Backbone resonance assignments of an artificially engineered TEM-1/PSE-4 Class A β-lactamase chimera

The rapid evolution of Class A β-lactamases, which procure resistance to an increasingly broad panel of β-lactam antibiotics, underscores the urgency to better understand the relation between their sequence variation and their structural and functional features. To date, more than 300 clinically-relevant β-lactamase variants have been reported, and this number continues to increase. With the aim of obtaining insights into the evolutionary potential of β-lactamases, an artificially engineered, catalytically active chimera of the Class A TEM-1 and PSE-4 β-lactamases is under study by kinetics and NMR. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments for the 30 kDa chimera cTEM-17m. Despite its high molecular weight, the data provide evidence that this artificially-evolved chimeric enzyme is well folded. The hydrolytic activity of cTEM-17m was determined using the chromogenic substrate CENTA, with K _M = 160 ± 35 μM and k _cat = 20 ± 4 s^?1, which is in the same range as the values for TEM-1 and PSE-4 β-lactamases.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

The role of tower height and guy wires on avian collisions with communication towers

Every year an estimated 4–5 million migratory birds collide with communication towers in the United States. We examined the relative risks that tower support systems and tower height pose to migrating and other birds. We collected data comparing tower support systems (guyed vs. unguyed) and tower height categories in Michigan during 20 days of the peak of songbird migration at 6 towers in September–October 2003, 23 towers in May 2004, 24 towers in September 2004, and 6 towers in both May and September 2005. We systematically and simultaneously searched for bird carcasses under each tower and measured carcass removal and observer detection rates each season. Of those towers, 21 were between 116 and 146 m above ground level (AGL, medium) and 3 were >305 m AGL (tall). During the five 20-day sample periods we found a mean of 8.2 bird carcasses per guyed medium tower and a mean of 0.5 bird carcasses under unguyed medium towers. During four 20-day sample periods we detected a mean of 34.7 birds per guyed tall tower. Using both parametric and nonparametric tests (Mann–Whitney U-test, Kruskal–Wallis test, and Tukey's Honestly Significant Difference multiple comparison procedure) we determined that unguyed medium towers were involved in significantly fewer fatalities than guyed medium towers. We detected 54–86% fewer fatalities at guyed medium towers than at guyed tall towers. We found 16 times more fatalities at guyed medium towers than at unguyed medium towers. Tall, guyed towers were responsible for 70 times as many bird fatalities as the unguyed medium towers and nearly five times as many as guyed medium towers. These findings will provide managers and regulators, such as the US Fish and Wildlife Service, with quantitative data; thereby, allowing them to effectively work with the Federal Communications Commission in siting and authorizing tower placement. © 2011 The Wildlife Society.     

The Early Activation of Toll-Like Receptor (TLR)-3 Initiates Kidney Injury after Ischemia and Reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.     

Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.     

Fabrication and Visualization of Capillary Bridges in Slit Pore Geometry

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and functionalized to render their top surfaces hydrophilic. The combination of a physical feature (the pillar) with a chemical boundary (the hydrophilic film on the top of the pillar) provides both a physical and chemical heterogeneity that pins the triple contact line, a necessary feature to create stable long but narrow capillary bridges. The substrates with the pillars are attached to glass slides and secured into custom holders. The holders are then mounted onto four axis microstages and positioned such that the pillars are parallel and facing each other. The capillary bridges are formed by introducing a fluid in the gap between the two substrates once the separation between the facing pillars has been reduced to a few hundred micrometers. The custom microstage is then employed to vary the height of the capillary bridge. A CCD camera is positioned to image either the length or the width of the capillary bridge to characterize the morphology of the fluid interface. Pillars with widths down to 250 µm and lengths up to 70 mm were fabricated with this method, leading to capillary bridges with aspect ratios (length/width) of over 100¹.