Diagnostic approaches for diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a cardiac dysfunction which affects approximately 12% of diabetic patients, leading to overt heart failure and death. However, there is not an efficient and specific methodology for DCM diagnosis, possibly because molecular mechanisms are not fully elucidated, and it remains asymptomatic for many years. Also, DCM frequently coexists with other comorbidities such as hypertension, obesity, dyslipidemia, and vasculopathies. Thus, human DCM is not specifically identified after heart failure is established. In this sense, echocardiography has been traditionally considered the gold standard imaging test to evaluate the presence of cardiac dysfunction, although other techniques may cover earlier DCM detection by quantification of altered myocardial metabolism and strain. In this sense, Phase-Magnetic Resonance Imaging and 2D/3D-Speckle Tracking Echocardiography may potentially diagnose and stratify diabetic patients. Additionally, this information could be completed with a quantification of specific plasma biomarkers related to related to initial stages of the disease. Cardiotrophin-1, activin A, insulin-like growth factor binding protein-7 (IGFBP-7) and Heart fatty-acid binding protein have demonstrated a stable positive correlation with cardiac hypertrophy, contractibility and steatosis responses. Thus, we suggest a combination of minimally-invasive diagnosis tools for human DCM recognition based on imaging techniques and measurements of related plasma biomarkers.

Parathormone Levels Are Independently Associated with the Presence of Left Ventricular Hypertrophy in Patients with Coronary Artery Disease.

BACKGROUND: Abnormalities of mineral metabolism and inflammation may affect the cardiovascular system. We have assessed the relationship of left ventricular hypertrophy (LVH) with inflammation and mineral metabolism. METHODS: LVH was measured in 146 outpatients with stable coronary artery disease (SCAD) using echocardiography. Calcidiol (a vitamin D metabolite), parathyroid hormone (PTH), fibroblast growth factor-23, high-sensitivity C-reactive protein, MCP-1 (monocyte chemoattractant protein-1), galectin-3, NGAL (neutrophil gelatinase-associated lipocalin), and sTWEAK (soluble TNF-related weak inducer of apoptosis) plasma levels were studied. RESULTS: LVH, defined as septal thickness ≥11 mm, was present in 19.9% of cases. These patients were older [75.0 (61.0-81.0) vs 64.0 (51.0-76.0) years; p=0.002], had higher prevalence of left ventricular ejection fraction (LVEF)>40%, and had higher PTH [84.7 (59.6-104.7) vs 63.2 (49.2-85.2) pg/ml; p=0.007], galectin-3 [9.6 (8.0-11.1) vs 8.3 (6.9-9.9) ng/ml; p=0.037], and NGAL (208.5±87.6 vs 173.9±73.4 ng/ml; p=0.031) plasma levels than those without LVH. Glomerular filtration rate was lower in patients with LVH than in those without it (65.1±20.0 vs 74.7±19.9 mL/min/1.73 m2; p=0.021). There were no significant differences in hypertension (79.3 vs 68.4%; p=0.363) or sex between both groups. Variables showing differences based on univariate analysis and hypertension were entered into a logistic regression analysis. Only age [odds ratio (OR) =1.052 (1.011-1.096); p=0.013], PTH plasma levels [OR=1.017 (1.003-1.031); p=0.021], and LVEF>40% [OR=7.595 (1.463-39.429); p=0.016] were independent predictors of LVH. CONCLUSIONS: In patients with SCAD, elevated PTH levels are independently associated with the presence of LVH. Further studies are needed to elucidate the role of PTH in the development of myocardial hypertrophy.

[Valvular heart disease: multidetector computed tomography evaluation]. / Enfermedad valvular cardíaca. Valoración con tomografía computarizada multidetector.

Heart valve disease is a clinical problem that has been studied with classical imaging techniques like echocardiography and MRI. Technological advances in CT make it possible to obtain static and dynamic images that enable not only a morphological but also a functional analysis in many cases. Although it is currently indicated only in patients with inconclusive findings at echocardiography and MRI or those in whom these techniques are contraindicated, multidetector CT makes it possible to diagnose stenosis or regurgitation through planimetry, to evaluate and quantify valvular calcium, and to show the functional repercussions of these phenomena on the rest of the structures of the heart. Given that multidetector CT is being increasingly used in the diagnosis of ischemic heart disease, we think it is interesting for radiologists to know its potential for the study of valvular disease.

pH control of the production of recombinant glucose oxidase in Aspergillus nidulans.

