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.     

Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells†

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost‐effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein‐based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500‐fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast‐synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.     

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.     

COSII genetic maps of two diploid Nicotiana species provide a detailed picture of synteny with tomato and insights into chromosome evolution in tetraploid N. tabacum

Using single-copy conserved ortholog set (COSII) and simple sequence repeat (SSR) markers, we have constructed two genetic maps for diploid Nicotiana species, N. tomentosiformis and N. acuminata, respectively. N. acuminata is phylogenetically closer to N. sylvestris than to N. tomentosiformis, the latter two of which are thought to contribute the S-genome and T-genome, respectively, to the allotetraploid tobacco (N. tabacum L., 2n = 48). A comparison of the two maps revealed a minimum of seven inversions and one translocation subsequent to the divergence of these two diploid species. Further, comparing the diploid maps with a dense tobacco map revealed that the tobacco genome experienced chromosomal rearrangements more frequently than its diploid relatives, supporting the notion of accelerated genome evolution in allotetraploids. Mapped COSII markers permitted the investigation of Nicotiana–tomato syntenic relationships. A minimum of 3 (and up to 10) inversions and 11 reciprocal translocations differentiate the tomato genome from that of the last common ancestor of N. tomentosiformis and N. acuminata. Nevertheless, the marker/gene order is well preserved in 25 conserved syntenic segments. Molecular dating based on COSII sequences suggested that tobacco was formed 1.0MYA or later. In conclusion, these COSII and SSR markers link the cultivated tobacco map to those of wild diploid Nicotiana species and tomato, thus providing a platform for cross-reference of genetic and genomic information among them as well as other solanaceous species including potato, eggplant, pepper and the closely allied coffee (Rubiaceae). Therefore they will facilitate genetic research in the genus Nicotiana.     

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.     

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca²⁺ from thapsigargin-sensitive stores. Here, we have studied Ca²⁺ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca²⁺]_i was elevated and agonist-induced [Ca²⁺]_i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca²⁺. Importantly, [Ca²⁺]_i increases and Ca²⁺ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca²⁺ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca²⁺ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca²⁺]_i increase and the other with a slower and prolonged [Ca²⁺]_i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca²⁺]_i increases, reflecting overall Ca²⁺ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca²⁺ storage and elevated basal [Ca²⁺]_i. S1P metabolism thus plays a role not only in acute Ca²⁺ mobilization but also in long-term regulation of Ca²⁺ homeostasis.     

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.