bluuebunny/crossref_metadata_2025 huggingface dataset:
Secondary structure and dynamics of micelle bound β‐ and γ‐synuclein
Authors: Sung Yoon‐hui , Eliezer David
Published: April 2006

We have used solution state NMR spectroscopy to characterize the secondary structure and backbone dynamics of the proteins β‐ and γ‐synuclein in their detergent micelle‐bound conformations. Comparison of the results with those previously obtained for the Parkinson's disease‐linked protein α‐synuclein shows that structural differences between the three homologous synuclein family members are directly related to variations in their primary amino acid sequences. An 11‐residue deletion in the lipid‐binding domain of β‐synuclein leads to the destabilization of an entire segment of the micelle‐bound helical structure containing the deletion site. The acidic C‐terminal tail region of γ‐synuclein, which displays extensive sequence divergence, is more highly disordered than the corresponding regions in the other two family members. The observed structural differences are likely to mediate functional variations between the three proteins, with differences between α‐ and β‐synuclein expected to revolve around their lipid interactions, while differences in γ‐synuclein function are expected to result from different protein–protein interactions mediated by its unique C‐terminal tail.
pK values of the ionizable groups of proteins
Authors: Thurlkill Richard L. , Grimsley Gerald R. , Scholtz J. Martin , Pace C. Nick
Published: April 2006

We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25°C are: 3.67±0.03 (α‐carboxyl), 3.67±0.04 (Asp), 4.25±0.05 (Glu), 6.54±0.04 (His), 8.00±0.03 (α‐amino), 8.55±0.03 (Cys), 9.84±0.11 (Tyr), and 10.40±0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N&T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N&T); His (6.54 this work vs. 6.3 N&T); α‐amino (8.00 this work vs. 7.5 N&T); Cys (8.55 this work vs. 9.5 N&T); and Tyr (9.84 this work vs. 9.6 N&T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins.
The IclR family of transcriptional activators and repressors can be defined by a single profile
Authors: Krell Tino , Molina‐Henares Antonio Jesús , Ramos Juan Luis
Published: April 2006

In the last decade enormous advances in life sciences have been possible due to the information obtained from DNA sequencing projects. The optimal interpretation and analysis of genome sequence data requires the precise annotation and classification of proteins deduced from open reading frames, which is usually done with the help of family‐specific signatures. Here we report a novel profile for the IclR type of transcriptional activators and repressors. In contrast to profiles for other families of transcriptional regulators, the new IclR profile is located outside the helix‐turn‐helix DNA‐binding motif. We provide evidence that the new profile is more specific than any of the existing signatures for this family of regulators. More than 500 representatives of this family were identified with this profile. A database on bacterial regulators (http://www.bactregulators.org) was built to compile and regroup the sequences with the aid of the new profile.
Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands
Authors: Rice Jeffrey J. , Schohn Aaron , Bessette Paul H. , Boulware Kevin T. , Daugherty Patrick S.
Published: April 2006

A bacterial display methodology was developed for N‐ and C‐terminal display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) display was directly compared to insertional fusion display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, bacterial display yielded the well‐characterized HPQ/M motif obtained previously using several alternative peptide display systems, as well as three additional motifs (LI/V CQNVCY, CGWMYF/YxEC, ERCWYVMHWPCNA). Using CPX display, a very high affinity streptavidin‐binding peptide was isolated having a dissociation rate constant koff = 0.002sec−1 even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N‐terminal display yields sequences with greater diversity, affinity, and modularity. CPX bacterial display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.
Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein
Authors: Beevers Andrew J. , Kukol Andreas
Published: April 2006

Human phospholemman (PLM) is a 72‐residue protein, which is expressed at high density in the cardiac plasma membrane and in various other tissues. It forms ion channels selective for K+, Cl−, and taurine in lipid bilayers and colocalizes with the Na+/K+‐ATPase and the Na+/Ca2+‐exchanger, which may suggest a role in the regulation of cell volume. Here we present the first structural data based on synthetic peptides representing the transmembrane domain of PLM. Perfluoro‐octaneoate‐PAGE of reconstituted proteoliposomes containing PLM reveals a tetrameric homo‐oligomerization. Infrared spectroscopy of proteoliposomes shows that the PLM peptide is completely α‐helical, even beyond the hydrophobic core residues. Hydrogen/deuterium exchange experiments reveal that a core of 20–22 residues is not accessible to water, thus embedded in the lipid membrane. The maximum helix tilt is 17° ± 2° obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.
Assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase
Authors: Bemporad Francesco , Taddei Niccolò , Stefani Massimo , Chiti Fabrizio
Published: April 2006

