Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.     

Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.     

Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus

Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non‐reducing polyketide synthase‐encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10‐fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non‐reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes.     

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background

Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results

In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion

A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.     

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.     

Oxylipins as developmental and host-fungal communication signals

Pathogenic microbes and their hosts have acquired complex signalling mechanisms to appraise themselves of the environmental milieu in the ongoing battle for survival. Several recent studies have implicated oxylipins as a novel class of host-microbe signalling molecules. Oxylipins represent a vast and diverse family of secondary metabolites that originate from the oxidation or further conversion of polyunsaturated fatty acids. Among the microbial oxylipins, the fungal oxylipins are best characterized and function as hormone-like signals that modulate the timing and balance between asexual and sexual spore development in addition to toxin production. Coupled with other studies that implicate a role for fungal oxylipins in pathogenesis by Aspergillus and Candida spp., these results suggest that host and microbial oxylipins might interfere with the metabolism, perception or signalling processes of each other.     

Influence of an additional 2-amino substituent of the 1-aminoethyl pharmacophore group on the potency of rimantadine against influenza virus A

We examined whether the incorporation of a second amino group into the 1-aminoethyl pharmacophore of rimantadine 2 and into the piperidine pharmacophore of the heterocyclic rimantadine 4 was compatible with anti-influenza virus A activity. The new synthetic molecules are capable of forming two hydrogen bonds within the receptor. We identified molecules 8 and 16, bearing the adamantyl and 1,2-diaminoethyl groups, which are equipotent to rimantadine 2 bearing the adamantyl and 1-aminoethyl pharmacophore groups. Interestingly, diamino compound 16 is a 4-fold more potent inhibitor than its parent monoamino heterocyclic rimantadine 4 propably because of additional hydrogen bonding interactions with the M2 protein receptor.     

High resolution footprinting of the hepatitis C virus polymerase NS5B in complex with RNA

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.