bluuebunny/crossref_metadata_2025 huggingface dataset:
Resolution of trisomic mosaicism in prenatal diagnosis: estimated performance of a 50K SNP microarray
Authors: Cross Jillian , Peters Greg , Wu Zhanhe , Brohede Jesper , Hannan Garry N.
Published: November 2007

Objective

To evaluate the ability of a DNA single nucleotide polymorphism (SNP) microarray to detect chromosome mosaicism for trisomy in prenatal samples in order to compare this with conventional cytogenetics.

Method

We created a dilution series of mock mosaic samples, by mixing measured amounts of fibroblast cells containing trisomy 8 from a male with aliquots of cells with a normal female karyotype. DNAs were extracted from these mosaic mixtures, then analysed on the Affymetrix 50K Xba SNP chip. Duplicate aliquots of each mosaic sample were probed using interphase FISH, with centromeric probes for chromosomes X, Y and 8, to estimate independently the proportion of male trisomy 8 in each sample. Data from the arrays were analysed using publicly available analysis tools. Statistical calculations were then performed using a Student's t‐test to determine if there was a significant difference between the copy numbers of each chromosome.

Results

These experiments using the Affymetrix 50K Xba SNP microarray showed mosaicism to be obvious at 20% and with additional statistical calculations, the lower limit for detection is about 10%.

Conclusion

The SNP microarray platform tested can detect mosaicism for trisomy in prenatal samples at levels comparable with conventional cytogenetic techniques in routine use. Copyright © 2007 John Wiley & Sons, Ltd.
Small intestinal atresia in a defined population: occurrence, prenatal diagnosis and survival
Authors: Hemming Victoria , Rankin Judith
Published: November 2007

Objective

To describe the prevalence, associated anomalies, prenatal diagnosis, and survival of cases of congenital small intestinal atresia (SIA).

Method

Data were extracted from the Northern Congenital Abnormality Survey (NorCAS) for cases delivered during 1991 to 2001.

Results

A total of 99 cases of SIA were notified from 372 717 registered births. Twenty‐five (25.8%) SIA cases were associated with a chromosomal anomaly. Of the 72 SIA cases with normal karyotype, 18 (25%) were associated with other structural anomalies. The total prevalence for all SIA cases over the 11 years was 2.66 per 10 000 registered births (95% CI 2.13, 3.18), and the livebirth prevalence was 2.37 per 10 000 livebirths (95% CI 1.88, 2.87). For jejunoileal atresia, the total prevalence increased from 0.54 per 10 000 births during 1991–1995 to 1.11 per 10 000 births during 1996–2001, and livebirth prevalence increased from 0.49 to 1.06 per 10 000 livebirths. The more proximal the lesion the prenatal diagnosis sensitivity was slightly better with 19 (44.2%) cases of duodenal atresia and 3 (42.8%) cases of jejunal atresia diagnosed prenatally by routine ultrasonography.

Conclusion

This population‐based study of SIA has confirmed several previous findings but, by considering subtypes separately, it also suggests a trend towards an increase in the prevalence of jejunoileal atresia (JIA). Copyright © 2007 John Wiley & Sons, Ltd.
A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double‐density gradient system
Authors: Kwon K. H. , Jeon Y. J. , Hwang H. S. , Lee K. A. , Kim Y. J. , Chung H. W. , Pang M. G.
Published: November 2007

Objectives

To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield.

Methods

Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer–Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X‐Y probes.

Results

For single‐density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H2O with 1.077 g/mL Percoll and 520 mOsm/kg H2O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double‐Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus.

Conclusion

The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods. Copyright © 2007 John Wiley & Sons, Ltd.
Quantitative aberrations of hypermethylated RASSF1A gene sequences in maternal plasma in pre‐eclampsia
Authors: Tsui Dana W. Y. , Chan K. C. Allen , Chim Stephen S. C. , Chan Lin‐wai , Leung Tak‐yeung , Lau Tze‐kin , Lo Y. M. Dennis , Chiu Rossa W. K.
Published: November 2007

Objective

To study if quantitative aberrations in circulating placental‐derived hypermethylated RASSF1A DNA in maternal plasma are associated with pre‐eclamptic pregnancies.

Method

Maternal plasma and placental tissues from third‐trimester pre‐eclamptic women and gestational‐age matched normotensive controls were studied. Real‐time PCR was performed to quantify RASSF1A concentrations before and after methylation‐sensitive restriction digestion in a duplex assay, where ß‐actin concentrations were quantified as an internal control to confirm complete enzyme digestion.