Among the many parameters that have been proposed to promote amyloid fibril formation is the π‐stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non‐aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation‐promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic β‐sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity.
Src kinase activation: A switched electrostatic network
Authors: Ozkirimli Elif , Post Carol Beth
Published: April 2006

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A‐loop); and movement of an α‐helix (αC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down‐regulated conformation of CD for the Src‐kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A‐loop motion is more complex requiring secondary structure changes as well as communication with αC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down‐regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.
Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study
Authors: Rajan Rahul S. , Li Tiansheng , Aras Mohini , Sloey Christopher , Sutherland Weston , Arai Hiromi , Briddell Robert , Kinstler Olaf , Lueras Alexis M.K. , Zhang Yu , Yeghnazar Heather , Treuheit Michael , Brems David N.
Published: April 2006

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N‐terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG‐GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG‐GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG‐GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG‐GCSF and 20kPEG‐GCSF. It was observed that even 5kPEG‐GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG‐GCSF. Biological activity measurements demonstrated that both 5kPEG‐GCSF and 20kPEG‐GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.
Sequential reorganization of β‐sheet topology by insertion of a single strand
Authors: Sagermann Martin , Baase Walter A. , Matthews Brian W.
Published: April 2006

Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%–50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the β‐sheet region in T4 lysozyme. Copies of two different β‐strands were inserted into two different loops of the β‐sheet, and the structures were determined. Not surprisingly, one insert “loops out” at its insertion site and forms a new small β‐hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the β‐sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C‐terminal end of the same β‐strand. Reasons as to why a non‐native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.
Structure and dynamics of the epidermal growth factor receptor C‐terminal phosphorylation domain
Authors: Lee Nam Y. , Hazlett Theodore L. , Koland John G.
Published: April 2006

The C‐terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR‐ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60‐ to 70‐kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C‐terminally truncated form of EGFR‐ICD yielded a slow component consistent with the rotational motion of the 38‐kDa kinase core. These findings suggested a structural arrangement of the EGFR‐ICD in which the C‐terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR‐ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62‐kDa EGFR‐ICD, suggesting an increase in the mobility of the C‐terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c‐Src and the phosphorylated EGFR C‐terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C‐terminal domain possessed a significant level of secondary structure in the form of α‐helices and β‐sheets, with a marginal change in β‐sheet content occurring upon phosphorylation.
Calorimetric and structural studies of the nitric oxide carrier S‐nitrosoglutathione bound to human glutathione transferase P1‐1
Authors: Téllez‐Sanz Ramiro , Cesareo Eleonora , Nuccetelli Marzia , Aguilera Ana M. , Barón Carmen , Parker Lorien J. , Adams Julian J. , Morton Craig J. , Lo Bello Mario , Parker Michael W. , García‐Fuentes Luis
Published: April 2006

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S‐nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1‐1 (hGSTP1‐1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild‐type hGSTP1‐1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild‐type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S‐nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix α2 of hGST P1‐1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild‐type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix α2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S‐nitrosylation of Cys47 by GSNO is a likely physiological scenario.
Modular organization of FDH: Exploring the basis of hydrolase catalysis
Authors: Reuland Steven N. , Vlasov Alexander P. , Krupenko Sergey A.
Published: April 2006

An abundant enzyme of liver cytosol, 10‐formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase‐homologous domain and a folate‐binding hydrolase domain, which are connected by an ∼100‐residue linker. The amino‐terminal hydrolase domain of FDH (Nt‐FDH) is a homolog of formyl transferase enzymes that utilize 10‐formyl‐THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+‐dependent oxidation of 10‐formyltetrahydrofolate (10‐formyl‐THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl‐tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt‐FDH and related 10‐formyl‐THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10‐formyl‐THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.
The crystal structure of YycH involved in the regulation of the essential YycFG two‐component system in Bacillus subtilis reveals a novel tertiary structure
Authors: Szurmant Hendrik , Zhao Haiyan , Mohan Michael A. , Hoch James A. , Varughese Kottayil I.
Published: April 2006

