Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real-Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro- from macro-adenomas and treated from non-treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas.     

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Reconsidering the sporulation characteristics of hypervirulent Clostridium difficile BI/NAP1/027

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and a major burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the BI/NAP1/027 type have become highly represented among clinical isolates. These so-called 'hypervirulent' strains are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Spores, formed during sporulation, play a pivotal role in disease transmission and it has been suggested that BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than 'non-epidemic' C. difficile types. Work in our laboratory has since provided credible evidence to the contrary suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased rate of sporulation. In this study, we analysed the sporulation rates of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28 BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which different C. difficile strains form spores. However, we clearly show that the sporulation rate of the BI/NAP1/027 type was no higher than that of non-BI/NAP1/027 strains. In addition, we observed substantial variation in sporulation characteristics within the BI/NAP1/027 type. This work highlights the danger of assuming that all strains of one type behave similarly without studying adequate sample sizes. Furthermore, we stress the need for more rigorous experimental procedures in order to quantify C. difficile sporulation more accurately in the future.     

Post-genomics of Neisseria meningitidis: an update

Neisseria meningitidis infection still remains a major life-threatening bacterial disease worldwide. The availability of bacterial genomic sequences generated a paradigm shift in microbiological and vaccines sciences, and post-genomics (comparative genomics, functional genomics, proteomics and a combination/evolution of these techniques) played important roles in elucidating bacterial biological complexity and pathogenic traits, at the same time accelerating the development of therapeutic drugs and vaccines. This article summarizes the most recent technological and scientific advances in meningococcal biology and pathogenesis aimed at the development and characterization of vaccines against the pathogenic meningococci.     

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Diplospory and obligate apomixis in Miconia albicans (Miconieae, Melastomataceae) and an embryological comparison with its sexual congener M. chamissois

Apomixis, or asexual reproduction through seeds, has been reported for species of the tribe Miconieae, Melastomataceae, but details of the process have yet to be described. We analyzed and compared sporogenesis and gametogenesis in the apomictic Miconia albicans and the sexual M. chamissois. The results point to some differences between species, which were related to the apomictic process. In M. albicans microsporogenesis, problems during meiosis and degeneration of its products led to total pollen sterility, while M. chamissois presented normal bicellular pollen grains in the mature anther. The absence or abnormality of meiosis in M. albicans megasporogenesis led to the formation of an unreduced embryo sac and also to egg cell parthenogenesis, which gave rise to the apomictic embryo. Embryo and endosperm development were autonomous, resulting in seeds and fruits independent of pollination and fertilization. Thus, in this species, apomixis can be classified as diplosporic and obligate. In contrast, meiosis was as expected in the sexual M. chamissois, and led to the development of a reduced embryo sac. Despite the divergent pathways, many embryological characteristics were similar between the studied species and other Melastomataceae and they seem to be conservative character states for the family.     

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation

Bis (cysteinyl) octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10^?3 M concentration was found to be very similar, but opposite to that of the K_ox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of β-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (≈ 90%). Similarly, operating under denaturing conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of β-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. © 1994 John Wiley & Sons, Inc. © 1994 John Wiley & Sons, Inc.