Propane Dehydrogenation on Single-Site [PtZn4] Intermetallic Catalysts

Benzene is an important air pollutant and a key chemical feedstock for the synthesis of cyclohexane. Because of the small difference of 0.6°C in their boiling points, the separation of benzene and cyclohexane is extremely challenging. Here, we report the high adsorption of benzene at low pressure and efficient separation of benzene/cyclohexane, achieved by the control of pore chemistry of two families of robust metal-organic frameworks, UiO-66 and MFM-300. At 298 K, UiO-66-CuII shows an exceptional adsorption of benzene of 3.92 mmol g−1 at 1.2 mbar and MFM-300(Sc) exhibits a high selectivity of 166 for the separation of benzene/cyclohexane (v/v = 1/1) mixture. In situ synchrotron X-ray diffraction and neutron powder diffraction, and multiple spectroscopic techniques reveal the binding mechanisms of benzene and cyclohexane in these materials. We also report the first example of direct visualization of reversible binding of benzene at an open Cu(II) site within metal-organic frameworks.

The human immunodeficiency virus (HIV) Vpr protein plays a critical role in AIDS pathogenesis, especially by allowing viral replication within nondividing cells such as mononuclear phagocytes. Most of the data obtained so far have been in experiments with endogenous Vpr protein; therefore the effects of extracellular Vpr protein remain largely unknown. We used synthetic Vpr protein to activate nuclear transcription factors activator protein-1 (AP-1) and NF-kappaB in the promonocytic cell line U937 and in primary macrophages. Synthetic HIV-1 Vpr protein activated AP-1, c-Jun N-terminal kinase, and MKK7 in both U937 cells and primary macrophages. Synthetic Vpr activated NF-kappaB in primary macrophages and to a lesser extent in U937 cells. Because synthetic Vpr activated AP-1 and NF-kappaB, which bind to the HIV-1 long terminal repeat, we investigated the effect of synthetic Vpr on HIV-1 replication. We observed that synthetic Vpr stimulated HIV-1 long terminal repeat in U937 cells and enhanced viral replication in chronically infected U1 promonocytic cells. Similarly, synthetic Vpr stimulated HIV-1 replication in acutely infected primary macrophages. Activation of transcription factors and enhancement of viral replication in U937 cells and primary macrophages were mediated by both the N-terminal and the C-terminal moieties of synthetic Vpr. Therefore, our results suggest that extracellular Vpr could fuel the progression of AIDS via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.

Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.

Kingdom-Agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.

Transforming growth factor beta (TGF-beta) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-beta/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-beta- and BMP-mediated transcriptional responses as well as TGF-beta-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-beta-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-beta signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-beta type I receptor ALK5/TbetaRI and inhibits the interaction between ALK5/TbetaRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-beta signaling.

Stem cell gene transfer—efficacy and safety in large animal studies

Radical Cyclic [3]Daisy Chains

Antimicrobial therapy promotes resistance emergence in target infections and in off-target microbiota. Off-target resistance emergence threatens patient health when off-target populations are a source of future infections, as they are for many important drug-resistant pathogens. However, the health risks of antimicrobial exposure in off-target populations remain largely unquantified, making rational antibiotic stewardship challenging. Here, we discuss the contribution of bystander antimicrobial exposure to the resistance crisis, the implications for antimicrobial stewardship, and some novel opportunities to limit resistance evolution while treating target pathogens.

The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

Anaesthesia and myocardial ischaemia/reperfusion injury