock-out T-lymphocytes demonstrated aberrant activation including widespread dysregulation in cytokine production and increased cellular apoptosis. Interestingly, an elevated proliferative signature defined by significant upregulation of cell cycle regulatory genes was also evident in MnSOD knock-out T-lymphocytes, but these cells did not show accelerated proliferative rates. Global disruption in T-lymphocyte DNA methylation and hydroxymethylation was also observed with increased mitochondrial O2•-, which was correlated to alterations in intracellular metabolite pools linked to the methionine cycle. Together, these results demonstrate a mitochondrial redox and metabolic couple that when disrupted may alter cellular processes necessary for proper T-lymphocyte activation., Graphical abstract Image 1, Highlights • Loss of MnSOD elevates steady-state mitochondrial superoxide in T-lymphocytes. • Elevated mitochondrial superoxide disrupts mitochondrial and glycolytic metabolism. • CD4+ and CD8+ T-lymphocytes show differential effects to increased superoxide. • Mitochondrial superoxide may affect T-lymphocytes via metabolic and epigenetic changes.

Paclitaxel (TAX) amount remaining to be absorbed from the intestine in the in situ closed loop, re-circulating absorption samples were estimated by the 3D view LC-diode array method. The Log ARA versus time graph (r2) of TAX loaded nanoparticles (NP)s explains that there is exponential disappearance of TAX from the intestine, which indicates TAX NPs follows first order absorption kinetic. The calculated Ka and t1/2 of TAX and TAX loaded NPs were 0.9, 1.02, 1.38, and 1.74/min and 46.20, 40.66, 30.09, and 23.88 min, respectively, shows the rapid absorption of TAX NPs from the intestine than the pure drug TAX. The 3D view chromatogram, counter view graph, UV, and IR spectrum of the degradation samples confirms the selectivity and specificity of the developed method. The peak purity curve of all the samples shows that the similarity curve did not intersect the threshold curve and the rectangular ratiograms, in addition, demonstrates the selectivity and specificity nature of the 3D view LC-method. The accuracy...

The drivers of abnormal DNA methylation in human cancers include widespread aberrant splicing of the DNMT3B gene, producing abnormal transcripts that encode truncated proteins that may act as dominant negative isoforms. To test whether reduced Dnmt3b dosage can alter tumorigenesis, we bred Dnmt3b(+/-) mice to Eµ-Myc mice, a mouse model susceptible to B-cell lymphomas. Eµ-Myc/Dnmt3b(+/-) mice showed a dramatic acceleration of lymphomagenesis, greater even than that observed in Eµ-Myc mice that express a truncated DNMT3B isoform found in human tumors, DNMT3B7. This finding indicates that Dnmt3b can act as a haploinsufficient tumor suppressor gene. Although reduction in both Dnmt3b dosage and expression of DNMT3B7 within the Eµ-Myc system had similar effects on tumorigenesis and DNA hypermethylation, different molecular mechanisms appear to underlie these changes. This study offers insight into how de novo DNA methyltransferases function as tumor suppressors and the sensitivity of Myc-induced lymphomas to DNA methylation.

Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1*) using Cre/Lox technology. Systemic activation of the IRP1* transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1* activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson’s disease or Friedreich’s ataxia. Electronic supplementary material The online version of this article (doi:10.1007/s00109-013-1008-2) contains supplementary material, which is available to authorized users.

Streptomyces are a major group of soil bacteria that produce wide range of bioactive compounds including antibiotics. Daunorubicin is a chemotherapeutic agent for treatment of certain types of cancer, which is produced as a secondary metabolite by S. peucetius. Owing to the significance of this drug in treating cancer, understanding the molecular mechanism of its biosynthesis will assist in the genetic manipulation of this strain for better drug yields. Additionally, the knowledge can also be applied to design hybrid antibiotics that can be made in vivo by transferring genes from one Streptomyces species to another. Biosynthesis of daunorubicin in S. peucetius is accomplished by the function of 30 enzyme-coding genes in a sequential and coordinated fashion. In addition to these enzymes, three transcriptional regulators DnrO, DnrN and DnrI regulate this multi-step process by forming a coherent feed forward loop regulatory circuit, consequently controlling the entire enzyme coding genes. Since daunorubicin is a DNA intercalating drug, maintaining an optimal intracellular drug concentration is pivotal to prevent self-toxicity. Commencement of daunorubicin biosynthesis also activates the feedback mechanisms mediated by the metabolite. At exceeding intracellular concentrations, daunorubicin intercalates into DNA sequences and impedes the binding of these transcription factors. This feedback repression is relieved by a group of self-resistance genes, which concurrently efflux the excess intracellular daunorubicin. This review will discuss the mechanistic role of each transcription factor and their interplay in initiating and maintaining the biosynthesis of daunorubicin in S. peucetius.

