A potentially important excretory-secretory product of Giardia lamblia

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.     

Ionic, structural, and temperature effects on DNA nanoparticles formed by natural and synthetic polyamines

We synthesized analogues of spermine and studied the effects of chemical structure, ionic strength, and temperature on lambda-DNA nanoparticle formation. Effective concentration of polyamines for DNA condensation (EC50) was lowest for hexamines (0.2 microM) and highest for spermine (tetramine, 4.2 microM). The EC50 value increased with [Na+]. Dynamic light scattering showed nanoparticles with hydrodynamic radii (R(h)) of 40-50 nm. Effect of temperature on R(h) was measured between 20 and 70 degrees C. For spermine, R(h) remained relatively stable until 50 degrees C and increased significantly at >60 degrees C. In contrast, the hexa- and penta-valent analogues exhibited a gradual increase in R(h) between 20 and 70 degrees C. The nanoparticles were mainly toroidal, as revealed by electron microscopy (EM). EM studies showed changes in morphology and size of condensed structures with an increase in temperature. A possible mechanism for the differential effects of temperature on DNA nanoparticles might involve different modes of DNA-polyamine interactions.     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.     

Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400: A NOVEL RING-CLEAVING ENZYME IN THE CROTONASE SUPERFAMILY*

The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 Å resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu¹⁴⁶ and Glu¹⁶⁸) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys¹¹¹ as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.Aromatic compounds comprise approximately one-quarter of the earth''s biomass (1) and are the second most abundant natural product next to carbohydrates. The majority of aromatic compounds in the environment are in the form of the organic polymer lignin that plays a structural role in cross-linking cell wall polysaccharides in plants. Despite the inherent thermostability of the aromatic ring, these naturally occurring compounds are efficiently mineralized by various microorganisms. Human-made aromatic compounds, such as those used in industrial processes, however, are often recalcitrant to microbial degradation due to their chemical complexity, decreased bioavailability, and increased thermostability. Moreover, bacteria have only been exposed to these compounds for a relatively short period of time. As a result, these compounds persist in the environment, where they can increase to toxic levels and cause irreversible damage to the biosphere.The common structural blueprint shared by natural and human-made aromatic compounds is the resonance-stabilized planar ring system. Microorganisms overcome the stability of these aromatic structures by employing specific ring-cleaving enzymes that form part of complex catabolic pathways. Until recently, two general classes of microbial processes were characterized that catalyze the degradation of aromatic compounds. These classifications, termed the aerobic and anaerobic pathways, were based primarily on the mode of initial activation and subsequent cleavage of the aromatic ring. The aerobic pathway, exemplified by the peripheral biphenyl and the central ben-cat pathway, relies on the extensive use of molecular oxygen for both the hydroxylation (activation) and cleavage of the aromatic ring (2–4). The anaerobic pathway, however, mediates a reductive dearomatization followed by a hydrolytic ring cleavage, as observed in the classical benzoate pathway (5–7). In both cases, the underlying mechanism incorporates an activation step that renders the ring susceptible to cleavage.Recently, a third aromatic degradation pathway was identified in Burkholderia xenovorans strain LB400 (LB400) (8–10) and Azoarcus evansii (11–13). This novel pathway, termed the box (benzoate oxidation) pathway, incorporates features of both the aerobic and anaerobic pathways, resulting in a hybrid pathway. Microarray analysis of the 9.7-Mb genome of LB400 revealed two paralogous copies of the box pathway, one encoded on chromosome 1 (box_c) and the second on the megaplasmid (box_m) (9). Knock-out studies confirm that both box pathways are capable of assimilating benzoate (10) yet are differentially regulated based on available carbon source and growth phase of the organism (9). Recent structural and biochemical characterization of benzoate CoA ligase (14) and aldeheyde dehydrogenase (15) from the box pathway in LB400 have provided valuable insight into the basis of substrate specificity and details describing the molecular mechanisms.A unique feature of the hybrid box pathway is the incorporation of both CoA ligation and hydroxylation prior to ring cleavage (16), suggesting that both strategies are important for ring activation. It is noteworthy that although CoA ligation is common in the activation of aromatic acids under anaerobic conditions, it has thus far been unseen in the aerobic degradation of aromatic compounds. Furthermore, investigation of the box pathway intermediates from the related A. evansii demonstrated that the thioesterified dihydrodiol intermediate was not oxidized and rearomatized as normally occurs in aerobic aromatic metabolism (11). Instead, it was shown to be directly cleaved without the requirement of molecular oxygen in a reaction that resulted in the loss of one unit of carbon and oxygen as formate (11). This critical ring cleavage step in the box pathway is catalyzed by BoxC (2,3-dihydro-2,3-dihydroxybenzoyl-CoA lyase/hydrolase) (11), which differs from traditional aerobic and anaerobic ring-cleaving enzymes in that oxygen is not used in catalysis, and the ring substrate is only partially reduced. Based on sequence analysis, BoxC is assigned to the crotonase superfamily. The cleavage reaction catalyzed by BoxC, however, suggests that BoxC defines a new mechanistic niche and intriguingly is one of the four outstanding crotonase superfamily members for which no structural information exists (17).A mechanism for BoxC from A. evansii was recently proposed based on the identification of chemical species using NMR and mass spectrometry (11). In the absence of structural information of BoxC, however, the mechanistic details, including the identity of the catalytic residues, remain undefined. To investigate the detailed molecular mechanism of BoxC, we carried out a structural and biophysical analysis complemented with molecular docking. The resulting data provide a compelling mechanistic model with the identification of key catalytic residues and active site structure that stabilize proposed transition state intermediates. Furthermore, the 1.5 Å resolution structure of BoxC reveals intriguing divergent architectural features with respect to other members of the crotonase superfamily. Overall, this study provides the first structural characterization of the novel BoxC family of enzymes and is interpreted with respect to the proposed molecular mechanism and divergence within the crotonase superfamily.     