The Bacillus subtilis YycFG two‐component signal transduction system is essential for cell viability, and the YycH protein is part of the regulatory circuit that controls its activity. The crystal structure of YycH was solved by two‐wavelength selenium anomalous dispersion data, and was refined using 2.3 Å data to an R‐factor of 25.2%. The molecule is made up of three domains, and has a novel three‐dimensional structure. The N‐terminal domain features a calcium binding site and the central domain contains two conserved loop regions.
Enhancing functional production of G protein‐coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen
Authors: André Nicolas , Cherouati Nadia , Prual Cécile , Steffan Tania , Zeder‐Lutz Gabrielle , Magnin Thierry , Pattus Franc , Michel Hartmut , Wagner Renaud , Reinhart Christoph
Published: April 2006

We have optimized the expression level of 20 mammalian G protein‐coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding‐competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.
Crystal structure of the conserved protein TTHA0727 from Thermus thermophilus HB8 at 1.9 Å resolution: A CMD family member distinct from carboxymuconolactone decarboxylase (CMD) and AhpD
Authors: Ito Kaori , Arai Ryoichi , Fusatomi Emiko , Kamo‐Uchikubo Tomomi , Kawaguchi Shin‐ichi , Akasaka Ryogo , Terada Takaho , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki
Published: April 2006

TTHA0727 is a conserved hypothetical protein from Thermus thermophilus HB8, with a molecular mass of 12.6 kDa. TTHA0727 belongs to the carboxymuconolactone decarboxylase (CMD) family (Pfam 02627). A sequence comparison with its homologs suggested that TTHA0727 is a distinct protein from alkylhydroperoxidase AhpD and γ‐carboxymuconolactone decarboxylase in the CMD family. Here we report the 1.9 Å crystal structure of TTHA0727 (PDB ID: 2CWQ) determined by the multiwavelength anomalous dispersion method. The TTHA0727 monomer structure consists of seven α‐helices (α1–α7) and one short 310‐helix. The crystal structure and the analytical ultracentrifugation revealed that TTHA0727 forms a hexameric ring structure in solution. The electrostatic potential distribution on the solvent‐accessible surface of the TTHA0727 hexamer showed that positively charged regions exist on the side of the ring structure, suggesting that TTHA0727 interacts with some negatively charged molecules. A structural homology search revealed that the structure of three α‐helices (α4–α6) is remarkably conserved, suggesting that it is the common structural motif for the CMD family proteins. In addition, the nine residues of the N‐terminal tag bound to the cleft region between α1 and α3 in chains A and B of TTHA0727, implying that this region is the putative binding/active site for some small molecules.
THE PROTOPLASMIC ULTRASTRUCTURE OF THE VEGETATIVE CELL OF MELOSIRA VARIANS C. A. AGARDH1
Authors: Crawford Richard M.
Published: April 2006

A detailed fine structural study of the nondividing vegetative cell of the centric diatom Melosira varians reveals peripheral organization of cytoplasm and organelles around a central vacuole. Polarity of the cell is such that the nucleus is found at the valve face of the epitheca. The unusual shape of the nucleus and its associated microtubular system and polar body are thought to relate to the migration of the nucleus during other phases of cell growth. Unusual discontinuities of the plastid lamellae are reported and the significance of the periplastid reticulum is discussed. Possible roles for the cytoplasmic vesicles and the membrane system in the cell are also considered.
ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1
Authors: Scott J. L. , Dixon P. S.
Published: April 2006

The differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup‐shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.
PERIDINIUM BALTICUM (LEVANDER) LEMMERMANN, AN UNUSUAL DINOFLAGELLATE WITH A MESOCARYOTIC AND AN EUCARYOTIC NUCLEUS123
Authors: Tomas Ronald N. , Cox Elenor R. , Steidinger Karen A.
Published: April 2006

Light and electron microscopy indicate that Peridinium balticum possesses 2 Feulgen‐positive, membrane‐bound nuclei which divide synchronously. One nucleus has the typical structure of dinocaryotic dinoflagellates, while the other nucleus has a structure typical of eucaryotic organisms. Connections between each nucleus and the endoplasmic reticulum are common. Membrane‐bound vesicles are intimately associated with the nuclear envelope of the eucaryotic nucleus.
AGGREGATION OF SOIL PARTICLES BY ALGAE1
Authors: Bailey Don , Mazurak Andrew P. , Rosowski James R.
Published: April 2006

