The use of phenyl-Sepharose for the affinity purification of proteinases

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effective retained by the media. Purification factors in the range of 2.9–60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts

Manufacturing of 100mm diameter GaSb substrates for advanced space based applications

Engineered substrates such as large diameter (100mm) GaSb wafers need to be ready years in advance of any major shift in DoD and commercial technology, and typically before much of the rest of the materials and equipment for fabricating next generation devices. Antimony based III-V semiconductors are of significant interest for advanced applications in optoelectronics, high speed transistors, microwave devices, and photovoltaics. GaSb demand is increasing due to its lattice parameter matching of various ternary and quaternary III-V compounds, as their bandgaps can be engineered to cover a wide spectral range. For these stealth and spaced based applications, larger format IRFPAs benefit clearly from next generation starting substrates. In this study, we have manufactured and tested 100mm GaSb substrates. This paper describes the characterization process that provides the best possible GaSb material for advanced IRFPA and SLS epi growth. The analysis of substrate by AFM surface roughness, particles, haze, GaSb oxide character and desorption using XPS, flatness measurements, and SLS based epitaxy quality are shown. By implementing subtle changes in our substrate processing, we show that a Sb-oxide rich surface is routinely provided for rapid desorption. Post-MBE CBIRD structures on the 100mm ULD GaSb were examined and reveals a high intensity, 6.6nm periodicity, low (15.48 arcsec) FWHM peak distribution that suggests low surface strain and excellent lattice matching. The Ra for GaSb is a consistent ~0.2-4nm, with average batch wafer warp of ~4 μm to provide a clean, flat GaSb template critical for next generation epi growth

Synthetic, non-intoxicating 8,9-dihydrocannabidiol for the mitigation of seizures

There can be a fine line between therapeutic intervention and substance abuse, and this point is clearly exemplified in herbal cannabis and its products. Therapies involving cannabis have been the treatment of last resort for some cases of refractory epilepsy, and this has been among the strongest medical justifications for legalization of marijuana. In order to circumvent the narcotic effects of 9-tetrahydrocannabinol (THC), many studies have concentrated on its less intoxicating isomer cannabidiol (CBD). However, CBD, like all natural cannabinoids, is a controlled substance in most countries, and its conversion into THC can be easily performed using common chemicals. We describe here the anticonvulsant properties of 8,9-dihydrocannibidiol (H2CBD), a fully synthetic analogue of CBD that is prepared from inexpensive, non-cannabis derived precursors. H2CBD was found to have effectiveness comparable to CBD both for decreasing the number and reducing the severity of pentylenetetrazole-induced seizures in rats. Finally, H2CBD cannot be converted by any reasonable synthetic route into THC, and thus has the potential to act as a safe, noncontroversial drug for seizure mitigation

Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S. Wats.)

Citation: Nakka, S., Godar, A. S., Wani, P. S., Thompson, C. R., Peterson, D. E., Roelofs, J., & Jugulam, M. (2017). Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S. Wats.). Frontiers in Plant Science, 8, 12. doi:10.3389/fpls.2017.00555Herbicides that inhibit hydroxyphenylpyruvate dioxygenase (HPPD) such as mesotrione are widely used to control a broad spectrum of weeds in agriculture. Amaranthus palmeri is an economically troublesome weed throughout the United States. The first case of evolution of resistance to HPPD-inhibiting herbicides in A. palmeri was documented in Kansas (KS) and later in Nebraska (NE). The objective of this study was to investigate the mechansim of HPPD-inhibitor (mesotrione) resistance in A. palmeri. Dose response analysis revealed that this population (KSR) was 10-18 times more resistant than their sensitive counterparts (MSS or KSS). Absorbtion and translocation analysis of [C-14] mesotrione suggested that these mechanisms were not involved in the resistance in A. palmeri. Importantly, mesotrione (>90%) was detoxified markedly faster in the resistant populations (KSR and NER), within 24 hours after treatment (HAT) compared to sensitive plants (MSS, KSS, or NER). However, at 48 HAT all populations metabolized the mesotrione, suggesting additional factors may contribute to this resistance. Further evaluation of mesotrione-resistant A. palmeri did not reveal any specific resistance-conferring mutations nor amplification of HPPD gene, the molecular target of mesotrione. However, the resistant populations showed 4- to 12-fold increase in HPPD gene expression. This increase in HPPD transcript levels was accompanied by increased HPPD protein expression. The significant aspects of this research include: the mesotrione resistance in A. palmeri is conferred primarily by rapid detoxification (non-target-site based) of mesotrione; additionally, increased HPPD gene expression (target-site based) also contributes to the resistance mechanism in the evolution of herbicide resistance in this naturally occurring weed species

Heme oxygenase-1 protects against Alzheimer’s amyloid-β1-42 induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer‟s disease (AD), catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediate cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentrationdependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent

Au/TiO2(110) interfacial reconstruction stability from ab initio

We determine the stability and properties of interfaces of low-index Au surfaces adhered to TiO2(110), using density functional theory energy density calculations. We consider Au(100) and Au(111) epitaxies on rutile TiO2(110) surface, as observed in experiments. For each epitaxy, we consider several different interfaces: Au(111)//TiO2(110) and Au(100)//TiO2(110), with and without bridging oxygen, Au(111) on 1x2 added-row TiO2(110) reconstruction, and Au(111) on a proposed 1x2 TiO reconstruction. The density functional theory energy density method computes the energy changes on each of the atoms while forming the interface, and evaluates the work of adhesion to determine the equilibrium interfacial structure.Comment: 20 pages, 11 figure

A New Limit on the Antiproton Lifetime

Measurements of the cosmic ray pbar/p ratio are compared to predictions from an inhomogeneous disk-diffusion model of pbar production and propagation within the Galaxy, combined with a calculation of the modulation of the interstellar cosmic ray spectra as the particles propagate through the heliosphere to the Earth. The predictions agree with the observed pbar/p spectrum. Adding a finite pbar lifetime to the model, we obtain the limit tau_pbar > 0.8 Myr (90 % C.L.).Comment: 13 pages, 3 encapsulated Postscript figures, uses AASTeX; accepted by Astrophysical Journal; minor change

The microtubule-associated protein DCAMKL1 regulates osteoblast function via repression of Runx2

Osteoblasts are responsible for the formation and mineralization of the skeleton. To identify novel regulators of osteoblast differentiation, we conducted an unbiased forward genetic screen using a lentiviral-based shRNA library. This functional genomics analysis led to the identification of the microtubule-associated protein DCAMKL1 (Doublecortin-like and CAM kinase–like 1) as a novel regulator of osteogenesis. Mice with a targeted disruption of Dcamkl1 displayed elevated bone mass secondary to increased bone formation by osteoblasts. Molecular experiments demonstrated that DCAMKL1 represses osteoblast activation by antagonizing Runx2, the master transcription factor in osteoblasts. Key elements of the cleidocranial dysplasia phenotype observed in Runx2+/− mice are reversed by the introduction of a Dcamkl1-null allele. Our results establish a genetic linkage between these two proteins in vivo and demonstrate that DCAMKL1 is a physiologically relevant regulator of anabolic bone formation