Scholarometer: A Social Framework for Analyzing Impact across Disciplines

The use of quantitative metrics to gauge the impact of scholarly publications, authors, and disciplines is predicated on the availability of reliable usage and annotation data. Citation and download counts are widely available from digital libraries. However, current annotation systems rely on proprietary labels, refer to journals but not articles or authors, and are manually curated. To address these limitations, we propose a social framework based on crowdsourced annotations of scholars, designed to keep up with the rapidly evolving disciplinary and interdisciplinary landscape. We describe a system called Scholarometer, which provides a service to scholars by computing citation-based impact measures. This creates an incentive for users to provide disciplinary annotations of authors, which in turn can be used to compute disciplinary metrics. We first present the system architecture and several heuristics to deal with noisy bibliographic and annotation data. We report on data sharing and interactive visualization services enabled by Scholarometer. Usage statistics, illustrating the data collected and shared through the framework, suggest that the proposed crowdsourcing approach can be successful. Secondly, we illustrate how the disciplinary bibliometric indicators elicited by Scholarometer allow us to implement for the first time a universal impact measure proposed in the literature. Our evaluation suggests that this metric provides an effective means for comparing scholarly impact across disciplinary boundaries.     

Laccase from a non-melanogenic,alkalotolerant gamma-proteobacterium JB isolated from industrial wastewater drained soil

A Gram-negative, alkalotolerant bacterium, isolated from the soil continually drained with industrial wastewater and identified as gamma-proteobacterium by partial 16S rRNA sequence analysis, produced a polyphenol oxidase, which showed laccase but not tyrosinase activity. The organism grew well from pH 6 to 10 and produced laccase maximally at pH 10. The enzyme was stable from pH 3 to 10.6 for at least 24 h and was optimally active at 55 °C and pH 6.5 in a 5 min assay.     

Occult breast carcinoma in reduction mammaplasty specimens: 14-year experience

Reduction mammaplasty is commonly performed for bilateral macromastia, congenital asymmetry, or as a contralateral symmetry procedure in breast reconstruction following mastectomy for cancer. Occult carcinoma has been detected in 0.06 percent to 0.4 percent of breast reduction specimens. The purpose of this study was to examine the incidence of breast cancer in breast reductions performed in one institution over a 14-year period. The authors reviewed their experience with 800 reduction mammaplasties performed between 1988 and 2001. Six cancers were detected (0.8 percent). Of these cancers, three were invasive (0.4 percent) and three were ductal carcinoma in situ (0.4 percent). Stratified by indication for surgery, there was a trend toward higher detection rates in the reconstruction group (1.2 percent) compared with the macromastia (0.7 percent) or congenital asymmetry (0 percent) groups. Mammography was performed preoperatively in these patients and all results were negative for masses or suspicious microcalcification. Pathological diagnosis was guided by gross specimen evaluation in two patients and specimen radiography in one patient. Reduction mammaplasty has a small but definite risk of finding cancer in the resection specimen.     

Ordering Dynamics in Neuron Activity Pattern Model: An Insight to Brain Functionality

We study the domain ordering kinetics in d = 2 ferromagnets which corresponds to populated neuron activities with both long-ranged interactions, V(r) ∼ r ⁻ⁿ and short-ranged interactions. We present the results from comprehensive Monte Carlo (MC) simulations for the nonconserved Ising model with n ≥ 2, interaction range considering near and far neighbors. Our model results could represent the long-ranged neuron kinetics (n ≤ 4) in consistent with the same dynamical behaviour of short-ranged case (n ≥ 4) at far below and near criticality. We found that emergence of fast and slow kinetics of long and short ranged case could imitate the formation of connections among near and distant neurons. The calculated characteristic length scale in long-ranged interaction is found to be n independent (L(t) ∼ t ^1/(n−2)), whereas short-ranged interaction follows L(t) ∼ t ^1/2 law and approximately preserve universality in domain kinetics. Further, we did the comparative study of phase ordering near the critical temperature which follows different behaviours of domain ordering near and far critical temperature but follows universal scaling law.     

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.