Chlorella pyrenoidosa Chick (Indiana University Number 252), Nostoc commune Vaucher (I.U. 584), and Oscillatoria prolifica (Grev.) Gomont (I.U. 1270) were grown separately on Peoria loess soil material to measure their effects on the water stability of soil aggregates. Each alga significantly (10% LSD) increased the percentage of soil aggregates after 6 weeks of incubation as compared with the soil without algae. Oscillatoria, Chlorella, and Nostoc increased water stability of aggregates >74 μ in diameter by 3.4, 1.1, and 0.6%, respectively. Nostoc and Oscillatoria produced measurable water stable aggregates in the 1000–2000 μ diameter range; Chlorella formed them in the 500–1000 μ range, while the control soil showed no aggregates >295 μ.
EFFECT OF INCUBATION TEMPERATURE ON THE MACROMOLECULAR AND LIPID CONTENT OF THE PHYTOFLAGELLATE OCHROMONAS DANICA1
Authors: Aaronson S.
Published: April 2006

Ochromonas danica grown in a chemically defined medium under controlled conditions at different incubation temperatures showed marked changes in chemical composition and secretory pattern. As the incubation temperature rose from 15 to 30 C, the cell number per unit volume of medium increased; the cell mass reached a maximum and then declined slightly, and the weight per single cell decreased. The chemical composition of the cells changed with increasing temperature: carbohydrates decreased dramatically; proteins quadrupled at 20 C and then declined 25%; lipids increased; and RNA increased to 25 C and then decreased to about 40% of maximum at 30 C. The extracellular secretion by the cells of carbohydrates seemed to increase and then decline; RNA decreased; proteins increased; and lipids remained approximately constant, as the temperature increased. The implications of these findings for thermal pollution are discussed.
PROTOTHECA: AN ALGA OR A FUNGUS?1
Authors: Nadakavukaren Mathew J. , McCracken Derek A.
Published: April 2006

Although Prototheca is generally considered to be a colorless member of the Chlorococcales, there have been a number of attempts to relate it to the fungi. However, our ultrastructural observations support the conclusion that it is indeed a nonphotosynthetic alga and not a fungus. One such structural feature that is of special interest is the presence of membrane‐bounded starch granules which are considered to be storage plastids. Fungi are not known to produce plastids.
Application of HEMA on intact mouse skin – effects on the immune system
Authors: Sandberg E. , Dahlgren Ulf I.
Published: April 2006

Previously, we have shown that 2‐hydroxyethylmethacrylate (HEMA) can bind to protein and that autoantibodies were induced in mice by immunization with a self‐protein in vitro conjugated with HEMA. The present study aimed to develop a model for HEMA‐induced sensitivity by the application of the substance on intact skin. Female BALB/c mice were painted on the dorsum of each ear with 50% HEMA in vehicle twice a week for 6 weeks. The anti‐CD3ε‐stimulated lymph node production and the spontaneous spleen‐cell production of the cytokines interleukin (IL)‐2, IL‐4, IL‐6, IL‐10 and interferon‐γ were assessed by enzyme‐linked immunosorbent assay. In another experiment, the cytokines were followed after subcutaneous HEMA injections. Animals painted with HEMA had a significantly higher IL‐6 production by anti‐CD3ε‐stimulated lymph node cells and significantly suppressed IL‐10 production by spleen cells compared to vehicle‐treated mice. This correlated to some extent with the spontaneous spleen‐cell production induced by subcutaneously administered HEMA. An injection of 20 µmol of HEMA induced an increased production of IL‐6, while injection of 40 µmol depressed both IL‐6 and IL‐10 production. Although there was no sign of inflammation on the ears, findings suggest that HEMA had penetrated the skin and induced a reaction in the immune system.
Primary prevention in health care employees: a prospective intervention study with a 3‐year training period
Authors: Löffler Harald , Bruckner Thomas , Diepgen Thomas , Effendy Isaak
Published: April 2006