AIMS: Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. METHODS AND RESULTS: Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg(-1) protein and a maximum volumetric productivity of 29.9 U l(-1) h(-1) were obtained at pH 5.5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. CONCLUSIONS: Specific GOX activity obtained at pH 5.5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies.

Improving extracellular production of food-use enzymes from Aspergillus nidulans.

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24).

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.

Purification and characterization of an alpha-L-rhamnosidase from Aspergillus nidulans.

An enzyme exhibiting alpha-L-rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase was shown to be N-glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4.5-6 and 60 degrees C, had an isoelectric point of 5. With rho-nitrophenyl-alpha-L-rhamnopyranoside as the substrate it showed Km and Vmax values of 0.27 mmol l-1 and 64.6 U mg-1, respectively. The enzyme was competitively inhibited by L-rhamnose (Ki 0.3 mmol l-1). Ca2+ (2 mmol l-1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l-1) inhibited it by 63%. Substrate specificity studies showed the alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides.

Carbon catabolite repression of the Aspergillus nidulans xlnA gene.

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.

Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.

Opposite patterns of expression of two Aspergillus nidulans xylanase genes with respect to ambient pH.

The Aspergillus nidulans xylanase genes xlnA and xlnB are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of D-xylose, xlnA is expressed under conditions of alkaline ambient pH while xlnB is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking A. nidulans mutants.

Signaling of ambient pH in Aspergillus involves a cysteine protease.

In Aspergillus nidulans, the regulation of gene expression in response to changes in ambient pH is mediated by the PacC zinc finger transcriptional regulator. At alkaline ambient pH, PacC is proteolytically processed to a functional form serving as an activator of alkaline-expressed genes and a repressor of acid-expressed genes. This activation of PacC occurs in response to a signal mediated by the products of the pal genes. Thus, the products of the palA, -B, -C, -F, -H, and -I genes constitute an alkaline ambient pH signal transduction pathway. How the pal signal transduction pathway senses ambient pH and transduces a signal to trigger PacC processing is a fascinating unresolved problem. We have cloned and sequenced the palB gene. The predicted palB gene product has similarity to the catalytic domain of the calpain family of calcium-activated cysteine proteases. We have shown, however, that the PalB protein does not catalyze the final step of proteolytic processing of PacC.

Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety.

Extremes of pH are an occupational hazard for many microorganisms. In addition to efficient pH homeostasis, survival effectively requires a regulatory system tailoring the syntheses of molecules functioning beyond the cell boundaries (permeases, secreted enzymes, and exported metabolites) to the pH of the growth environment. Our previous work established that the zinc finger PacC transcription factor mediates such pH regulation in the fungus Aspergillus nidulans in response to a signal provided by the products of the six pal genes at alkaline ambient pH. In the presence of this signal, PacC becomes functional, activating transcription of genes expressed at alkaline pH and preventing transcription of genes expressed at acidic pH. Here we detect two forms of PacC in extracts, both forming specific retardation complexes with a PacC-binding site. Under acidic growth conditions or in acidity-mimicking pal mutants (defective in ambient pH signal transduction), the full-length form of PacC predominates. Under alkaline growth conditions or in alkalinity-mimicking pacCc mutants (independent of the ambient pH signal), a proteolysed version containing the amino-terminal approximately 40% of the protein predominates. This specifically cleaved shorter version is clearly functional, both as an activator for alkaline-expressed genes and as a repressor for acid-expressed genes, but the full-length form of PacC must be inactive. Thus, PacC proteolysis is an essential and pH-sensitive step in the regulation of gene expression by ambient pH. Carboxy-terminal truncations, resulting in a gain-of-function (pacCc) phenotype, bypass the requirement for the pal signal transduction pathway for conversion of the full-length to the proteolyzed functional form.

The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH.

The pH regulation of gene expression in Aspergillus nidulans is mediated by pacC, whose 678 residue-derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth at alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic glutamine repeat. Nine pacC+/- mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth and conidiation. A PacC fusion protein binds DNA with the core consensus GCCARG. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction.

Molecular characterization of a fungal secondary metabolism promoter: transcription of the Aspergillus nidulans isopenicillin N synthetase gene is modulated by upstream negative elements.

