Chemo-selective hydrolysis of the iminic moiety in salicylaldehyde semicarbazone promoted by ruthenium

ortho-Hydroxybenzaldehyde semicarbazone (salicylaldehyde semicarbazone) undergoes chemo-selective hydrolysis of the iminic carbon nitrogen double bond through its reaction with [RuCl₂(dmso)₄] in ethanol in the presence of water, yielding free salicylaldehyde and semicarbazide that remains coordinated to the ruthenium ion as a bidentate N,O-donor to afford [RuCl₂(dmso)₂(semicarbazide)] · 2H₂O complex. The ruthenium-semicarbazide complex has been characterized by ¹H NMR and FTIR spectroscopies and X-ray diffraction methods. Related semicarbazones, derived from p-hydroxybenzaldehyde and benzaldehyde, were not hydrolyzed under the same conditions, suggesting a significant role of the structural o-hydroxy motive in the reaction. Theoretical studies were performed in order to gain further insight on the mechanism of reaction. Results support the hypothesis that the ortho-hydroxy moiety, in the keto tautomeric form, participates in the chemo-selective hydrolysis promoted by [RuCl₂(dmso)₄].     

Chronic exposure of rabbits to 0.5 and 5 mW/cm2 2450-MHz CW Microwave Radiation

Two groups of 16 male New Zealand rabbits were exposed to 2450-MHz continuous wave microwave fields in two experiments of 90 days each. The incident power densities of the first and second experiment were 0.5 and 5 mW/cm², respectively. During each study, 16 animals were adapted to a miniature anechoic chamber exposure system for at least 2 weeks, then 8 of them were exposed for 7 h daily, 5 days a week for 13 weeks, and the other 8 animals were sham exposed. The rabbits were placed in acrylic cages, and each was exposed from the top in an individual miniature anechoic chamber. Thermography showed a maximum specific absorption rate of 5.5 W/kg in the head and 7 W/kg in the back at 5-mW/cm² incident power density. After each 7-h exposure session, the animals were returned to their home cages. Food consumption in the exposure chamber and body mass were measured daily. Blood samples were taken before exposure and monthly thereafter for hematological, morphological, chemical, protein electrophoresis, and lymphocyte blast transformation studies. Eyes were examined for cataract formation. Finally, pathological examinations of 28 specimens of organs and tissues of each rabbit were performed. Statistically, there was a significant (P < .01) decrease only of food consumption during the 5-mW/cm² exposure; other variables were not significantly different between exposed and control groups.     

Evidence for chaotic behavior in driven ventricles.

Toad ventricles were externally driven by periodic pulses while monophasic action potential (MAP) signals were recorded in seven excised and seven in situ ventricles. As the frequency was slowly increased in steps, the stimulated tissue displayed several dynamic characteristics. Hierarchies of periodic behavior, like phase-locking and period-doubling sequences leading to chaos, were observed. Results showed that subharmonic bifurcations (order one and two) and chaotic-like behavior may systematically occur in the MAP signal within a definite frequency interval in the 1:1 phase locking regime. The chaotic, or more cautiously expressed, chaotic-like behavior is characterized by the power spectrum, the autocorrelation function, the Poincaré map, and the reconstructed 2-D phase portrait. It is concluded that (a) bifurcations of order one and two and the characteristic irregular behavior are evidences of local universal chaotic dynamics in cardiac tissue; (b) there are no qualitative differences in the dynamics of the in situ and excised ventricles; and (c) fibrillation seems to be related to chaotic behavior, but whether they are similar or equivalent phenomena still remains to be seen.     