Irritant contact dermatitis is a mayor problem in health care employees. Because educational programs have shown convincing success in certain occupations (e.g. in hairdressers), this study investigates the effect of a special training program in health care trainees. 521 trainees from 14 nursing schools in Central Germany were randomly divided in 2 groups, (i) an intervention group with a regular teaching protocol regarding all aspects of primary prevention and (ii) a control group without any further teaching. Morphological changes of the hands, use of hand care creams and knowledge regarding skin care were evaluated regularly during their 3 years lasting training period (1999–2002). In the intervention group, we found at the end of the 3‐year training period a significant better skin condition of the hands than in the control group: a 3‐year prevalence of morphological skin changes of 66.7% versus 89.3%. The unteached trainees (control group) had an odds ratio (OR) of 4.8 [95% confidential interval (CI): 2.9–7.8] for developing any skin changes on the hands after 3 years. Besides the effect of the teaching, the history of hand dermatitis before the study start was an independent risk factor for development of further hand dermatitis [OR 1.9, 95% CI: 1.0–3.6). Age and sex showed no influence on the skin condition. Atopic constitution had an influence on the development of skin changes only at the evaluation after 18 month. The observed effect in the intervention group may best be explained by different behaviour of the trainees, e.g. the amount of hand washing was reduced, while procedure of hand disinfection remained unchanged compared with the control group. However, the amount of used skin care cream did not differ between the both groups. This study shows that primary prevention of skin disease by regularly teaching during the training period of medical employees can effectively reduce the risk of development of irritant skin changes of the hands. Therefore, teaching and continuous motivation regarding preventive individual and collective measures during the training of medical staff should be an obliged part of the curriculum.
The local lymph node assay and skin sensitization: a cut‐down screen to reduce animal requirements?
Authors: Kimber Ian , Dearman Rebecca J. , Betts Catherine J. , Gerberick G. Frank , Ryan Cindy A. , Kern Petra S. , Patlewicz Grace Y. , Basketter David A.
Published: April 2006

The local lymph node assay (LLNA), an alternative approach to skin‐sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut‐down assay comprising a single high‐dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.
Induction of systemic resistance in tomato by N‐acyl‐L‐homoserine lactone‐producing rhizosphere bacteria
Authors: SCHUHEGGER REGINA , IHRING ALEXANDRA , GANTNER STEPHAN , BAHNWEG GüNTHER , KNAPPE CLAUDIA , VOGG GERD , HUTZLER PETER , SCHMID MICHAEL , VAN BREUSEGEM FRANK , EBERL LEO , HARTMANN ANTON , LANGEBARTELS CHRISTIAN
Published: April 2006

N‐acyl‐L‐homoserine lactone (AHL) signal molecules are utilized by Gram‐negative bacteria to monitor their population density (quorum sensing) and to regulate gene expression in a density‐dependent manner. We show that Serratia liquefaciens MG1 and Pseudomonas putida IsoF colonize tomato roots, produce AHL in the rhizosphere and increase systemic resistance of tomato plants against the fungal leaf pathogen, Alternaria alternata. The AHL‐negative mutant S. liquefaciens MG44 was less effective in reducing symptoms and A. alternata growth as compared to the wild type. Salicylic acid (SA) levels were increased in leaves when AHL‐producing bacteria colonized the rhizosphere. No effects were observed when isogenic AHL‐negative mutant derivatives were used in these experiments. Furthermore, macroarray and Northern blot analysis revealed that AHL molecules systemically induce SA‐ and ethylene‐dependent defence genes (i.e. PR1a, 26 kDa acidic and 30 kDa basic chitinase). Together, these data support the view that AHL molecules play a role in the biocontrol activity of rhizobacteria through the induction of systemic resistance to pathogens.
Direct in situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water deficits
Authors: THOMAS TYLER R. , MATTHEWS MARK A. , SHACKEL KEN A.
Published: April 2006

Vitis vinifera L. berries are non‐climacteric fruits that exhibit a double‐sigmoid growth pattern, and at the point known as ‘veraison’, which is just before the beginning of the second period of rapid fruit growth, these berries undergo several abrupt physiological changes. Cell pressure probe was used to examine the in situ turgor (P) of cells in the mesocarp during berry development and in response to plant water deficits. Initial tests comparing attached and detached berries demonstrated that cell P was stable for up to 48 h after detachment from the vine, provided that water loss from the berry was prevented. Cell P at pre‐dawn was on the order of 0.25 MPa pre‐veraison (PreV) and was reduced by an order of magnitude to 0.02 MPa post veraison (PostV). Cell P declined slightly but significantly with depth from the berry surface PreV, but not PostV. When water was withheld from potted vines, cell P declined about 0.2 Mpa, as pre‐dawn vine water potential declined about 0.6 MPa over 12 d, whereas cell P was completely insensitive to a 1.10 MPa decrease in pre‐dawn vine water potential after veraison. Rewatering of stressed plants also resulted in a 24 h recovery of cell P before, but not after veraison. The substantial decline in cell P around veraison is consistent with the decline in berry firmness that is known to occur at this time, and the PostV insensitivity of P to changes in vine water status is consistent with current hypotheses that the PostV berry is hydraulically isolated from the vine. The fact that a measurable P of about 0.02 MPa and typical cell hydraulic/osmotic behaviour were exhibited in PostV berries, however, indicates that cell membranes remain intact after veraison, contrary to many current hypotheses that veraison is associated with a general loss of membrane function and cellular compartmentation in the grape berry. We hypothesize that cell P is low in the PostV berry, and possibly other fleshy fruits, because of the presence of regulated quantities of apoplastic solutes.
From English to Italian: Aesthetic Restorative Course By Distance Learning
Authors: Millar B , Reynolds PA. , Paganelli P , Gastaldi G
Published: April 2006

Aims  To provide a new online course translated into Italian entitled ‘Aesthetic Restorative Dentistry’ based on selected units from the e‐MClinDent (Master of Clinical Dentistry in Prosthodontics) of the University of London, UK.

Materials and Methods  The M.Clin.Dent. (Prosthodontics) is intended for dental practitioners who wish to enhance their knowledge of fixed and removable prosthodontics. The online four year course is run through the External Programme of London University. http://www.londonexternal.ac.uk/dentistry. To make the course more available to countries where English is not the first language, and as part of the Anglo‐Italian Collaboration, a pilot short course selected 8 of the 72 modules of the degree. Copyright permission was granted from London University. Two units were translated by an Italian restorative specialist working in the UK and checked for accuracy by the Italian Director. The units were uploaded onto the virtual learning environment (WebCT) with rich media and references hyperlinked. Assessment is by online multiple choice questionnaires and online assignments marked by Italian staff. This poster describes the process and progress of the development of the short course.

Results  Following initial consultations between the two Schools, four sections were identified:

 Section 1 ‐ Treatment options: Diagnosis, treatment planning and options,

 Section 2 – Techniques: Tooth preparation, impressions, soft tissue management, temporary restorations

 Section 3 – Materials

 Section 4 – Optional hands‐on training for credit rated certification

 The first two Units have been translated, verified and uploaded. An online feedback questionnaire was devised to assess the educational, assessment and presentational issues. Initial response from prospective Italian students has been positive and the remaining Units are being processed.

Conclusions  The planning, translation and production of the first two Units has brought a very positive response. Completion of the short course and launch in 2006 with translation into other languages such as Spanish is planned.
An Approach to Interdisciplinary Training in Postgraduate Education
Authors: Brodin P , Espeland L , Stenvik A
Published: April 2006

Aims  A primary goal for clinical graduate training is to provide the student with the expertise required for specialist treatment in the actual discipline. At the same time there is an increasing need for a broader perspective on specialist care and the students should be aware of the limitations inherent in own specialty. In order to plan treatment in the best interest of the patient, and to be prepared to take part in treatments involving other specialties, the students should be exposed to interdisciplinary cooperation throughout the training. An approach to joint academic and clinical training with the purpose of providing graduate students with a broader perspective on specialist care is described and discussed.

Material and methods  During their first year graduate students in the 7 different disciplines complete a joint Core Curriculum consisting of 8 different courses to stimulate a scientific approach to their profession and understanding of basic biologic mechanisms. To create a learning environment focusing on the development of interdisciplinary competence, a joint clinic has been established. Teams of students from different disciplines have been organized in order to establish ‘partnership’ for the treatment of patients with complex problems. The students also take part in the sessions held by a faculty Team of experts for assessment and treatment planning of referred patients with complex problems. Furthermore, faculty members conduct courses and seminars for students from other disciplines and students also participate in selected parts of the regular program in other disciplines.

Results  Formal evaluation has so far been conducted for the Core Curriculum. Most students respond that they are satisfied with the courses, and the curriculum has also been adjusted based on the comments. The students report that treatment of patients in need of interdisciplinary treatment has been facilitated by having ‘partners’ in other disciplines. Participation in the Team of expert's sessions has been appreciated, and the attendance at interdisciplinary courses and seminars has been good.

