A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps

Vacuolar-type H⁺-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H⁺-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M_r 64 000 membrane polypeptide that specifically catalyzes Mg²⁺-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H⁺-translocation is phylogenetically more deeply rooted than previously thought.     

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps.

Vacuolar-type H(+)-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H(+)-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M(r) 64? omitted?000 membrane polypeptide that specifically catalyzes Mg(2+)-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H(+)-translocation is phylogenetically more deeply rooted than previously thought.     

Investigation of optical attenuation imaging using optical coherence tomography for monitoring of scars undergoing fractional laser treatment

We demonstrate the use of the near‐infrared attenuation coefficient, measured using optical coherence tomography (OCT), in longitudinal assessment of hypertrophic burn scars undergoing fractional laser treatment. The measurement method incorporates blood vessel detection by speckle decorrelation and masking, and a robust regression estimator to produce 2D en face parametric images of the attenuation coefficient of the dermis. Through reliable co‐location of the field of view across pre‐ and post‐treatment imaging sessions, the study was able to quantify changes in the attenuation coefficient of the dermis over a period of ～20 weeks in seven patients. Minimal variation was observed in the mean attenuation coefficient of normal skin and control (untreated) mature scars, as expected. However, a significant decrease (13 ± 5%, mean ± standard deviation) was observed in the treated mature scars, resulting in a greater distinction from normal skin in response to localized damage from the laser treatment. By contrast, we observed an increase in the mean attenuation coefficient of treated (31 ± 27%) and control (27 ± 20%) immature scars, with numerical values incrementally approaching normal skin as the healing progressed. This pilot study supports conducting a more extensive investigation of OCT attenuation imaging for quantitative longitudinal monitoring of scars.

En face 2D OCT attenuation coefficient map of a treated immature scar derived from the pre‐treatment (top) and the post‐treatment (bottom) scans. (Vasculature (black) is masked out.) The scale bars are 0.5 mm.     

Plant ABC proteins--a unified nomenclature and updated inventory

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.     

Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis> for sugars monitoring

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. The authors wish to dedicate this work to Prof. Ignacy Gryczynski, University of North Texas, TX, USA, for his outstanding contribution to the development of new sensing methodologies.     

Myeloid dendritic cells display downregulation of C-type lectin receptors and aberrant lectin uptake in systemic lupus erythematosus

Introduction  

There is a growing body of evidence implicating aberrant dendritic cell function as a crucial component in the immunopathogenesis of systemic lupus erythematosus. The purpose of the present study was to characterize the phagocytic capacity and expression of receptors involved in pathogen recognition and self-nonself discrimination on myeloid dendritic cells from patients with lupus.     

Efficacy of immobilized polyplexes and lipoplexes for substrate‐mediated gene delivery

Non‐viral gene delivery by immobilization of complexes to cell‐adhesive biomaterials, a process termed substrate‐mediated delivery, has many in vitro research applications such as transfected cell arrays or models of tissue growth. In this report, we quantitatively investigate the efficiency of gene delivery by surface immobilization, and compare this efficiency to the more typical bolus delivery. The ability to immobilize vectors while allowing cellular internalization is impacted by the biomaterial and vector properties. Thus, to compare this efficiency between vector types and delivery methods, transfection conditions were initially identified that maximized transgene expression. For surface delivery from tissue culture polystyrene, DNA complexes were immobilized to pre‐adsorbed serum proteins prior to cell seeding, while for bolus delivery, complexes were added to the media above adherent cells. Mathematical modeling of vector binding, release, and cell association using a two‐site model indicated that the kinetics of polyplex binding to cells was faster than for lipoplexes, yet both vectors have a half‐life on the surface of approximately 17 min. For bolus and surface delivery, the majority of the DNA in the system remained in solution or on the surface, respectively. For polyplexes, the efficiency of trafficking of cell‐associated polyplexes to the nucleus for surface delivery is similar or less than bolus delivery, suggesting that surface immobilization may decrease the activity of the complex. The efficiency of nuclear association for cell‐associated lipoplexes is similar or greater for surface delivery relative to bolus. These studies suggest that strategies to enhance surface delivery for polyplexes should target the vector design to enhance its potency, whereas enhancing lipoplex delivery should target the material design to increase internalization. Biotechnol. Bioeng. 2009;102: 1679–1691. © 2008 Wiley Periodicals, Inc.     

Proteasome inhibitors impair RANKL‐induced NF‐κB activity in osteoclast‐like cells via disruption of p62, TRAF6, CYLD,and IκBα signaling cascades

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK‐mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG‐132, MG‐115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast‐like cells. Proteosome inhibition also blocked RANKL‐induced NF‐κB activation, IκBα degradation and nuclear translocation of p65. The disruption in RANK‐signaling correlated dose‐dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG‐132 > MG‐115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK‐mediated signaling cascades (p62, TRAF6, CYLD, and IκBα) underscores proteasome‐mediated inhibition of osteolytic bone conditions. J. Cell. Physiol. 220: 450–459, 2009. © 2009 Wiley‐Liss, Inc.     

UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.