Does loss of heterozygosity in critical genome regions predict a local relapse in patients after laryngectomy?

BACKGROUND: Patients, who had an upper aerodigestive tract malignancy, have a high incidence of succeeding tumor development. This has been attributed to the role of "field cancerization" in carcinogenesis. The aim of this study was analysis of loss of heterozygosity (LOH) in the regions frequently lost during the course of head and neck squamous cell carcinomas (HNSCC), especially at early stages, which could answer the clinicians' question, if LOH analysis has any "predictive" value in relation to tumor occurrence. MATERIAL AND METHODS: Sixty-five larynx cancer patients were examined for loss of heterozygosity on 3p, 7q, 8p, 9p and 18q chromosomal arms with the use of 12 microsatellite markers. The material from a single patient consisted of blood, tumor, safe margin and one or two clinically unchanged mucosal samples. During follow up, the material from brush specimens (14 patients) as well as laryngeal swabs (4 patients) was also examined. RESULTS: The highest frequency of LOH was detected for marker D3S1234 in tumor tissues (29%). Analysis of margin samples (b) revealed low LOH frequencies (2-5%) and complete retention of heterozygosity for markers: D3S1234, D7S486, D8S261, D8S264, D9S171 and D18S46. Similarly, for normal appearing mucosa from upper part of larynx (c) frequencies of LOH were low (2-6%), with the complete retention of heterozygosity for markers: D3S1284, D3S1304, D3S1234, D8S264 and D9S1870. We did not detect any LOH in the material of normal appearing mucosa from tracheostoma region (d). During follow up, LOH was detected for eight markers, with the highest incidence for markers D18S46 (six cases), D7S486 (four cases) and D3S1300 (three cases). CONCLUSIONS: The data, obtained during this investigation, did not reveal the predictive value of LOH with respect to local relapse occurrence in laryngeal cancer patients. However, time of follow up did not reach 5 years, so that further clinical monitoring should be conducted.     

Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation

Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.     

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.     

Macrophyte communities in unimpacted European streams: variability in assemblage patterns, abundance and diversity

Macrophytes are an important component of aquatic ecosystems and are used widely within the Water Framework Directive (WFD) to establish ecological quality. In the present paper we investigated macrophyte community structure, i.e., composition, richness and diversity measures in 60 unimpacted stream and river sites throughout Europe. The objectives were to describe assemblage patterns in different types of streams and to assess the variability in various structural and ecological metrics within these types to provide a basis for an evaluation of their suitability in ecological quality assessment. Macrophyte assemblage patterns varied considerably among the main stream types. Moving from small-sized, shallow mountain streams to medium-sized, lowland streams there was a clear transition in species richness, diversity and community structure. There was especially a shift from a predominance of species-poor mosses and communities dominated by liverwort in the small-sized, shallow mountain streams to more species-rich communities dominated by vascular plants in the medium-sized, lowland streams. The macrophyte communities responded to most of the features underlying the typological framework defined in WFD. The present interpretation of the WFD typology may not, however, be adequate for an evaluation of stream quality based on macrophytes. First and most important, by using this typology we may overlook an important community type, which is characteristic of small-sized, relatively steep-gradient streams that are an intermediate type between the small-sized, shallow mountain streams and the medium-sized, lowland streams. Second, the variability in most of the calculated metrics was slightly higher when using the pre-defined typology. The consistency of these results should be investigated by analysing a larger number of sites. Particularly the need of re-defining the typology to improve the ability to detect impacts on streams and rivers from macrophyte assemblage patterns should be investigated. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Macrophyte communities of European streams with altered physical habitat

The impact of altering hydro-morphology on three macrophyte community types was investigated at 107 European stream sites. Sites were surveyed using standard macrophyte and habitat survey techniques (Mean Trophic Rank Methodology and River Habitat Survey respectively). Principal Components Analysis shows the macrophyte community of upland streams live in a more structurally diverse physical habitat than lowland communities. Variables representing the homogeneity and diversity of the physical environment were used to successfully separate un-impacted from impacted sites, e.g. homogeneity of depth and substrate increased with decreasing quality class for lowland sites (ANOVA p < 0.05). Macrophyte attribute groups and structural metrics such as species richness were successfully linked to hydro-morphological variables indicative of impact. Most links were specific to each macrophyte community type, e.g., the attribute group liverworts, mosses and lichens decreased in abundance with increasing homogeneity of depth and decreasing substrate size at lowland sites but not at upland sites. Elodea canadensis, Sparganium emersum and Potamogeton crispus were indicative of impacted lowland sites. Many of the indicator species are also known to be tolerant to other forms of impact. The potential for a macrophyte tool indicative of hydro-morphological impact is discussed. It is concluded one could be constructed by combining indicator species and metrics such as species richness and evenness.     