Conclusions  Based on the experience over the last 5 years, the interdisciplinary aspects of graduate training should be expanded to stimulate a holistic approach also to specialist treatment.
Different Structures of European Postgraduate Programmes
Authors: Eaton KA
Published: April 2006

This paper considered the ever growing range of European postgraduate programmes on dental topics that can lead to the award of a postgraduate degree or diploma. These programmes may be centred on general dentistry, specific clinical disciplines, research or other topics such as practice management or applied dental informatics. Some cover all these areas. Historically they have been run within dental schools or other academic institutions. However, recently some have been (and are) provided as distance learning courses.

The resulting degrees or diplomas are frequently required for career progression in academic dentistry or, in countries where dental specialties are recognised, prior to recognition as a specialist. In the last decade, a number of European specialist societies, including those for Periodontology and Children's Dentistry have developed curricula for 3‐year Masters courses, approved training centres and externally validated this training. The paper concluded that there has been considerable growth in both the number and range of postgraduate dental programmes in Europe and that, although the providing institutions are in competition, it is essential that they work together to ensure that the quality of education provided is at a high level. Such collaboration may be facilitated by organisations such as the Association for Dental Education in Europe and other Pan‐European groups.
PhD Programmes
Authors: Beertsen W
Published: April 2006

The Berlin Communiqué (2003) on The Bologna Process mentions the importance of research as an integral part of Higher Education across Europe and calls for the inclusion of doctoral level qualifications as the Third Cycle (after the Bachelor‐Master system). It is expected that incorporation of the PhD‐training programme will reinforce the link between the European Research Area and the European Higher Education Area. To facilitate this process it is important to define common standards and to remove legal and other obstacles that might prohibit co‐operation among EU countries.

Not all dental faculties in Europe are using a similar format for attaining the doctoral level. The varying format highlights the differences rather than the similarities among members of the EU. Based on the outcome of a trans‐continental questionnaire, an attempt was made to outline the contours of a common PhD programme for dentistry that might serve as a template for European compliance.
Genetic architecture of the F7 gene in a Spanish population: implication for mapping complex diseases and for functional assays
Authors: Sabater‐Lleal M , Almasy L , Martínez‐Marchán E , Martínez‐Sánchez E , Souto R , Blangero J , Souto Jc , Fontcuberta J , Soria JM
Published: April 2006

Delineating the genetic variability of loci coding for complex diseases helps to understand the individual variation in disease susceptibility and drug response. We present the allelic architecture of the F7 gene. This gene is the major determinant of FVII plasma levels, and these plasma levels constitute an important intermediate risk factor for cardiovascular disease. As part of the Genetic Analysis of Idiopathic Thrombophila Project, we completely re‐sequenced the F7 locus (promoter, exons, introns, and 3′‐untranslated region) in 40 unrelated individuals. We found 49 polymorphisms with only two amino acid changes suggesting that regulatory non‐coding and intronic variants are responsible for the FVII variability. These results are important for mapping susceptibility alleles of complex diseases, because differences in pair‐wise linkage disequilibrium patterns between DNA variants and haplotype frequency distributions may help to detect disease‐associated alleles. In addition, we present the results of an in silico search that established genomic comparisons among different species. In conclusion, our study of the F7 DNA sequence variations is an example of a strategy for analyzing the genetic architecture of a quantitative trait locus. Furthermore, it provides a model for future analyses of genetic factors that contribute to the susceptibility of complex diseases in humans.
Views of female breast cancer patients who donated biologic samples regarding storage and use of samples for genetic research
Authors: Kaphingst KA , Janoff JM , Harris LN , Emmons KM
Published: April 2006

Although social and ethical issues related to the storage and use of biologic specimens for genetic research have been discussed extensively in the medical literature, few empiric data exist describing patients' views. This qualitative study explored the views of 26 female breast cancer patients who had consented to donate blood or tissue samples for breast cancer research. Participants generally did not expect personal benefits from research and had few unprompted concerns. Few participants had concerns about use of samples for studies not planned at the time of consent. Some participants did express concerns about insurance or employment discrimination, while others believed that current privacy protections might actually slow breast cancer research. Participants were generally more interested in receiving individual genetic test results from research studies than aggregate results. Most participants did not want individual results of uncertain clinical significance, although others believed that they should be able to receive such information. These data examined the range of participants' views regarding the storage and use of biologic samples. Further research with different and diverse patient populations is critical to establishing an appropriate balance between protecting the rights of human subjects in genetic research and allowing research to progress.