A molecular perspective: biology of the emerging pathogen Batrachochytrium dendrobatidis

Ten years after the first discovery of the chytrid pathogen Batrachochytrium dendrobatidis (Bd), the catastrophic effect of Bd on wild amphibian populations is indisputable. However, a number of persistent questions remain about Bd's origin and mechanisms of pathogenicity. Here we discuss the promise of genetic and genomic tools for answering these previously intractable questions about the biology and evolutionary history of Bd. Full genomes of 2 Bd strains have recently been sequenced, and Bd research on this species using population genetics, phylogenetics, proteomics, comparative genomics and functional genomics is already underway. We review some of the insights gleaned from the first studies using these genome-scale approaches focusing particularly on Bd's genomic architecture, patterns of global genetic variation, virulence factors and genetic interactions with hosts. Avenues of future research promise to be particularly fruitful and highlight the need for integrative studies that unite genetic, ecological and spatial data in both Bd and its amphibian hosts.     

Increasing ω-3 Desaturase Expression in Tomato Results in Altered Aroma Profile and Enhanced Resistance to Cold Stress

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the ω-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.Tomato (Solanum lycopersicum) breeding has often focused on improving yield, fruit size, and disease resistance, while organoleptic properties have largely been neglected. However, consumer demand for higher nutritional and flavor characteristics in tomato fruits is growing. Despite the complexity of this trait, with multiple biosynthetic pathways contributing, quantitative trait loci that affect volatile composition have been recently identified (Tieman et al., 2006; Mathieu et al., 2009). While proper tomato flavor requires low sugar and acid concentrations, tomato aroma is determined by the contribution of over 400 volatile compounds. The importance of each volatile is determined by both its concentration and its odor threshold (Baldwin et al., 2000). A group of approximately 30 compounds participate, either in a positive or a negative manner, in the properties of tomato aroma. Among them, straight-chain C6 aldehydes and alcohols, such as hexanal, (Z)-hex-3-enal, its isomer (E)-hex-2-enal, and (Z)-hex-3-enol, are the most important to tomato flavor, imparting in a correct proportion some of the unique fresh notes to tomato fruit aroma. Indeed, most appreciated tomato varieties have a higher (Z)-hex-3-enal/hexanal ratio than others less demanded by consumers (Carbonell-Barrachina et al., 2006). Therefore, modifying the (Z)-hex-3-enal/hexanal ratio may be important in the aroma perception of tomato fruits, and since the odor threshold for (Z)-hex-3-enal is low, small changes in the concentration of this compound may exert an important variation in the tomato fruit aroma.These C6 aldehydes and alcohols belong to the complex group of oxylipins, biologically active compounds derived from the oxygenation of unsaturated fatty acids. From the different fatty acids present in plants, hexanal is produced from linoleic acid (18:2), while linolenic acid (18:3) is the precursor of hexenals and derived alcohols. 18:2 and 18:3 are the most abundant fatty acids in plant membrane lipids. In contrast to the biosynthetic pathways of other components of the tomato aroma, the enzymes that participate in the biosynthesis of hexenals and hexanal have been identified and characterized to a large extent (Feussner and Wasternack, 2002). The high specificity of many of the enzymes involved is a feature of this pathway that determines the final products obtained. The first step of this pathway is the production by a specific lipoxygenase (LOX) of the fatty acid hydroperoxide (HPO), derived either from 18:2 or 18:3. According to the position of oxygen insertion, either at the carbon atom 9 or at the carbon atom 13 of the fatty acid backbone, LOXs are classified as 9-LOX or 13-LOX, respectively. In tomato, there are five genes that encode LOXs (TomLoxAto -E) that are differentially expressed during fruit ripening (Chen et al., 2004). TomLoxA, TomLoxB, and TomLoxE are mainly found in fruits and, although their substrate and product specificity is not clear, likely belong to the 9-LOX group based on their sequence similarities and expression (Griffiths et al., 1999; Chen et al., 2004). On the other hand, TomLoxC and TomLoxD are 13-LOX and show differential expression. While TomLoxC is found in fruits, TomLoxD is mainly expressed in leaves and in response to wounding (Heitz et al., 1997; Chen et al., 2004). Interestingly, the major LOX activity in tomato fruit, close to 95%, has 9-LOX specificity (Hatanaka et al., 1992), and no further enzymatic processing of 9-HPOs has been reported. Since the enzymes responsible for HPO modification in fruits have a preference for 13-HPOs, 9-HPOs accumulate in tomato fruits (Matthew et al., 1977). However, minor 13-LOX activity produces a small quantity of 13-HPOs in the fruits that are further cleaved to C6 aldehydes by the action of 13-hydroperoxide lyases (HPLs). From the aldehydes produced by 13-HPL, (Z)-hex-3-enal, derived from 18:3, contributes the most valuable notes to tomato fruit aroma (Boukobza et al., 2001).Addition of exogenous 18:3 increases the level of (Z)-hex-3-enal produced by tomato fruit homogenates (Boukobza et al., 2001), suggesting that the enzymes required for the synthesis of this aroma compound are fully functional in fruit tissues and that the abundance of 18:3 may be a limiting step in (Z)-hex-3-enal production. Contrary to the situation in leaves, tomato fruit is more abundant in 18:2, precursor of hexanal, which may represent up to 80% of its fatty acid content (Galliard et al., 1977). Conversion of 18:2 to 18:3 is carried out by membrane-bound ω-3 desaturases. In Arabidopsis (Arabidopsis thaliana), three genes, FAD3, FAD7, and FAD8, encode the enzymes that participate in the synthesis of hexadecatrienoic acid (16:3) and 18:3 from dienoic fatty acids. FAD3 catalyzes the desaturation reaction of 18:2 that takes place in the endoplasmic reticulum. It uses phospholipids as acyl substrates and NADH, NADH-cytochrome b₅ reductase, and cytochrome b₅ as electron donors. In contrast, FAD7 and FAD8 are located at the chloroplast, providing the majority of the trienoic fatty acids present in the chloroplastic membranes (Wallis and Browse, 2002). They use primarily glycolipids as acyl carriers and NAD(P)H, ferredoxin-NAD(P) reductase, and ferredoxin as electron donors.Metabolic engineering offers an ideal solution to improve the aroma in tomato fruit by increasing the levels of (Z)-hex-3-enal that provides the highly valued fresh notes. To this end, one possible strategy would be to increase the 13-LOX activity specifically involved in the generation of short-chain aldehyde precursors. However, several independent efforts to overexpress the responsible 13-LOX gene led to cosuppression and the consequent depletion of this specific activity (Leon et al., 2002; Chen et al., 2004). A different approach to address this question is to alter the balance between hexenals and hexanal by overexpressing the ω-3 desaturase to increase the content of 18:3, the hexenal precursor. In addition, tomato being a cold-sensitive crop, modifying the unsaturation level of fatty acids present in membrane lipids could contribute to improve the cold tolerance of tomato plants. It is known that modification of the unsaturation degree of the fatty acids is a significant adaptive feature in response to temperature stress (Somerville and Browse, 1991; Iba, 2002). This increase in the trienoic fatty acids present in membrane lipids upon exposure to chilling temperatures is supposed to maintain the required membrane fluidity and to reduce membrane damage, thus ensuring the numerous processes that take place at cell membranes. This capacity of the plants to withstand chilling temperature is not constant but increases noticeably upon exposure to progressively lower temperatures (Guy, 1990). Interestingly, this cold acclimation increases the desaturase activity and the percentage of unsaturated fatty acids (Steponkus et al., 1993). Since most trienoic acids are present in the thylakoid membranes, where the photosynthetic machinery is found, variation of their unsaturation degree at low temperatures could play an important role in maintaining the photosynthetic capacity of the plants.We report here that overexpression of ω-3 desaturases FAD3 and FAD7 in transgenic tomato plants results in a modification of the fatty acid composition, with a major increase of the unsaturation ratio 18:3/18:2 in leaves and fruits. This altered fatty acid profile leads to changes in the ratio of the aroma compounds (Z)-hex-3-enal/hexanal in both tissues. Moreover, transgenic tomato plants with higher levels of FAD3 and FAD7 desaturases are more tolerant to chilling temperatures.     

Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes

Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid-base catalysis, and are >15 A from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.     

Quantitation of adenovirus type 5 empty capsids

Adenovirus empty capsids are immature intermediates that lack DNA and viral core proteins. Highly purified preparations of empty and full capsids were generated by subjecting purified adenovirus preparations to repeated cesium chloride gradient separations. PAGE results revealed that empty capsids contain at least five bands that correspond to proteins absent from the mature virus proteome. Peptide mapping by matrix-assisted laser desorption/ionization time-of-flight MS revealed that three of these bands correspond to varying forms of L1 52/55kDa, a protein involved in the encapsidation of the viral DNA. One band at around 31kDa was found to include precursors to proteins VI and VIII. These precursors correspond to proteins that have not been cleaved by the adenovirus-encoded protease and are not present in the mature full capsids. The precursor to protein VIII (pVIII), a capsid cement protein, is used in this study as a marker in reverse-phased HPLC (RP-HPLC) analyses of adenovirus for the quantitation of empty capsids. A novel calculation method applied to the integration of RP-HPLC chromatograms allowed for the generation of a percentage empty capsid value in a given adenovirus preparation. The percentage empty capsid values generated to date by this method show a high degree of precision and good agreement with a cesium chloride gradient/SDS-PAGE quantitation method of empty capsids. The advantage of this method lies in the accurate, precise, and rapid generation of the percentage of empty capsids in a given purified virus preparation without relying on tedious and time-consuming cesium chloride gradient separations and extractions.     

