Synthesis, structure and cytotoxicity studies of diisopropylammonium and triethylammonium salts of triphenylphosphinegold(I) sulfanylcarboxylates

Compounds of the type [HQ][Au(PPh₃)(xspa)] and [HP][Au(PPh₃)(xspa)] {HQ = diisopropylammonium; HP = triethylammonium; H₂xspa = 3-aryl-2-sulfanylpropenoic acids [x: p = 3-phenyl-, f = 3-(2-furyl)-, t = 3-(2-thienyl)-, -o-py = 3-(2-pyridyl)-, Clp = 3-(2-chlorophenyl)-, -o-mp = 3-(2-methoxyphenyl)-, -p-mp = 3-(4-methoxyphenyl)-, -o-hp = 3-(2-hydroxyphenyl)-, -p-hp = 3-(4-hydroxyphenyl)-, diBr-o-hp = 3-(3,5-dibromo-2-hydroxyphenyl]} were synthesized and characterized by IR and NMR (¹H, ¹³C and ³¹P) spectroscopy and by FAB mass spectrometry. The structures of [HQ][Au(PPh₃)(Clpspa)] and [HQ][Au(PPh₃)(-o-mpspa)] show that the crystal contains hydrogen-bonded diisopropylammonium cations and [Au(PPh₃)(xspa)]⁻ anions. The anions in the two compounds have different structures, with the carboxylate group either coordinated or not coordinated to the gold atom, respectively. The in vitro antitumour activities against the HeLa-229, A2780 and A2780cis cell lines were determined for all complexes. The diisopropylammonium derivatives were generally found to be more active, in particular against the A2780cis cell line, and showed a high ability to circumvent the cellular resistance to cisplatin.     

Southern limit of distribution of the soft-shell clam <Emphasis Type="Italic">Mya arenaria</Emphasis> on the Atlantic East Coast

The invasive soft-shell clam Mya arenaria was recorded for the first time in the Tagus estuary, central Portugal. Propagules of the soft-shell clam were probably unintentionally introduced into the Tagus estuary through ballast water. Mya arenaria was observed in muddy sands in the upper intertidal zone. The assemblage in which the soft-shell clam was observed was composed of typical estuarine species such as the gastropod Hydrobia ulvae, the bivalve Scrobicularia plana and several polychaete species. The population density of Mya arenaria in the finding area was 40 ind m⁻² with a fresh biomass of 163.5 g FW m⁻².     

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54–1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54–1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.Filamentous fungi have an extraordinary ability to secrete proteins, secondary metabolites, and organic acids to the culture medium. The secreted proteins play important roles in nutrition, substrate colonization, or pathogenicity (1). This high secretory capacity has made filamentous fungi attractive for the commercial production of extracellular proteins (2), especially for the food and beverage industries (3), which have been using the compounds secreted by filamentous fungi for decades. Examples are provided by Aspergillus oryzae, which has been important for the production of traditional fermented foods and beverages in Japan and is used in modern biotechnology because of its ability to secrete large amounts of proteins (4) or Aspergillus niger, which has been widely used in biotechnology for the production of organic acids, food ingredients, and industrial enzymes (5). Because A. oryzae, A. niger, and Penicillium chrysogenum belong to the same fungal family, the latter microorganism might be considered of interest for the secretion of extracellular proteins.Although the understanding of the molecular basis of the secretion process in filamentous fungi is still limited (1), it is generally accepted that the secretion pathway in these microorganisms does not differ greatly from that present in yeasts and higher eukaryotes and protein secretion is believed to occur mainly at hyphal tips (6). The classical secretory pathway of proteins is driven by a canonical N-terminal signal peptide. These proteins enter the endoplasmic reticulum, where they are properly folded and modified (glycosylation, phosphorylation, etc.) and subsequently reach the Golgi compartment packed in transport vesicles. In this compartment, proteins can undergo further additional modifications such as glycosylation and peptide processing. Following this step, proteins are packed in secretory vesicles directed to the plasma membrane for secretion, or targeted to the vacuole either to become resident proteins or to undergo proteolytic degradation (7). In addition to the classical endoplasmic reticulum-Golgi pathway, it has been suggested that various kinds of mechanistically distinct nonclassical export routes may exist (8, 9). Cytoplasmic, nuclear and signal-peptide-containing proteins have been shown to reach the cell surface by nonconventional transport pathways (10). In yeasts, other mechanisms of secretion, which drive proteins lacking the signal peptide outside the plasma membrane, have also been described (11).P. chrysogenum is a filamentous fungus well-known by its ability to synthesize β-lactam antibiotics such as benzylpenicillin and isopenicillin N (12). Because the isolation of the wild-type strain NRRL 1951 from an infected cantaloupe in Peoria, Illinois in 1943 (13), this microorganism has undergone artificial selection by mutagenesis during industrial strain improvement programs, which gave rise to the improved-producing Wisconsin 54–1255 strain (hereafter named Wis 54-1255) (14). This strain became a laboratory model strain and was used for the genome sequencing project (15) and the intracellular proteome reference map (16). P. chrysogenum Wis 54–1255 was the ancestor of penicillin high-producing mutants, such as the AS-P-78 strain developed by Antibióticos S.A (León, Spain). The mutagenesis processes undergone by the P. chrysogenum strains during the industrial selection have introduced several important modifications in their metabolic networks (16).The recent advances in the Proteomics tools and the availability of genome sequences, has allowed an analysis of the secretomes of a few filamentous fungi, but the available information is still scarce (17–19). However, because of the availability of several fungal genomes and diverse prediction programs for secretory proteins, an integrated platform for annotation of fungal secretomes (Fungal Secretome Database) has been established and implemented in a web-based database (20). This database has been proposed as an integrated environment for the study of secretory proteins in the fungal kingdom.In order to fully characterize P. chrysogenum and to establish how the modifications acquired during the industrial strain improvement programs affected the wild type plant pathogenicity, analysis of the secreted proteins present in the culture broths was carried out. Using two-dimensional gel electrophoresis (2-DE)1 gels coupled to peptide mass fingerprint (PMF) and tandem MS we describe here for the first time the extracellular proteome of P. chrysogenum and the differences found in secreted protein among the wild type and two improved strains of this microorganism. Results reveal the nutritional versatility of this filamentous fungus and its potential interest for other biotechnological purposes different from antibiotic production, because nonpenicillin producer strains have been previously developed (21) that lack the penicillin biosynthesis genes (22) and can be used for other biotechnological uses.     

