Towards stress freezing in birefringent orthotropic composite models

Birefringent composite models are fabricated using epoxy resin reinforced with unidirectionally oriented glass fibers. The mechanical and photoelastic properties of the material at room temperature are determined. To explore the possibility of application of stress-freezing technique to birefringent composite models, the behavior and properties of this material are studied at elevated temperature (at stress-freezing temperature of the resin). The properties of the material at room and at elevated temperatures are reported. The feasibility of stress freezing glass-fiber-reinforced epoxy composites with low-fiber-volume fraction is discussed.

Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.

Regulation of Growth by Drosophila FoxO Transcription Factor

Forkhead box class O (FoxO) transcription factors are members of the forkhead box transcription factor superfamily, with orthologues in various species such as human, worm and fly. FoxO proteins are key regulators of growth, metabolism, stress resistance and, consequently, life span. FoxOs integrate signals from different pathways, e.g. the growth controlling Insulin-TOR signaling pathway and the stress induced JNK and Hippo signaling pathways. FoxO proteins have evolved to guide the cellular response to varying energy and stress conditions by inducing the expression of genes involved in the regulation of growth and metabolism. This work has aimed to deepen the understanding of how FoxO executes its biological functions. A particular emphasis has been laid to its role in growth control. Specifically, evidence is presented indicating that FoxO restricts tissue growth in a situation when TOR signaling is high. This finding can have implications in a human condition called Tuberous sclerosis, manifested by multiple benign tumors. Further, it is shown that FoxO directly binds to the promoter and regulates the expression of a Drosophila Adenylate cyclase gene, ac76e, which in turn modulates the fly s development and growth systemically. These results strengthen FoxOs position among central size regulators as it is able to operate at the level of individual cells as well as in the whole organism. Finally, an attempt to reveal the regulatory network upstream of FoxO has been carried out. Several putative FoxO activity regulators were identified in an RNAi screen of Drosophila kinases and phosphatases. The results underscore that FoxO is regulated through an elaborate network, ensuring the correct execution of key cellular processes in metabolism and response to stress. Overall, the evidence provided in this study strengthens our view of FoxO as a key integrator of growth and stress signals.

The role of MicroRNAs in stress response in the resurrection plant Tripogon loliiformis

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.

Comment on "Pseudoelasticity of Cu-Zr nanowires via stress-induced martensitic phase transformations"

Recently, a novel stress-induced phase transformation in an initial < 100 >/{100} B2-CuZr nanowire has been reported for the first time [Sutrakar and Mahapatra, Mater. Lett. 63, 1289 (2009)]. Following this, a martenisitic phase transformation in Cu-Zr nanowire was shown [Cheng et al., Appl. Phys. Lett. 95, 021911 (2009)] using the same idea (Sutrakar and Mahapatra, Mater. Lett. 63, 1289 (2009)]. The pseudoelastic recovery of the bct phase of Cu-Zr by unloading has also been shown [Cheng et al., Appl. Phys. Lett. 95, 021911 (2009)]. They also tested the epitaxial bain path [Alippi et al., Phys. Rev. Lett. 78, 3892 (1997)] and reported that the bct phase in the nanowire is metastable, whereas the bulk counterpart is unstable. This aspect is re-examined in this comment with corrected results.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

Nucleotide sequence of the 3' terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins

The 3prime terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3prime terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

The structure of glycosaminoglycans and their interactions with proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three-dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.