Growing laccase utilization in numerous biotechnological applications encourages the search for high benefit/cost ratio purification methods such as Three Phase Partitioning (TPP). TPP was employed to the crude extract of Pleurotus ostreatus , gave yielded nearly 161% with 27.8 fold purity. The specific activity of the TPP purified laccase was 91 U/mg and the molecular mass of isozymes was estimated to be 72 kDa and 40 kDa. An extracellular laccase acted optimally at pH 6.0 and exhibited an optimum temperature of 45 °C. The enzyme was able to sustain its activity even at elevated temperatures (50–60 °C) for extended periods (2–3 h). Laccase displayed a high affinity towards syringaldazine and oxidizes other phenolic compounds. This laccase was activated by Mn 2+ and glycerol, but completely inhibited by the classic laccase inhibitors, oxalic acid, Hg 2+ , Pb 2+ and Fe 2+ . The purified laccase could efficiently decolorize triphenyl methane (TPM), azo and indigo dyes without addition of redox mediators. The biodegradation of malachite green (MG) was monitored by UV–vis, IR-spectroscopy and HPLC. The thermostable and rapid dye decolorizing property makes it an attractive and promising biocatalysts for the environment biotechnological applications.

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34+ cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34+ cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.

DnrO is a transcription factor that regulates biosynthesis of secondary metabolite daunorubicin (DNR) in Streptomyces peucetius. DNR is a DNA-intercalating drug widely used in cancer chemotherapy. Binding of DnrO close to its promoter fulfils dual functions, namely activation of dnrN and repression of dnrO. DnrN protein binds to a sequence close to the dnrI promoter to activate it, which is essential for turning on biosynthetic genes. In this study, we analyzed the inhibition of DNA-DnrO complex formation by DNR and its effect on dnrO and dnrN expression. The intracellular concentration of drug required to alter the expression of these two genes was determined in vitro. Based on the results, a model is proposed which describes the modulation of dnrN and dnrO expression by intracellular stoichiometric concentration of the drug DNR and protein DnrO. This regulatory mechanism would maintain optimal intracellular drug concentrations in S. peucetius. This would imply that the organism has an adaptive mechanism to escape the cytotoxicity of DNR in addition to its self-resistance.

Iron-sulfur cluster-dependent interconversion of iron regulatory protein 1 (IRP1) between its RNA binding and cytosolic aconitase (c-acon) forms controls vertebrate iron homeostasis. Cluster removal from c-acon is thought to include oxidative demetallation as a required step, but little else is understood about the process of conversion to IRP1. In comparison with c-acon(WT), Ser(138) phosphomimetic mutants of c-acon contain an unstable [4Fe-4S] cluster and were used as tools to further define the pathway(s) of iron-sulfur cluster disassembly. Under anaerobic conditions cluster insertion into purified IRP1(S138E) and cluster loss on treatment with NO regulated aconitase and RNA binding activity over a similar range as observed for IRP1(WT). However, activation of RNA binding of c-acon(S138E) was an order of magnitude more sensitive to NO than for c-acon(WT). Consistent with this, an altered set point between RNA-binding and aconitase forms was observed for IRP1(S138E) when expressed in HEK cells. Active c-acon(S138E) could only accumulate under hypoxic conditions, suggesting enhanced cluster disassembly in normoxia. Cluster disassembly mechanisms were further probed by determining the impact of iron chelation on acon activity. Unexpectedly EDTA rapidly inhibited c-acon(S138E) activity without affecting c-acon(WT). Additional chelator experiments suggested that cluster loss can be initiated in c-acon(S138E) through a spontaneous nonoxidative demetallation process. Taken together, our results support a model wherein Ser(138) phosphorylation sensitizes IRP1/c-acon to decreased iron availability by allowing the [4Fe-4S](2+) cluster to cycle with [3Fe-4S](0) in the absence of cluster perturbants, indicating that regulation can be initiated merely by changes in iron availability.