Comparative immunomodulatory effect of scopoletin on tumoral and normal lymphocytes

Some coumarins possess enhancing effects on lymphocyte mitogen responsiveness. In this investigation, the activity of scopoletin, a coumarin that has been isolated from different plants and in this case specifically from T. cordata Mill., was evaluated. For this purpose, normal T lymphocytes and a hyperproliferative T lymphoma cell line were used. Scopoletin was found to exert a dual action on tumoral lymphocytes exhibiting both a cytostatic and a cytotoxic effect. These effects varied with the concentrations analysed and the time of cell incubation (EC(50): 251+/-15 microg/ml) and were associated to the induction of apoptosis. Scopoletin induced cell proliferation on normal T lymphocytes (Proliferation stimulation index: 1 microg/ml scopoletin: 1.26+/-0.1; 10 microg/ml scopoletin: 3+/-0.25; 100 microg/ml scopoletin: 1.86+/-0.08); this stimulatory action was found to be due to the interaction with kinase C (PKC) protein. These results indicate that scopoletin could be a potential antitumoral compound to be used for cancer treatment.     

Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity

In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, tubacin), other structural related molecules (niltubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity.     

A combination of five short tandem repeats of chromosome 15 significantly improves the identification of Prader-Willi syndrome etiology in the Argentinean population

Prader-Willi syndrome (PWS) is a multisystemic disorder caused by the loss of expression of paternally transcribed genes in the PWS critical region of chromosome 15. Various molecular mechanisms are known to lead to PWS: deletion 15q11-q13 (75% of cases), maternal uniparental disomy (matUPD15) (23%) and imprinting defects (2%). FISH and microsatellite analysis are required to establish the molecular etiology, which is essential for appropriate genetic counseling and care management. We characterized an Argentinean population, using five microsatellite markers (D15S1035, D15S11, D15S113, GABRB3, D15S211) chosen to develop an appropriate cost-effective method to establish the parental origin of chromosome 15 in nondeleted PWS patients. The range of heterozygosity for these five microsatellites was 0.59 to 0.94. The average heterozygosity obtained for joint loci was 0.81. The parental origin of chromosome 15 was established by microsatellite analysis in 19 of 21 non-deleted PWS children. We also examined the origin of the matUPD15; as expected, most of disomies were due to a maternal meiosis I error. The molecular characterization of this set of five microsatellites with high heterozygosity and polymorphism information content improves the diagnostic algorithm of Argentinean PWS children, contributing significantly to adequate genetic counseling of such families.     

Design and synthesis of 3,4-methylenedioxy-6-nitrophenoxyacetylhydrazone derivatives obtained from natural safrole: new lead-agents with analgesic and antipyretic properties

In this work, we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and antipyretic properties of new 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazone derivatives (3), designed exploring molecular hybridization and isosteric replacement approaches between nimesulide (1) and carbanalogue NAH series (2) developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (4) as starting material. The evaluation of the antinociceptive properties of this series led us to discover a new potent prototype of analgesic and antipyretic agent, that is, NAH derivative 3c, named LASSBio-891, which showed to be more potent than dipyrone used as standard.     

Design of new dopamine D2 receptor ligands: Biosynthesis and pharmacological evaluation of the hydroxylated metabolite of LASSBio-581

LASSBio-581 is a N-phenylpiperazine derivative designed for the treatment of schizophrenia. In this study, four strains of filamentous fungi were screened for their capabilities to biotransform LASSBio-581. Cunninghamella echinulata ATCC 9244 was chosen to scale up the biosynthesis of the p-hydroxylated metabolite of LASSBio-581. The chemical structure of the metabolite was confirmed by NMR, LC–MS and X-ray crystallography. Binding studies performed on brain homogenate indicated that the p-hydroxylated metabolite can be considered more selective for dopamine receptors than LASSBio-581, and, therefore, can be used to design new selective dopamine inhibitors.     

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.