Improvements in G protein-coupled receptor purification yield light stable rhodopsin crystals

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins and are the target of approximately half of all therapeutic agents. Agonist ligands bind their cognate GPCRs stabilizing the active conformation that is competent to bind G proteins, thus initiating a cascade of intracellular signaling events leading to modification of the cell activity. Despite their biomedical importance, the only known GPCR crystal structures are those of inactive rhodopsin forms. In order to understand how GPCRs are able to transduce extracellular signals across the plasma membrane, it is critical to determine the structure of these receptors in their ligand-bound, active state. Here, we report a novel combination of purification procedures that allowed the crystallization of rhodopsin in two new crystal forms and can be applicable to the purification and crystallization of other membrane proteins. Importantly, these new crystals are stable upon photoactivation and the preliminary X-ray diffraction analysis of both photoactivated and ground state rhodopsin crystals are also reported.     

Hypothyroidism during treatment with tyrosine kinase inhibitors

Tyrosine kinase inhibitors are relatively new targeted therapy drugs used for the treatment of metastatic clear cell kidney carcinoma, gastrointestinal stromal tumours, thyroid carcinoma and pancreatic neuroendocrine tumours during the progression of the disease. Hypothyroidism or thyroid dysfunction is often a side effect of this treatment. Therefore, monitoring of thyroid hormone levels before the beginning and during the treatment of tyrosine kinase inhibitors is a necessity. Hypothyroidism correlates with objective response to the treatment. Sunitinib. This is the most described tyrosine kinase inhibitor which causes hypothyroidism. The mechanism of hypothyroidism is still unclear. Sorafenib. Symptoms of hypothyroidism occur in 18% of patients treated with sorafenib due to metastatic renal cell carcinoma. Imatinib. Hypothyroidism is one of the most frequent side effects of the treatment. Emergent tracheotomy was necessary due to larynx swelling during marked hypothyroidism. Motesanib. Hypothyroidism or increased TSH level is diagnosed in 22% to 69% of patients with metastatic differentiated or medullary thyroid carcinomas. The management of patients with thyroid dysfunction and related symptoms such as fatigue is undoubtedly a challenge to an oncologist.     

Macrozoobenthos of two Antarctic glacial coves: a comparison with non-disturbed bottom areas

There are only few studies on shallow Antarctic benthic communities associated with habitats affected by intense mineral sedimentation inflow. The analysis of macrofaunal communities associated with two shallow, isolated glacial coves was performed in Admiralty Bay (King George Island) and compared with non-disturbed sites. Multivariate analyses (hierarchical classification, nMDS) clearly separated glacial cove communities (two assemblages) from the sites situated outside both basins (two assemblages). The community influenced by the streamflow of glacial discharge of meltwater situated in the area with sandy–clay–silt sediments had a very low species richness, diversity and abundance. It was dominated by eurytopic, motile deposit feeding polychaetes such as Mesospio moorei, Tharyx cincinnatus and Leitoscoloplos kerguelensis as well as the bivalve Yoldia eightsi. The second glacial community of the area located at a grater distance from the outlet of the stream was characterized by sandy–clay–silt and clay–silt deposits and showed also a low diversity and species richness. The most abundant here were peracarid crustaceans, with the dominant opportunistic feeder Cheirimedon femoratus. Community from the non-disturbed area with silty–clay–sand, and silty–sand sediments had higher species richness and diversity. The assemblage of fauna from the sandy bottom has values of those two indexes similar to those found in the disturbed areas.     

Structure and reorientational dynamics of angiotensin I and II: a microscopic physical insight

We present a study of structural analysis and reorientational dynamics of Angiotensin I (AngI) and Angiotensin II (AngII) in aqueous solution. AngI is a decapeptide that acts as a precursor to the octapeptide AngII in the Renin-Angiotensin-Aldosterone system for blood pressure regulation. Experimental structural characterization of these peptides, carried out with circular dichroism and infrared spectroscopy, showed that the angiotensins are mostly disordered but exhibit a measurable population of ordered structures at room temperature. Interestingly, these change from the unordered polyproline-like conformation for AngI to a more compact and ordered conformation for AngII as the length of the peptide is decreased. Anisotropy decay measurements with picosecond time resolution indicate slower overall tumbling and a greater amplitude of internal motion in AngI compared to AngII, consistent with more compact and less flexible structure of the active form of the peptide. To model the microscopic behavior of the peptides, 2-μs molecular dynamics simulation trajectories were generated for AngI and AngII, at 300?K using the OPLS-AA potential and SPC water. The structures sampled in the simulations mostly agree with the experimental results, showing the prevalence of disordered structures, turns, and polyproline helices. Additionally, the computational results predict fewer sampled conformations, tighter side-chain packing and marked increase of Phe8 solvent accessibility upon AngI truncation to AngII. Our combined approach of experiment and extensive computer simulation thus yields new information on the conformational dynamics of the angiotensins, helping provide insight into the structural basis for the potency of AngI relative to AngII.