Key role of two terminal domains in the bidirectional polymerization of FtsA protein

The effect of two different truncations involving either the 1C domain or the simultaneous absence of the S12-13 β-strands of the FtsA protein from Streptococcus pneumoniae, located at opposite terminal sides in the molecular structure, suggests that they are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA(+) polymers during the assembling process. Consequently, they block the growth of the FtsA(+) polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA(+) polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.     

X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.     

Evidence suggesting that ghrelin O-acyl transferase inhibitor acts at the hypothalamus to inhibit hypothalamo-pituitary-adrenocortical axis function in the rat

Production of n-octanoyl-modified ghrelin (GHREL), an active form of the peptide requires prohormone processing protease and GHREL O-acyltransferase (GOAT), as well as n-octanoic acid. Recently a selective GOAT antagonist (GO-CoA-Tat) was invented and this tool was used to study the possible role of endogenous GHREL in regulating HPA axis function in the rat. Administration of GOAT inhibitor (GOATi) resulted in a notable decrease in plasma ACTH, aldosterone and corticosterone concentrations at min 60 of experiment. Octanoic acid (OA) administration had no effect on levels of studied hormones. Plasma levels of unacylated and acylated GHREL remained unchanged for 60min after either GOATi or OA administration. Under experimental conditions applied, no significant changes were observed in the levels of GOAT mRNA in hypothalamus, pituitary, adrenal and stomach fundus. After GOATi injection hypothalamic CRH mRNA levels were elevated at 30 min and pituitary POMC mRNA levels at 60 min. Both GOATi and OA lowered basal, but not K(+)-stimulated CRH release by hypothalamic explants and had no effect on basal or CRH-stimulated ACTH release by pituitary slices. Neither GOATi nor OA affected corticosterone secretion by freshly isolated or cultured rat adrenocortical cells. Thus, results of our study suggest that in the rat endogenous GHREL exerts tonic stimulating effect on hypothalamic CRH release. This effect could be demonstrated by administering rats with selected inhibitor of ghrelin O-acyltransferase, the enzyme responsible for GHREL acylation, a process which is absolutely required for both GHSR-1a binding and its central endocrine activities.     

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.     

Melanopsin is highly resistant to light and chemical bleaching in vivo

Melanopsin is the photopigment of mammalian intrinsically photosensitive retinal ganglion cells, where it contributes to light entrainment of circadian rhythms, and to the pupillary light response. Previous work has shown that the melanopsin photocycle is independent of that used by rhodopsin (Tu, D. C., Owens, L. A., Anderson, L., Golczak, M., Doyle, S. E., McCall, M., Menaker, M., Palczewski, K., and Van Gelder, R. N. (2006) Inner retinal photoreception independent of the visual retinoid cycle. Proc. Natl. Acad. Sci. U.S.A. 103, 10426-10431). Here we determined the ability of apo-melanopsin, formed by ex vivo UV light bleaching, to use selected chromophores. We found that 9-cis-retinal, but not all-trans-retinal or 9-cis-retinol, is able to restore light-dependent ipRGC activity after bleaching. Melanopsin was highly resistant to both visible-spectrum photic bleaching and chemical bleaching with hydroxylamine under conditions that fully bleach rod and cone photoreceptor cells. These results suggest that the melanopsin photocycle can function independently of both rod and cone photocycles, and that apo-melanopsin has a strong preference for binding cis-retinal to generate functional pigment. The data support a model in which retinal is continuously covalently bound to melanopsin and may function through a reversible, bistable mechanism.     

Application of phenotypic microarrays to environmental microbiology

Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting.     

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.     

Long-term follow-up in adult patients with low-grade glioma (WHO II) postoperatively irradiated. Analysis of prognostic factors

AimTo report the long-term follow-up of a cohort of adult patients with LGG post-operatively irradiated in one institution, and to identify prognostic factors for progression free survival.BackgroundThere is little consensus about the optimal treatment for low-grade glioma (LGG), and the clinical management of LGG is one of the most controversial areas in neurooncology. Radiation therapy is one option for treatment of patients with LGG, whereas other options include postoperative observation.Materials and methodsBetween 1975 and 2005, 180 patients with LGG (WHO II) received postoperative irradiation after non radical (subtotal or partial) excision. Patients had to be 18 years of age or older, and have histologic proof of supratentorial fibrillary (FA), protoplasmic (PA) or gemistocytic astrocytoma (GA). Radiotherapy was given within 3–10 weeks after surgery. Treatment fields were localized and included the preoperative tumor volume, with a 1–2 cm margin, treated to a total dose of 50–60 Gy in 25–30 fractions over 5–6 weeks.ResultsActuarial ten-year progression free survival (APFS) in the whole group was 19%. The worse prognosis was observed in patients with GA. Ten-year APFS rates for GA, PA and FA were 10%, 18% and 22%, respectively.ConclusionThe findings from our long-term cohort of 180 patients with LGG confirmed by uni- and multivariate analysis demonstrated that only astrocytoma histology significantly determined the prognosis. The best survival was observed in patients with the fibrillary variant, and the worst for the gemistocytic one.