Photodegradation illuminates the role of polyanions in prion infectivity

Understanding the mechanism by which prion infectivity is encoded by the misfolded protein PrP^Sc remains a high priority within the prion field. Work from several groups has indicated cellular cofactors may be necessary to form infectious prions in vitro. The identity of endogenous prion conversion cofactors is currently unknown, but may include polyanions and/or lipid molecules. In a recent study, we manufactured infectious hamster prions containing purified PrP^Sc, co-purified lipid and a synthetic photocleavable polyanion. The polyanion was incorporated into infectious PrP^Sc complexes and then specifically degraded by exposure to ultraviolet light. Light-induced in situ degradation of the incorporated polyanion had no effect on the specific infectivity of the samples as determined by end-point dilution sPMCA and scrapie incubation time assays. Furthermore, prion strain properties were not changed by polyanion degradation, suggesting that intact polyanions are not required to maintain the infectious properties of hamster prions. Here, we review these results and discuss the potential roles cofactors might play in encoding prion infectivity and/or strain properties.Key words: prion, polyanion, photodegradation, incorporation, PrPThe prion diseases are infectious diseases that are believed to be caused by the conformational change of a host-encoded protein, PrP^C, to a pathogenic conformer PrP^Sc. The controversial “protein-only” hypothesis posits that the infectious agent is composed solely of the misfolded conformer PrP^Sc. There have been many attempts to create infectious prions from purified recombinant PrP protein. However, all of the samples generated in these experiments display relatively low levels of specific infectivity when inoculated intracerebrally into wild-type animals.^1–4 Several lines of evidence suggest that cellular cofactors, such as polyanionic molecules, facilitate the formation of the infectious conformation.^5–14The first in vitro PrP conversion assay used radiolabeled PrP^C substrate purified from mammalian cells mixed with a stoichiometric excess of unlabeled PrP^Sc. This cell free assay produced a protease-resistant, radioactive product termed PrP-res.¹⁵ These pioneering studies showed for the first time that PrP could be specifically transformed in vitro, but the yield using purified substrates was low. Using a modification of the cell free assay in which crude brain homogenate replaced purified PrP^C as the substrate, our laboratory was able to amplify PrP^Sc 6-fold over input prion seed, suggesting that non-PrP constituents of crude brain homogenate might be required for efficient PrP^Sc formation in vitro.¹⁶ Using this system, we discovered that nuclease treatment of hamster brain homogenates abolished PrP^Sc amplification in vitro, and that reconstituting the nuclease-treated reactions with purified mammalian RNA rescued the amplification process.⁵ PrP^Sc amplification could also be obtained by adding certain synthetic homopolymeric nucleic acids to immunopurified PrP^C.⁶ Taken together, these surprising results argue that non-proteinaceous, host-encoded cofactors such as RNA molecules might facilitate prion conversion through a structural (as opposed to encoding) mechanism.⁸ The high efficiency of the serial protein misfolding amplification (sPMCA) technique developed by Soto and colleagues has allowed researchers to amplify prion infectivity as well as PrP^Sc molecules.^17,18 Using sPMCA, we showed that infectious PrP^Sc molecules could be formed from immunopurified PrP^C, co-purified lipid and synthetic RNA molecules. Moreover, even unseeded reactions containing these defined components were capable of generating prions with high specific infectivity in a prion-free environment, showing for the first time that wild type infectious prions could be produced de novo.⁷Additional studies in this purified system showed that PrP^C molecules undergo a time-dependent conformational change upon interaction with RNA. When this change occurs, PrP^C adopts an intermediate conformation that mimics some of the characteristics of PrP^Sc, such as detergent insolubility and reactivity to PrP^Sc-specific antibodies, but remains sensitive to proteinase K digestion.⁸ When incubated with a heterogeneous size mixture of homopolymeric [³²P] poly(A) molecules during PMCA, hamster PrP^C molecules incorporated a specific size subset (1–2.5 kb) of the RNA molecules into nuclease-resistant complexes. The physical interaction between RNA and PrP^Sc was confirmed by fluorescence microscopy experiments showing that fluorescein-labeled RNA molecules became integrated into nuclease-resistant complexes with PrP^Sc molecules. Interestingly, neuropathologic analysis of scrapie-infected hamsters revealed that endogenous RNA molecules stained with acridine orange co-localized with large extracellular PrP aggregates.⁸ Taken together, these studies suggest that PrP interacts specifically with polyanionic molecules in vitro and in situ, and raised the possibility that polyanions might be a necessary component of infectious prions.Jeong et al. investigated whether endogenous RNA molecules might be required for prion infectivity by treating scrapie brain homogenates with LiAlH4 (lithium aluminum hydride), a strong reducing agent that can cleave the phosphodiester bond in RNA molecules.