The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5'-end of the mRNA, with a major start at -106 relative to the initiation codon. This indicates that the actual length of the non-transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying beta-galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis-acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis-acting elements is involved in sucrose repression. Strikingly, it is located outside the non-transcribed 525bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5'-deletion up to -56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total-protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA-binding activity that specifically shifts a promoter fragment located between -654 and -455 (relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.

Characterisation of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes.

Acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) from some plectomycete fungi is possibly involved in an accessory step of penicillin biosynthesis, in addition to its role in primary metabolism. We present the characterisation of the gene encoding this enzyme in Penicillium chrysogenum, which we designated acuA. Sequencing of genomic and cDNA clones showed that the coding region was interrupted by five introns, located at the same positions as those present in the Aspergillus nidulans homologue. This supports the possibility that the gene acquired its definitive mosaic organisation before the Penicillium/Aspergillus divergence. The mature transcript encodes a polypeptide with an M(r) of 74,287 which is 89.4% identical to its A. nidulans counterpart.

Expression of fungal genes involved in penicllin biosynthesis.

Carbon catabolite repression and pH regulation are regulatory circuits with a wide domain of action in the Plectomycetes. Penicillin biosynthesis is one of the pathways which are under their control. The conclusions obtained so far, which are based on studies of the genetic and molecular regulation of the penicillin pathway of Aspergillus nidulans, would have been much harder to produce using an organism such as Penicillium chrysogenum (the industrial penicillin producer). However, A. nidulans and P. chrysogenum are close in terms of their phylogeny and one can reasonably predict that the conclusions about A. nidulans, which are summarized in this review and which are of unquestionable biotechnological relevance, will be extrapolable to the industrial organism.

Comparative studies of the quinic acid (qa) cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region.

The organization of the quinic acid (qa) genes in Neurospora crassa has been compared to that in several other Neurospora species. This gene cluster was found to be highly conserved in all species examined. However, there are numberous restriction fragment length polymorphisms that distinguish the heterothallic and homothallic species. Catabolic dehydroquinase assays indicated that qa-2 gene expression in the homothallic species is subject to induction by quinic acid, as is the case in N. crassa. The qa-x-qa-2 intergenic region of the homothallic species N. africana was cloned and sequenced. Conserved qa activator (qa-1F) binding sites have been identified in this region. When the qa-x-qa-2 intergenic region of N. crassa was replaced with its N. africana counterpart, qa-2 gene expression was reduced; however repression by glucose appeared normal. Furthermore, the N. africana start site for qa-2 transcription (which differs from the N. crassa start site) was utilized in the transformant. The overall evidence suggests that a weakening of the -120 activator binding site in the qa-x-qa-2 intergenic region may be responsible for these differences.

Light-controlled adaptation kinetics in Phycomyces: evidence for a novel yellow-light absorbing pigment.

When sporangiophores of the fungus Phycomyces blakesleeanus adapt from high to low fluence rate, dark adaptation (sensitivity recovery) can be accelerated by dim subliminal light [Galland et al. (1989) Photochem. Photobiol. 49, 485-491]. We measured fluence rate-response curves for this acceleration under the following conditions. After sporangiophores were initially adapted symmetrically to a fluence rate of 1 W m-2 (447 nm), they were exposed to unilateral subliminal light (subthreshold for phototropism) of variable wavelength and fluence rate, and then to unilateral test light (447 nm) of fluence rate either 10(-3) or 10(-5) W m-2. The duration of the subliminal light was chosen so that phototropism would not occur during this period. Phototropic latencies could be shortened by subliminal light that was less intense than the test light by several orders of magnitude. In experiments with the final unilateral light of fluence rate 10(-3) W m-2, the 447 nm subliminal light had a threshold (for the acceleration effect) of about 10(-11) W m-2. Yellow light of wavelength 575 nm, which itself is extremely ineffective for phototropism was extremely effective in shortening phototropic latencies in response in response to the test light. At 575 nm, the threshold was about 2 x 10(-12) W m-2. Conversely, near-UV light of wavelength 347 nm, which is highly effective for phototropism, was relatively ineffective (threshold approximately 7 x 10(-8) W m-2) in shortening the phototropic latency. Our results suggest the presence of a novel yellow-light absorbing pigment in Phycomyces that specifically regulates dark adaptation.(ABSTRACT TRUNCATED AT 250 WORDS)