Exercise and physical strenuous activity have been demonstrated to increase the serum TNF-alpha and IL-6. Regular physical training is expected to attenuate such a response. This study was undertaken to understand the impact of regular exercise training on IL-6 and TNF-alpha in athletes and non-athletes. Ten athletes, who have been on regular training for the past 6 months, and 10 age- and sex-matched subjects (non-athlete group) who had no practice of regular exercise, were recruited. Both were subjected to undergo the same frequency level of strenuous exercise. Blood samples were collected; one before strenuous exercise and the other after the exercise. Plasma cytokines, IL-6 and TNF-alpha, were estimated using Sandwich ELISA method. All participants in the study were male with the athletes' age being 18.00+/-1.3years (mean+/-SD) and the non-athletes were aged 20.00+/-0.6years (mean+/-SD). Majority of the athletes and non-athletes demonstrated a rise in IL-6 and a fall in TNF-alpha levels. Further, the athletes showed a lesser magnitude of change in the cytokine levels following a longer duration of exercise than non-athletes. Athletes appear to have an attenuated cytokine response. Regular physical training has been demonstrated to attenuate the immune response to exercise in either direction.

© Springer-Verlag Wien 2015, Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same., Fundação para a Ciência e Tecnologia (Portugal) is acknowledged for funding (Grant Nos. SFRH/BD/65709/2009, PTDC/BIA-PRO/104378/2008 and PEst-OE/EQB/LA0004/2011). Katia Conceição and Vasanthakumar Ramu were recipients of Marie Curie IAPP fellowships. Marie Curie Industry-Academia Partnerships and Pathways (European Commission) is acknowledged for funding (FP7-PEOPLE-2007-3-1-IAPP. Project 230654). The financial contributions of the Deutsche Forschungsgemeinschaft (to MM), the Agence Nationale de la Recherche (projects TRANSPEP, ProLipIn, membrane DNP and the LabEx Chemistry of Complex Systems), the University of Strasbourg, the CNRS, the Région Alsace and the RTRA International Center of Frontier Research in Chemistry are gratefully acknowledged.

Background: Fanconi anemia (FA) is caused by mutations in one of seventeen genes that make up the FA DNA double strand break (DSB) repair pathway. Recently, two individuals with biallelic germline BRCA1 mutations, each consisting of one null and one hypomorphic mutation, were identified and noted to have features consistent with FA, including congenital anomalies and increased chromosomal breakage of lymphocytes on exposure to diepoxybutane (Domchek et al. Cancer Discov. 2013 Apr; (4):399-405; and Sawyer et al. Cancer Discov. Epub. 2014 Dec 3.), adding BRCA1 as the newest FA gene. However, neither patient developed bone marrow failure (BMF), making the bone marrow effects of BRCA1 deficiency still unclear. Methods: To test the hypothesis that Brca1 is also essential in hematopoiesis, we developed a conditional mouse model with Mx-1 Cre-mediated Brca1 deletion and examined the effects of Brca1 deficiency on hematopoiesis in this model. Results: At baseline, Brca1-/- mice have macrocytic anemia and leukopenia. Further, by 6 months of age, 30% and 50% of the Brca1-/- mice develop spontaneous BMF or hematologic malignancies (HM), respectively. Brca1-/- mice develop a diverse range of HM, including T-cell lymphomas and acute myeloid leukemias, suggesting a defect in an early hematopoietic progenitor population. Methylcellulose colony forming assays also demonstrate a defect in progenitor cell function with Brca1-/- bone marrow cells forming fewer colonies (44.4±31.9) than Brca1+/+ cells (200.3±30.5, p=0.004) at baseline, and show FA-like hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC) (mean colony survival % at 10 nM MMC 40% versus 82% and at 50 nM 1% vs 56%). Spectral karyotyping of bone marrow cells from mice that developed BMF demonstrated chromatid exchanges and breaks. Similarly, multiple chromosomal translocations were seen in the myeloid leukemia cells, implicating genomic instability in the pathogenesis of these disorders. Conclusions: Taken together, our results show that loss of Brca1 in murine bone marrow causes hematopoietic defects and MMC sensitivity similar to that seen in humans with FA, providing strong evidence that Brca1 is critical for normal hematopoiesis and that Brca1 is a bona fide FA gene. This novel mouse model provides the opportunity to gain functional insight into the key stage(s) of hematopoiesis that require Brca1 and the effects of Brca1 haploinsufficiency, as seen in humans, on hematopoiesis. Further, as nearly all of the single gene FA mouse models to-date have failed to recapitulate the bone marrow phenotype of human FA, this model will be critical for deepening our understanding of the pathogenesis of FA. Disclosures No relevant conflicts of interest to declare.