The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer

The functional properties of glycoproteins are strongly influenced by their profile of glycosylation, and changes in this profile are seen in malignancy. In mucin-type O-linked glycosylation these changes can result in the production of mucins such as MUC1, carrying shorter sialylated O-glycans, and with different site occupancy. Of the tumor-associated sialylated O-glycans, the disaccharide, sialyl-Tn (sialic acid alpha2,6GalNAc), is expressed by 30% of breast carcinomas and is the most tumor-specific. The ST6GalNAc-I glycosyltransferase, which can catalyze the transfer of sialic acid to GalNAc, shows a highly restricted pattern of expression in normal adult tissues, being largely limited to the gastrointestinal tract and absent in mammary gland. In breast carcinomas, however, a complete correlation between the expression of RNA-encoding ST6GalNAc-I and the expression of sialyl-Tn is evident, demonstrating that the expression of sialyl-Tn results from switching on expression of hST6GalNAc-I. Endogenous or exogenous expression of hST6GalNAc-I (but not ST6GalNAc-II) always results in the expression of sialyl-Tn. This ability to override core 1/core 2 pathways of O- linked glycosylation is explained by the localization of ST6GalNAc-I, which is found throughout the Golgi stacks. The development of a Chinese hamster ovary (CHO) cell line expressing MUC1 and ST6GalNAc-I allowed the large scale production of MUC1 carrying 83% sialyl-Tn O-glycans. The presence of ST6GalNAc-I in the CHO cells reduced the number of O-glycosylation sites occupied in MUC1, from an average of 4.3 to 3.8 per tandem repeat. The availability of large quantities of this MUC1 glycoform will allow the evaluation of its efficacy as an immunogen for immunotherapy of MUC1/STn-expressing tumors.     

Structure-function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH(2)-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (E(max)) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.     

Tumour-host interactions: implications for developing anti-cancer therapies

Cancers are a complex set of proliferative diseases that arise in most cases through multi-step pathways involving an accumulation of genetic and epigenetic changes. These steps include inactivation of tumour suppressor genes and activation of oncogenes. However, in addition to genetic mutations in the tumour cells themselves, the local host environment can act as a critical modulator of cancer progression, having either tumour-suppressive or tumour-promoting effects depending on the stage and site of cancer development. Because stromal cells can have these opposing functions during cancer development and progression, a recurring theme throughout this review will be that of balance: maintaining the normal functions of these co-opted cells, yet selectively inhibiting their pro-tumourigenic functions. To achieve this equilibrium, we need to understand the molecular mechanisms by which normal cells become modified by cancer cells before we can hope to target these functions selectively. Here, we will discuss recent efforts to address these key challenges and offer perspectives on the translation of discoveries made in model systems to the clinic.     

Novel missense mutations of theDeleted-in-AZoospermia-Like (DAZL) gene in infertile women and men

Background  

TheDeleted-in-AZoospermia-Like (DAZL) gene has homologs required for germ cell development in many organisms. Recently, we showed that there are several common polymorphisms within theDAZL gene that are associated with age at ovarian failure/menopause and sperm count.     

C-reactive protein and vulnerability to mental stress-induced myocardial ischemia

Myocardial ischemia provoked in the laboratory during mental stress (MSI) in patients with stable coronary artery disease (CAD) predicts subsequent clinical events. The pathophysiology of MSI differs from that of exercise ischemia, and the mechanisms tying MSI to poor prognosis are not known. C-reactive protein (CRP) is a risk marker for cardiovascular events in patients with CAD, but little is known regarding the relationship of CRP to MSI. The purpose of this study was to examine the association of CRP to risk of MSI in CAD patients. Eighty-three patients with stable CAD underwent simultaneous single-photon emission computed tomography (SPECT) imaging with technetium-99m tetrofosmin myocardial perfusion imaging (MPI) and transthoracic echocardiography (TTE), at rest and during MS induced by laboratory mental stress. Serum CRP levels were measured 24 h after MS. MSI was defined by the presence of a new perfusion defect on SPECT and/or new regional wall motion abnormality on TTE during MS. Of the 83 patients, 30 (36%) developed MSI. There was no difference in gender, sex, BMI, histories of diabetes, hypertension, smoking, lipid profile, medications used (including statins, beta-blockers, ACE inhibitors, and aspirin), or hemodynamic response during MS between those with and without MSI. In univariate logistic regression analysis, each unit (1 mg/L) increase in CRP level was associated with 20% higher risk of MSI (OR 1.2, 95% CI 1.01-1.39, P=.04). This relationship remained in multivariate models. These data suggest that levels of CRP may be a risk marker for MSI in patients with CAD.