Results

The median concentrations of hypermethylated RASSF1A were 4.3‐fold higher in maternal plasma of pre‐eclamptic subjects than in controls. There was no significant difference between the extent of RASSF1A hypermethylation in placental tissues obtained from pre‐eclamptic and control pregnancies.

Conclusion

This study demonstrated the potential utility of hypermethylated RASSF1A sequences in maternal plasma as a gender‐ and polymorphism‐independent marker for pre‐eclampsia. Copyright © 2007 John Wiley & Sons, Ltd.
Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor
Authors: Ahrendt Angela J. , Tollaksen Sandra L. , Lindberg Carl , Zhu Wenhong , Yates John R. , Nevin Kelly P. , Babnigg György , Lovley Derek R. , Giometti Carol S.
Published: November 2007

Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2‐DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty‐six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.
Comparative proteomics of industrial lager yeast reveals differential expression of the cerevisiae and non‐cerevisiae parts of their genomes
Authors: Caesar Robert , Palmfeldt Johan , Gustafsson John S. , Pettersson Ellinor , Hashemi Sayed Hossein , Blomberg Anders
Published: November 2007

The proteomes of three industrial lager beer strains, CMBS33, OG2252 and A15, were analysed under standardised laboratory growth conditions. Protein spots in the 2‐DE pattern of the lager strains were subjected to MS/MS to identify protein variants. We found the protein composition of the three lager strains to be qualitatively rather similar, while being substantially different from the Saccharomyces cerevisiae strain BY4742. Database searches using several fully sequenced genomes from the Saccharomyces genera indicated that the non‐cerevisiae proteins in the 2‐D pattern of lager strains were most closely related to S. bayanus. For many proteins the regulation of the bayanus‐like protein and its cerevisiae counterpart varied in a strain‐dependent manner, e.g. the bayanus‐like form of Tdh3p was roughly eight‐fold more abundant than the cerevisiae form in the OG2252 strain. We also found differential regulation of cerevisiae‐ and bayanus‐like proteins during various stress conditions like low temperature growth, and adaptation to high temperatures or high salinity, e.g. for Arg1p, Sti1p and Pdc1p. Our data on the differential regulation of the two genomes in these hybrid strains may have important industrial implications for strain improvement and strain protection.
Mass spectrometry‐based prokaryote gene annotation
Authors: Ishino Yoko , Okada Hitomi , Ikeuchi Masahiko , Taniguchi Hisaaki
Published: November 2007

MS combined with database searching has become the preferred method for identifying proteins present in cell or tissue samples. The technique enables us to execute large‐scale proteome analyses of species whose genomes have already been sequenced. Searching mass spectrometric data against protein databases composed of annotated genes has been widely conducted. However, there are some issues with this technique; wrong annotations in protein databases cause deterioration in the accuracy of protein identification, and only proteins that have already been annotated can be identified. We propose a new framework that can detect correct ORFs by integrating an MS/MS proteomic data mapping and a knowledge‐based system regarding the translation initiation sites. This technique can provide correction of predicted coding sequences, together with the possibility of identifying novel genes. We have developed a computational system; it should first conduct the probabilistic peptide‐matching against all possible translational frames using MS/MS data, then search for discriminative DNA patterns around the detected peptides, and lastly integrate the facts using empirical knowledge stored in knowledge bases to obtain correct ORFs. We used photosynthetic bacteria Synechocystis sp. PCC6803 as a sample prokaryote, resulting in the finding of 14 N‐terminus annotation errors and several new candidate genes.
Identification of intra‐ and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis
Authors: Winger Alison M. , Taylor Nicolas L. , Heazlewood Joshua L. , Day David A. , Millar A. Harvey
Published: November 2007

Redox active proteins in plant mitochondria were examined using 2‐D oxidant/reductant diagonal‐SDS‐PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in‐gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O‐acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox‐functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant‐specific redox modulations of mitochondrial proteins.
A proteomic kinetic analysis of IGROV1 ovarian carcinoma cell line response to cisplatin treatment
Authors: Le Moguen Karen , Lincet Hubert , Marcelo Paulo , Lemoisson Edwige , Heutte Natacha , Duval Marilyne , Poulain Laurent , Vinh Joëlle , Gauduchon Pascal , Baudin Bruno
Published: November 2007

Ovarian cancer is one of the leading causes of mortality by gynecological cancer. Despite good response to surgery and initial chemotherapy, essentially based on cisplatin (cis‐diamino‐dichloro‐platinum(II) (CDDP)) compounds, frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been described as implicated in CDDP resistance, however they are not sufficient to exhaustively account for this resistance emergence. We applied a proteomic approach based on 2‐DE coupled with MS (MALDI‐TOF/TOF) to identify proteins associated with chemoresistance induced by CDDP. A kinetic analysis of IGROV1 cell behavior following treatment with CDDP and subsequent statistical analysis revealed time and/or concentration‐dependent modifications in protein expression. We evidenced events such as decreased amino‐acid and nucleotide synthesis potentially associated with cell cycle blockade, and variations that may be related to resistance acquisition, such as possible enhanced glycolysis and increased proliferating potential. Moreover, overexpressions of aldehyde dehydrogenase 1 and both cytokeratins 8 and 18 were consistent with our previous findings, demonstrating that expression of these proteins was increased in cisplatin‐resistant IGROV1‐R10 as compared to IGROV1 parental cells. Identification of such proteins could allow improved understanding of the mechanisms leading to cell death or survival and, thus, to the acquisition of chemoresistance.
Proteomic analysis identified N‐cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells
Authors: Sosa María Soledad , Girotti María Romina , Salvatierra Edgardo , Prada Federico , de Olmo Juan Antonio López , Gallango Silvia Juárez , Albar Juan Pablo , Podhajcer Osvaldo Luis , Llera Andrea Sabina
Published: November 2007

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2‐DE proteomic analysis of human melanoma cells following antisense‐mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N‐cadherin (N‐CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N‐CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral‐mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi‐transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N‐CAD and CLU and increased secretion of HSP27. Transient re‐expression of SPARC in SPARC‐downregulated cells reverted extracellular N‐CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N‐CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.
Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum
Authors: Wuhrer Manfred , Stam Jord C. , van de Geijn Fleur E. , Koeleman Carolien A. M. , Verrips C. Theo , Dolhain Radboud J. E. M. , Hokke Cornelis H. , Deelder André M.
Published: November 2007

All four subclasses of human serum IgG contain a single N‐glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core‐fucosylated and partially truncated oligosaccharides, that may carry a bisecting N‐acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N‐glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N‐glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96‐well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano‐LC‐MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N‐glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass‐specific analysis of IgG glycosylation from clinical samples.
SPINE: A method for the rapid detection and analysis of protein–protein interactions in vivo
Authors: Herzberg Christina , Weidinger Lope Andrés Flórez , Dörrbecker Bastian , Hübner Sebastian , Stülke Jörg , Commichau Fabian M.
Published: November 2007

The detection and analysis of protein–protein interactions is one of the central tasks of proteomics in the postgenomic era. For this purpose, we present a procedure, the Strep–protein interaction experiment (SPINE) that combines the advantages of the Strep‐tag protein purification system with those of reversible in vivo protein crosslinking by formaldehyde. Using two Bacillus subtilis regulator proteins, we demonstrate that this method is well suited to isolate protein complexes with high purity and virtually no background. Plasmids allowing the high‐level expression of proteins carrying an N‐ or C‐terminal Strep‐tag in B. subtilis were constructed.
Membrane proteomic analysis of human mesenchymal stromal cells during adipogenesis
Authors: Jeong Ju Ah , Ko Kyung‐Min , Park Hyung Soon , Lee Jinsook , Jang Cholsoon , Jeon Choon‐Ju , Koh Gou Young , Kim Hoeon
Published: November 2007

Mesenchymal stromal cells (MSCs) have proven useful for cell and immune therapy, but the molecular constituents responsible for their functionalities, in particular, those on the plasma membrane, remain largely unknown. Here we employed both gel and nongel based MS to analyze human MSCs' membrane proteome before and after adipogenesis. 2‐DE of cells that were pretreated with membrane impermeable fluorescent dyes revealed that both the whole cell proteome and the cell surface subproteome were independent of donors. LC coupled with tandem MS analysis of the plasma membrane‐containing fraction allowed us to identify 707 proteins, approximately half of which could be annotated as membrane‐related proteins. Of particular interest was a subset of ectodomain‐containing membrane‐bound proteins that encompass most known surface markers for MSCs, but also contain a multitude of solute carriers and ATPases. Upon adipogenic differentiation, this proteomic profile was amended to include several proteins involved in lipid metabolism and trafficking, at the expense of, most noticeably, ectoenzymes. Our results here provide not only a basis for future studies of MSC‐specific molecular mechanisms, but also a molecular inventory for the development of antibody‐based cell isolation and identification procedures.
Carbohydrate restriction, prostate cancer growth, and the insulin‐like growth factor axis
Authors: Freedland Stephen J. , Mavropoulos John , Wang Amy , Darshan Medha , Demark‐Wahnefried Wendy , Aronson William J. , Cohen Pinchas , Hwang David , Peterson Bercedis , Fields Timothy , Pizzo Salvatore V. , Isaacs William B.
Published: November 2007

BACKGROUND

Recent evidence suggests carbohydrate intake may influence prostate cancer biology. We tested whether a no‐carbohydrate ketogenic diet (NCKD) would delay prostate cancer growth relative to Western and low‐fat diets in a xenograft model.

METHODS

Seventy‐five male SCID mice were fed a NCKD (84% fat–0% carbohydrate–16% protein kcal), low‐fat (12% fat–72% carbohydrate–16% protein kcal), or Western diet (40% fat–44% carbohydrate–16% protein kcal). Low‐fat mice were fed ad libitum and the other arms fed via a modified‐paired feeding protocol. After 24 days, all mice were injected with LAPC‐4 cells and sacrificed when tumors approached 1,000 mm3.

RESULTS

Despite consuming equal calories, NCKD‐fed mice lost weight (up to 15% body weight) relative to low‐fat and Western diet‐fed mice and required additional kcal to equalize body weight. Fifty‐one days after injection, NCKD mice tumor volumes were 33% smaller than Western mice (rank‐sum, P = 0.009). There were no differences in tumor volume between low‐fat and NCKD mice. Dietary treatment was significantly associated with survival (log‐rank, P = 0.006), with the longest survival among the NCKD mice, followed by the low‐fat mice. Serum IGFBP‐3 was highest and IGF‐1:IGFBP‐3 ratio was lowest among NCKD mice while serum insulin and IGF‐1 levels were highest in Western mice. NCKD mice had significantly decreased hepatic fatty infiltration relative to the other arms.

CONCLUSIONS

In this xenograft model, despite consuming more calories, NCKD‐fed mice had significantly reduced tumor growth and prolonged survival relative to Western mice and was associated with favorable changes in serum insulin and IGF axis hormones relative to low‐fat or Western diet. Prostate 68: 11–19, 2008. © 2007 Wiley‐Liss, Inc.
The conformation of fusogenic B18 peptide in surfactant solutions
Authors: Rocha Sandra , Lúcio Marlene , Pereira M. Carmo , Reis Salette , Brezesinski Gerald
Published: November 2007

The interaction of B18 peptide with surfactants has been studied by circular dichroism spectroscopy and fluorescence measurements. B18 is the fusogenic motif of the fertilization sea urchin protein. The peptide forms an α‐helix structure when interacting with positively or negatively charged surfactants below and above the critical micellar concentration (CMC). The α‐helix formation is due to binding of surfactant monomers rather than the formation of surfactant micelles on the peptide. Fluorescence measurements show that the CMC of the negatively charged surfactant increases in the presence of B18, supporting the fact that there is a strong interaction between the peptide and monomers. Nonionic surfactant monomers have no effect on the peptide structure, whereas the micelles induce an α‐helical conformation. In this case the helix stabilization results from the formation of surfactant micelles on the peptide. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Combination of ellipsometry, laser scanning microscopy and Z‐scan fluorescence correlation spectroscopy elucidating interaction of cryptdin‐4 with supported phospholipid bilayers
Authors: Miszta Adam , Macháň Radek , Benda Aleš , Ouellette Andre J. , Hermens Wim Th. , Hof Martin
Published: November 2007

The present study has two main objectives. The first is to characterize antimicrobial peptide (AMP) cryptdin‐4 (Crp‐4) interactions with biological membranes and to compare those interactions with those of magainin 2. The second is to combine the complementary experimental approaches of laser scanning microscopy (LSM), ellipsometry, and Z‐scan fluorescence correlation spectroscopy (FCS) to acquire comprehensive information on mechanisms of AMP interactions with supported phospholipid bilayers (SPBs)—a popular model of biological membranes. LSM shows appearance of inhomogeneities in spatial distribution of lipids in the bilayer after treatment with Crp‐4. Ellipsometric measurements show that binding of Crp‐4 does not significantly change the lipid structure of the bilayer (increase in adsorbed mass without a change in thickness of adsorbed layer). Furthermore, Crp‐4 slows the lateral diffusion of lipids within the membrane as shown by Z‐scan FCS. All changes of the bilayer induced by Crp‐4 can be partially reversed by flushing the sample with excess of buffer. Bilayer interactions of magainin 2 are significantly different, causing large loss of lipids and extensive damage to the bilayer. It seems likely that differences in peptide mode of action, readily distinguished using these combined experimental methods, are related to the distinctive β‐sheet and α‐helical structures of the respective peptides. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Molecular interaction studies of peptides using steady‐state fluorescence intensity. Static (de)quenching revisited
Authors: Ribeiro Marta M. B. , Franquelim Henri G. , Castanho Miguel A. R. B. , Veiga Ana Salomé
Published: November 2007

Protein–protein interactions, as well as peptide–peptide and peptide–protein interactions are fields of study of growing importance as molecular‐level detail is avidly pursued in drug design, metabolic regulation and molecular dynamics, among other classes of studies. In membranes, this issue is particularly relevant because lipid bilayers potentiate molecular interactions due to the high local concentration of peptides and other solutes.

However, experimental techniques and methodologies to detect and quantify such interactions are not abundant. A reliable, fast and inexpensive alternative methodology is revisited in this work.

Considering the interaction of two molecules, at least one of them being fluorescent, either intrinsically (e.g. Trp residues) or by grafting a specific probe, changes in their aggregation state may be reported, as long as the fluorophore is sensitive to local changes in polarity, conformation and/or exposure to the solvent. The interaction will probably lead to modifications in fluorescence intensity resulting in a decrease (‘quenching’) or enhancement (‘dequenching’). Although the presented methodology is based on static quenching methodologies, the concept is extended from quenching to any kind of interference with the fluorophore.

Equations for data analysis are shown and their applications are illustrated by calculating the binding constant for several data‐sets. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Preparation and characterization of a novel exendin‐4 human serum albumin fusion protein expressed in Pichia pastoris
Authors: Huang Yan‐Shan , Chen Zhi , Chen Yi‐Qiong , MA Guo‐Chang , Shan Jian‐Feng , Liu Wei , Zhou Lin‐Fu
Published: November 2007

A novel recombinant exendin‐4 human serum albumin fusion protein (rEx‐4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex‐4 is a 39‐amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half‐life (t1/2 = 2.4 h). To extend the half‐life of Ex‐4 molecule in vivo, we designed a genetically engineered Ex‐4/HSA fusion protein. Between Ex‐4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a three‐step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex‐4 for interaction with GLP‐1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx‐4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx‐4/HSA had a much longer plasma half‐life. Therefore, rEx‐4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Concurrent purification of two defense proteins from French bean seeds: a defensin‐like antifungal peptide and a hemagglutinin
Authors: Leung Edwin H. W. , Wong Jack H. , Ng T. B.
Published: November 2007

A purification protocol is described herein for concurrent isolation of two defense proteins including a 6‐kDa defensin‐like antifungal peptide and a 60‐kDa dimeric hemagglutinin from seeds of the French bean (Phaseolus vulgaris). It involved ion‐exchange chromatography on SP‐Sepharose, affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography on Q‐Sepharose, and gel filtration on Superdex Peptide (for defensin‐like antifungal peptide) or Superdex 200 (for hemagglutinin). Both antifungal and hemagglutinating activities were adsorbed on SP‐Sepharose and then on Affi‐gel blue gel. Hemagglutinin was subsequently unadsorbed and defensin‐like antifungal peptide adsorbed on Q‐Sepharose. The antifungal activity of the antifungal peptide was stable in the temperature range of 0–90 °C for 20 min, in the pH range of 4–10, and after exposure to trypsin (1 mg/ml) at 37 °C for 1 h. The hemagglutinin was stable from 10 to 80 °C, from pH 1 to 12, and after treatment with trypsin at 37 °C for 2 h. It inhibited [methyl‐3H]thymidine incorporation into breast cancer (MCF‐7), leukemia (L1210), hepatoma (HepG2) and human embryonic liver (WRL68) cells with an IC50 of 6.6, 7, 13 and 15 µM, respectively, and elicited maximal mitogenic response from mouse splenocytes at 1 µM concentration. It curtailed HIV‐1 reverse transcriptase activity with an IC50 of 1.9 µM, but was devoid of antifungal activity. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
The minimal fusion peptide of simian immunodeficiency virus corresponds to the 11 first residues of gp32
Authors: Lorin Aurélien , Lins Laurence , Stroobant Vincent , Brasseur Robert , Charloteaux Benoit
Published: November 2007