From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe. chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the Mycobacterium tuberculosis KAPA synthase with both L- and D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 M-1 s(-1)) is similar to 5 times slower than that with L-alanine (k = 399.4 M-1 s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed D-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L- alanine and pimeloyl-CoA (k = 16.9 x 10(4) M-1 s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, D-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (K-i = 114.83 mu M) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 mu M), the next enzyme of the pathway

Summary The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75+ 0.11 M 71 s 71 . The Km values for 7-keto-8aminopelargonic acid (KAPA) and SAM are 2.83 mM and 308.28 mM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 mmoles/min/ml and 0.003 s 71 , respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme. IUBMB Life, 58: 225–233, 2006

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation., info:eu-repo/semantics/published

Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the iron–sulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1/c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis -aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate/isocitrate metabolism.

A simple, efficient and stereospecific approach for the homologation of urethane-protected alpha-amino acids to beta-amino acids by the Arndt-Eistert method employing Fmoc-/Boc-alpha-amino acid and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate mixture for the acylation of diazomethane synthesizing the key intermediates Fmoc-/Boc-alpha-aminodiazomethanes as crystalline solids is described.

The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the1H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.

A simple and concomitant esterification method for the synthesis of methyl, ethyl, t-butyl, benzyl, and 9-fluorenylmethyl esters of Fmoc-/Boc-/Z-beta-homoamino acids employing Fmoc-/Boc-/Z-alpha-aminodiazoketones by Wolff rearrangement is described. The method offers good yield with purity.

The synthesis of C-protected esters of Boc- / Z-α,α-dialkylamino acids is accomplished by using alkyl / aryl chloroformate in presence of DMAP as a catalyst. The reaction proceeds through mixed carboxylic-carbonic anhydride, which was monitored by IR. The reaction was clean and complete in about 2 hr. All the esters prepared have been obtained in good yield and are fully characterized.

The antiviral properties of 5′-noraristeromycin (3) have been attributed to its inhibition of S-adenosylhomocysteine hydrolase. As part of an effort to establish the limiting structural parameters possible for the biological properties of 3, a ring-extended analog possessing 8-aminoimidazo[4,5-g]quinazoline as the base (7) has been prepared and found to be less active than 3.

Amides of Z- and Boc protected amino acids have been prepared by using p-toluenesulphonyl chloride (TsCI) for the activation of carboxyl group. The resulting mixed carboxylic-sulphonic anhydride intermediate was treated in situ with an excess of 25 percent ammonia solution. All the amino acid amides prepared were obtained as crystalline solids in good yield and purity.

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.

Synthesis and biochemical screening against guanase of analogues of the naturally occurring guanase inhibitor azepinomycin (2) are reported. Compound e-amino-5,6,7,8,-tetrahydro-4H-imidazo[4,5-e][1,4]diazepine-5,8-dione (3) was synthesized in six steps commencing with 1-benzyl-5-nitroimidazole-4-carboxylic acid (5). Compound 3 and its synthetic precursor 3-benzyl-6-(N-benzyloxycarbonyl)amino-5,6,7,8-tetrahydro-4H-imidazo[4,5-e][1,4]diazepine-5,8-dione (12) were screened against rabbit liver guanase. Both were found to be moderate inhibitors of the enzyme with K1′s in the range of 10−4 M.

An efficient, three-step synthesis of 5'-amino-5'-deoxyaristeromycin (5), from a protected form of aristeromycin (6), is described. Compound 5 was evaluated against a large number of viruses. It showed weak activity towards vaccinia, herpes simplex virus 2, and cytomegalovirus. No other activity was observed. Compound 5 displayed some cytotoxicity towards the host cell lines human foreskin fibroblast, Daudi, and human T-lymphocyte (CEM).

The synthesis of Fmoc protected amino acid hydroxamates using Fmoc-amino acid chlorides and magnesium oxide is described. The method is simple and efficient, results in complete conversion, and gives good yields and satisfactory purity.

Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed.