Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.     

Cross talk between the KNOX and ethylene pathways is mediated by intron-binding transcription factors in barley

In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.     

Do digger wasps time their provisioning activity to avoid cuckoo wasps (Hymenoptera: Crabronidae and Chrysididae)?

Strategies adopted by parasitoids and kleptoparasites co-evolve with the defensive adaptations of their hosts, and vice-versa. Hedychrum rutilans and Hedychrum nobile are brood parasites of, respectively, Philanthus triangulum and Cerceris arenaria, two digger wasps that share most aspects of their nesting biology (solitary females dig aggregated nests in the ground and mass-provision the brood with paralyzed insects). We tested the hypothesis that similarity in the hosts’ nesting habits corresponds to similar defensive strategies against these cuckoo wasps. Peak provisioning activity by P. triangulum occurred in late afternoon (and early morning in 1 year) while peak H. rutilans activity was in early afternoon. In contrast, peak provisioning by C. arenaria and peak H. nobile activity occurred in early afternoon. Thus, P. triangulum (as previously found in other populations) appears to have timed its provisioning to avoid its brood parasite whereas C. arenaria did not, rejecting our hypothesis. The daily activity of both chrysidid wasps was positively correlated to air temperature. Host nest density positively affected only H. rutilans activity, in agreement with previous reports for other populations of this species, whereas the daily pattern of activity of the host was the key correlate with the activity of H. nobile. Mortality due to cuckoo wasps was low for both digger wasps, although it was somewhat higher at sites with many nests of P. triangulum. We suggest that perhaps the degree to which digger wasps time their activity to avoid cuckoo wasps is related to the degree of specialization in host choice by their brood parasite. H. rutilans is more highly dependent on P. triangulum, which may have had a greater effect on the timing of provision by its host.     

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

Amino Acids - The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the...     

A non-pollen palynomorphs contribution to the local environmental history in the Ligurian Apennines: a preliminary study

As part of a regional research project on wetlands, a analysis was carried out on non pollen palynomorphs (NPP) from the upper sediments of a mountain peat bog in the Ligurian Apennines, northwest Italy. The aim of this project was to see if NPPs could yield useful results on local ecology and human activity in connection with the use of resources. More than 60 NPP types were found, among which a new type is described. A good agreement was observed between pollen assemblages and different NPP types, which are still an under-exploited source of information. Coprophilous fungi suggest the presence of livestock at the site after cal. a.d. 770–1160, while various algae and other water-demanding organisms show an increasing eutrophication of the environment in the most recent phase after cal. a.d. 1500. This approach, which is new for this region, confirms the hypothesis that local farming practices, such as management of pasture and woodland, were not recently established here. The site features and both NPP variety and significance show that further improvement of this additional tool could contribute to answering some questions about past cultural landscapes.     

Solid phase subtractive cloning in differentially expressed genes identification

We developed an array-based subtractive hybridization system for one-step high-throughput subtraction. We printed subtractor RNA up to 10.000 times obtaining an excellent contact surface using a little amount of RNA. During hybridization cDNA, common to subtractor and target samples, remains attached to slide immobilized RNA, leaving free in solution target specific cDNA which after retrieval is cloned.     

PNA zipper as a dimerization tool: Development of a bZip mimic

The article describes the use of a PNA duplex (PNA zipper) as a tool to dimerize or bring in close proximity two polypeptides or protein domains. The amino acid sequence to be dimerized is covalently bound to complementary PNA sequences. Annealing of the PNA strands results in dimer formation. To test the ability of the “PNA‐zipper” as a dimerization tool, we designed a GCN4 mimetic, where the leucine‐zipper dimerization domain was replaced by the PNA zipper, whereas the basic DNA‐binding domain was covalently attached to the PNA. The molecule was assembled by chemical ligation of the peptide corresponding to the DNA‐binding domain of GCN4 modified with a succinyl thioester with two complementary PNAs harboring a cysteine residue. Electromobility‐shift experiments show the ability of the PNA zipper‐GCN4 to bind selected DNA duplexes. The PNA zipper‐GCN4 binds both the TRE and CRE DNA sites, but it does not bind TRE and CRE mutants containing even a single base mutation, as the native GCN4. The ability to fold upon complexation with DNA was investigated by CD. A good correlation between the ability of the PNA zipper‐GCN4 to fold into α helices and the ability to bind DNA was found. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 434–441, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

FIRST RECORD OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA) IN THE PO DELTA LAGOONS,MEDITERRANEAN SEA (ITALY)1

Molecular outcomes led us to report the first occurrence of the invasive alien species Gracilaria vermiculophylla (Ohmi) Papenf. in the Mediterranean Sea. This species was recorded for the first time in the Po Delta lagoons in May and October 2008, probably introduced by the importation of the Manila clam Tapes philippinarum. At present, G. vermiculophylla is spread only near some clam‐farming areas, but its diffusion is expected to increase with the colonization of other lagoons where aquaculture is spread, as already observed for other alien species such as Agardhiella subulata and Solieria filiformis. The present study supplies further information on the morphology of this species, the ecological characteristics of the colonized areas, and the most probable introduction vector, confirming that the species spreading occurs in eutrophic and turbid coastal systems.