Characterization of the residual structure in the unfolded state of the Delta131Delta fragment of staphylococcal nuclease

The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular dynamics simulations to characterise the residual structure of the Delta131Delta fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that Delta131Delta under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild-type staphylococcal nuclease. Only rarely, however, all these interactions are simultaneously realized to generate conformations with an overall native topology.     

Multipoint identity-by-descent computations for single-point polymorphism and microsatellite maps

We used the LOKI software to generate multipoint identity-by-descent matrices for a microsatellite map (with 31 markers) and two single-nucleotide polymorphism (SNP) maps to examine information content across chromosome 7 in the Collaborative Study on the Genetics of Alcoholism dataset. Despite the lower information provided by a single SNP, SNP maps overall had higher and more uniform information content across the chromosome. The Affymetrix map (578 SNPs) and the Illumina map (271 SNPs) provided almost identical information. However, increased information has a computational cost: SNP maps require 100 times as many iterations as microsatellites to produce stable estimates.     

Taphonomy of Eemian marine molluscs and acorn barnacles from eastern Arkhangelsk region, northern Russia

Marine Eemian deposits along the Pyoza river and its tributary Varchuska, Arkhangelsk region, constitute successions of muddy and sandy facies with rich macrobenthic fauna dominated by bivalves and barnacles. Taphonomic features formed by abrasion, disarticulation, dissolution, fragmentation, bioerosion and encrustation define taphofacies for a palaeoenvironmental model. Five bivalve taphofacies and three barnacle taphofacies could be distinguished. Both bivalves and barnacles are poorly preserved in foreshore/shoreface environments, as the shells were subjected to extensive transportation by currents. The shells were best preserved in offshore environments, where rapid episodic sedimentation enabled within-habitat preservation, in some cases even preservation in life position. Barnacles are absent from the most clay-rich offshore deposits, probably because of clogging of filters by turbidity and lack of suitable substrate. Such dissimilarities suggest that the number and distribution of taphofacies may depend on which fossil groups are used. Interspecific variability may exist within the individual taphofacies. The barnacles, for example, tend to be better preserved than the mussel Mytilus edulis, although both are fixosessile suspension feeders. This indicates that not only life habits but also intrinsic shell properties influence preservation. Thus, taphofacies analyses should combine data on taphonomic features, specific life habit and shell properties to determine overall preservation patterns. In that way, taphofacies analyses may form a powerful tool for palaeoenvironmental analyses of marine deposits.     

Production of Recombinant Proteins with Pichia pastoris in Integrated Processing

Devices and methods for Integrated Bioprocessing have been developed for production of recombinant proteins with the yeast Pichia pastoris. In doing so cross flow filtration techniques for cell separation and product concentration are connected directly to high instrumented cultivation processes. These are equipped with on‐line measuring techniques for substrates and products, e.g., glycerol, methanol and pyruvate as well as recombinant proteins, e.g., the chemokines 1–8del MCP‐1 and vMIP‐II. Complex automation structures allow for process development at virtual plants which can be used as the basis for establishing and implementing fully automated real processes. Experiments for determination of reaction kinetics, optimization of productivity in high‐cell density cultures and Integrated Bioprocessing are outlined, along with detailed illustration of the realization of the methods at industrial pilot plant scale.     

Within-season flowering interruptions are common in the water-limited Sky Islands

Within-season breaks in flowering have been reported in a wide range of highly variable ecosystems including deserts, tropical forests and high-elevation meadows. A tendency for interruptions in flowering has also been documented in southwestern US “Sky Island” plant communities, which encompass xeric to mesic conditions. Seasonal breaks in flowering have implications for plant reproductive success, population structure, and gene flow as well as resource availability for pollinators and dependent animals. Most reports of multiple within-season flowering events describe only two distinct flowering episodes. In this study, we set out to better quantify distinct within-season flowering events in highly variable Sky Islands plant communities. Across a >1,200 m elevation gradient, we documented a strong tendency for multiple within-season flowering events. In both distinct spring and summer seasons, we observed greater than two distinct within-season flowering in more than 10 % of instances. Patterns were clearly mediated by the different climate factors at work in the two seasons. The spring season, which is influenced by both temperature and precipitation, showed a mixed response, with the greatest tendency for multiple flowering events occurring at mid-elevations and functional types varying in their responses across the gradient. In the summer season, during which flowering across the gradient is limited by localized precipitation, annual plants exhibited the fewest within-season flowering events and herbaceous perennial plants showed the greatest. Additionally, more distinct events occurred at lower elevations. The patterns documented here provide a baseline for comparison of system responses to changing climate conditions.     

Mapping the degradation pathway of a disease-linked aspartoacylase variant

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.     

Characterization of a Novel Polyomavirus Isolated from a Fibroma on the Trunk of an African Elephant (Loxodonta africana)

Viruses of the family Polyomaviridae infect a wide variety of avian and mammalian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. In this study a novel polyomavirus, the African elephant polyomavirus 1 (AelPyV-1) found in a protruding hyperplastic fibrous lesion on the trunk of an African elephant (Loxodonta africana) was characterized. The AelPyV-1 genome is 5722 bp in size and is one of the largest polyomaviruses characterized to date. Analysis of the AelPyV-1 genome reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. In the area preceding the VP2 start codon three putative open-reading frames, possibly coding for an agnoprotein, could be localized. A regulatory, non-coding region separates the 2 coding regions. Unique for polyomaviruses is the presence of a second 854 bp long non-coding region between the end of the early region and the end of the late region. Based on maximum likelihood phylogenetic analyses of the large T antigen of the AelPyV-1 and 61 other polyomavirus sequences, AelPyV-1 clusters within a heterogeneous group of polyomaviruses that have been isolated from bats, new world primates and rodents.     

Bowl-shaped structures in a Pleistocene clastic carbonate wedge on the Island of Rhodes,Greece

Abstract

Bowl-shaped structures are for the first time reported from the Greek Island of Rhodes. They occur in Pleistocene deposits of the Cape Arkhangelos Formation in the Rhodes Synthem. The regularity of their three-dimensional appearance is the argument for a biological origin. This gives the reason to assign the structures to Piscichnus waitemata for which we issue a formal diagnosis. They were formed on a clastic carbonate wedge that built into a coastal, steep-sided basin. Associate trace fossils are Thalassinoides suevicus and Bichordites monastiriensis.     

Therapeutic targeting of the p53 tumor suppressor gene

The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.     

Cloning and characterization of two alternatively spliced rat alpha-amidating enzyme cDNAs from rat medullary thyroid carcinoma

The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.