¹⁹ Interestingly, treatment of hamster scrapie brain homogenates with LiAlH₄ caused an ∼3-fold increase in scrapie incubation period measured by bioassay, suggesting that RNA may be an important component of infectious prions and therefore may play a role in stabilizing PrP^Sc structure. However, LiAlH₄ is not a specific reagent, and can damage a variety of other macromolecules, including proteins. Therefore, the decrease in infectivity measured in this study cannot be specifically ascribed to degradation of the polyanion.¹⁹We recently reinvestigated the potential role of polyanion in maintaining prion infectivity by using a more targeted approach.²⁰ Specifically, we utilized a synthetic oligonucleotide that could be selectively hydrolyzed by treatment with ultraviolet (UV) light. The photocleavable oligonucleotide was synthesized by inserting a photocleavable linker in between every fives bases of a poly(dT) 100-mer. Exposure to UV light quantitatively converted the oligonucleotide into five base fragments. During incubation with excess recombinant PrP, the photocleavable oligonucleotide became incorporated into a nuclease-resistant nucleoprotein complex, but remained sensitive to photocleavage. This novel system allowed us to study the role of a polyanion molecule incorporated into infectious prions in situ (Fig. 1).Open in a separate windowFigure 1Selective photodegradation of an incorporated polyanion in situ.We used PMCA to create PrP^Sc molecules that contained either the photocleavable oligonucleotide or a non-photocleavable control analog. After treatment with UV light, the infectivity of each sample was measured using a combination of end-point dilution sPMCA and animal bioassays. The end-point dilution PMCA assay showed a ∼1 log decrease in the seeding ability of PrP^Sc samples treated with UV light, but this effect was not specific since a similar decrease was measured in samples containing the control nucleic acid. In the bioassay, there was no change in the incubation periods of animals inoculated with PrP^Sc samples treated either in the presence or absence of UV light. Neuropathological analysis of inoculated animals also showed no differences in neurotropism between the two groups. Degradation of the nucleic acid had no effect on the molecular migration or structural stability of PrP^Sc samples as determined by SDS-PAGE and urea denaturation assays, respectively. There were also no differences in the molecular migration or glycosylation profile of the PrP^Sc molecules produced in the brains of animals inoculated with light- versus mock-treated inocula, and urea denaturation assays showed no differences in PrP^Sc stability. These results collectively demonstrate that the presence of intact polyanion molecules is not required to maintain the infectious, biochemical or strain properties prions generated in vitro.These results are consistent with the stringent “protein-only” hypothesis, but do not yet provide definitive proof. The purified PrP^C molecules used as substrate in these experiments contain a stoichiometric amount of co-purified lipid⁷ that may play a role in the generation of prion infectivity.⁹ Also, although the efficacy of photocleavage conditions was carefully confirmed in control reactions, it is possible that some intact oligonucleotide survived UV treatment at a level below detection. Alternatively, the remnant five base nucleic acid fragments may remain incorporated within the PrP^Sc molecule and play a role in maintaining the infectious conformation. Even in this scenario, our results would place a significant geometric constraint on the role of incorporated polyanion. While polyanions ≥40 bases facilitate the formation infectious prions in vitro,⁸ our results suggest that polyanions >5 bases are not necessary to maintain the infectious properties the prion. The exact role polyanions play in prion formation is still unclear, but it is tempting to speculate that they may serve as scaffolds that facilitate prion conversion by (a) bringing PrP^C and PrP^Sc seed together for templating to occur or (b) acting as a catalyst which is necessary to reduce the activation energy of refolding to the PrP^Sc form. Future studies will need to be performed to differentiate between these two hypotheses. It is also possible that polyanions are completely dispensable for maintaining PrP^Sc structure, and it is the co-purified lipid molecules that serve this role instead. Consistent with this possibility, we recently discovered that mouse PrP^Sc can be serially propagated in vitro in the absence of nucleic acids.²¹ Finally, it is possible that either polyanions or lipids can function equally well as stabilizers of the infectious PrP^Sc conformation. More work is required to distinguish between these possibilities.Generating high levels of specific infectivity solely using purified recombinant PrP remains the ultimate proof of the “protein-only” hypothesis. To date, evidence suggests that cellular cofactors are necessary to create infectious prions but may or may not be required to maintain infectivity once formed. Significantly, Wang et al. showed that bona fide prions could be formed from recombinant PrP, synthetic lipid and RNA molecules.⁹ Although no completely pure preparations of misfolded PrP possessing significant levels of specific infectivity have yet been produced, it should eventually be possible to produce such a preparation if the “protein-only” hypothesis is correct. On the other hand, a rigorous refutation of the hypothesis would require demonstrating that PrP^Sc and infectivity can be dissociated.     

IRG and GBP Host Resistance Factors Target Aberrant, “Non-self” Vacuoles Characterized by the Missing of “Self” IRGM Proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.     

Breast fine-needle aspiration malondialdehyde deoxyguanosine adduct in breast cancer

This study has analysed the generation of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adduct [M?dG], a biomarker of oxidative stress and lipid peroxidation, in breast fine-needle aspirate samples of 22 patients with breast cancer, at different clinical stages, in respect to 13 controls. The multivariate analysis show that M(1)dG adduct was higher in cases than in controls (Mean Ratio (MR) = 5.26, 95% CI = 3.16-8.77). Increased M?dG was observed in women with a tumour grade 3 and a pathological diameter 2 (MR = 7.61, 95% CI = 3.91-14.80 and MR = 5.75, 95% CI = 3.13-10.59, respectively). A trend with increasing tumour grade and pathological diameter was present (MR = 1.98, 95% CI = 1.57-2.50 and MR = 2.44, 95% CI = 1.71-3.48, respectively). Not significant effects of age and smoking habit were found (MR = 1.58, 95% CI = 0.92-2.72 and MR = 1.68, 95% CI 0.88-3.20, respectively). An increment over the background frequency of M?dG can contribute to breast cancer development. Increasing severity of breast tumour can influence DNA damage level.     

Membrane-bound glucosamine acetyltransferase in coleoptile segments of Avena sativa

The in vivo metabolism of D-[U-¹⁴C]glucosamine and the in vitro properties of glucosamine acetyltransferase (EC 2.3.1.3), the first committed enzyme in the metabolism of exogenously supplied D-glucosamine, were studied in coleoptile segments of Avena sativa L. cv. Sole II. D-[U-¹⁴C]glucosamine was taken up by oat coleoptile segments and sequentially metabolised to radioactive N-acetylglucosamine, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, N-acetylglucosamine residues were incorporated into glycoproteins and glycolipids of the cells. All glucosamine acetyltransferase activity was found to be membrane-bound. The enzyme was solubilized by either digitonin or CHAPS. The specificities and the kinetics of the membrane-bound and soluble glucosamine acetyltransferase were determined. The effects of ions, nucleotides, nucleoside diphosphate amino sugars, coenzymes and group-specific chemical probes on the rate of membrane-bound and CHAPS-solubilized enzyme were investigated. Our data indicate that UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine do not exert a feed-back control on the glucosamine acetyltransferase either in vivo or in vitro. Further, some nucleotides and the metal ions Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺ and Co²⁺ affect the activity of the enzyme in vitro.     

Prevalence of sleep disorders and their impact on academic performance in medical students/University of Duhok

Full-time students experiencing high levels of stress due to a high bulk of teaching materials and academic performance demands are the most susceptible population class for different types of sleep disorders. The current study examined the prevalence of sleep disorders and their impacts on academic performance of a random sample of medical college students. In this regard, a random sample of 316 medical students of a large public university in Iraq participated in a cross-sectional study. The participants completed the SLEEP-50 self-reported questionnaire and questions about socio-demographic factors. The variables set included sleep apnea, insomnia, narcolepsy, restless legs syndrome, circadian rhythm sleep disorder, sleepwalking, nightmares, grade point average, and some socio-demographic characteristics. The study showed that to some extent, the students suffer from different types of sleep disorders with no substantial difference between males and females. Students with worse level of sleep disorders had a lower grade point average compared with those with normal sleep patterns (p = 0.001). The study confirmed that students with sleep disorders had poorer academic performance at college.

     

No correlation between <Emphasis Type="Italic">NF1</Emphasis> mutation position and risk of optic pathway glioma in 77 unrelated NF1 patients

Neurofibromatosis type 1 (NF1) is a common monogenic disorder whereby affected individuals are predisposed to developing CNS tumors, including optic pathway gliomas (OPGs, occurring in ~15 to 20 % of cases). So far, no definite genotype–phenotype correlation determining NF1 patients at risk for tumor formation has been described, although enrichment for mutations in the 5′ region of the NF1 gene in OPG patients has been suggested. We used whole exome sequencing, targeted sequencing, and copy number analysis to screen 77 unrelated NF1 patients with (n = 41) or without (n = 36; age ≥10 years) optic pathway glioma for germline NF1 alterations. We identified germline NF1 mutations in 69 of 77 patients (90 %), but no genotype–phenotype correlation was observed. Our data using a larger patient cohort did not confirm the previously reported clustering of mutations in the 5′ region of the NF1 gene in patients with OPG. Thus, NF1 mutation location should not currently be used as a clinical criterion to assess the risk of developing OPGs.