We had previously predicted successfully the minimal fusion peptides (FPs) of the human immunodeficiency virus 1 (HIV‐1) gp41 and the bovine leukemia virus (BLV) gp30 using an original approach based on the obliquity/fusogenicity relationship of tilted peptides. In this paper, we have used the same method to predict the shortest FP capable of inducing optimal fusion in vitro of the simian immunodeficiency virus (SIV) mac isolate and of other SIVs and human immunodeficiency virus (HIV‐2) isolates. In each case, the 11‐residue‐long peptide was predicted as the minimal FP. For the SIV mac isolate, liposome lipid‐mixing and leakage assays confirmed that this peptide is the shortest peptide inducing optimal fusion in vitro, being therefore the minimal FP. These results are another piece of evidence that the tilted properties of FPs are important for the fusion process and that our method can be used to predict the minimal FPs of other viruses. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Interfacial properties and structural analysis of the antimicrobial peptide NK‐2
Authors: Olak Claudia , Muenter Annabel , Andrä Jörg , Brezesinski Gerald
Published: November 2007

The structure of the antimicrobial peptide NK‐2 has been studied at the air–water interface and in different solutions using spectroscopic methods such as circular dichroism (CD) and infrared reflection absorption spectroscopy (IRRAS) as well as specular X‐ray reflectivity (XR). NK‐2 adopts an unordered structure in water, buffer, and in the presence of monomeric cationic and noncharged amphiphiles. However, it forms a stable α‐helix in 2,2,2‐trifluoroethanol (TFE) and in micellar solutions of anionic, cationic as well as nonionic amphiphiles, whereas only in sodium dodecyl sulfonate solutions the α‐helical structure can also be found below the critical micellar concentration (cmc). The amphiphilic molecule NK‐2 is surface active and forms a Gibbs monolayer at the air–buffer interface. In contrast, no adsorption was observed if NK‐2 is dissolved in water. During the adsorption process in buffer solutions, NK‐2 undergoes a conformational transition from random coil in bulk to α‐helix at the interface. This change of the peptide's secondary structure is known to be associated with its antimicrobial activity. A comparison of the experimental IRRA spectra with the simulated spectra indicates that the adsorbed NK‐2 α‐helix lies flat at the interface. This is confirmed by XR measurements which show that the thickness of the NK‐2 layer is ∼17 Å, which is the average diameter of a α‐helix, indicating that only a monomolecular adsorption layer is formed. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
A novel bradykinin‐like peptide from skin secretions of the frog, Rana nigrovittata
Authors: Liu Xiuhong , You Dewen , Chen Lihua , Wang Xu , Zhang Keyun , Lai Ren
Published: November 2007

A bradykinin‐like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin‐N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin‐like peptides identified from skin secretions of other amphibians. Ranakinin‐N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin‐N was found to exert concentration‐dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin‐N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15‐nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide‐coding region resulted in the absence of a dibasic site for trypsin‐like proteinases and an unusual ‐AEVA‐ insertion in the N‐terminal part of ranakinin‐N. The ‐AEAV‐ insertion resulted in neutral net charge at the N‐terminus of ranakinin‐N. Ranakinin‐N is the first reported bradykinin‐like peptide with a neutral net charge at the N‐terminus. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Activity‐dependent neurotrophic factor, ADNF, determines the structure characteristics of Colivelin, a fusion protein of ADNF9 and Humanin analog
Authors: Arakawa Tsutomu , Niikura Takako , Arisaka Fumio , Kita Yoshiko
Published: November 2007

A 24‐amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)‐related cell toxicities at sub‐nM‐uM concentrations. Activity‐dependent neurotrophic factor (ADNF) is a glia‐derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD‐related and amyotrophic lateral sclerosis‐related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9‐amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is ∼103–107 fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM‐active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate‐buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self‐associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel‐like activity, and the role of the amidated C‐terminus
Authors: Dos Santos Cabrera Marcia Perez , Arcisio‐Miranda Manoel , Broggio Costa Sabrina Thais , Konno Katsuhiro , Ruggiero José Roberto , Procopio Joaquim , Ruggiero Neto João
Published: November 2007

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C‐terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C‐terminus introduced a negative charge at an all‐positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel‐like activity on anionic bilayers with a well‐defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans‐negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.
Stability in a high humidity environment of TCO thin films deposited at low temperatures
Authors: Minami Tadatsugu , Kuboi Takeshi , Miyata Toshihiro , Ohtani Yuusuke
Published: November 2007

The stability of transparent conducting Al‐ and Ga‐doped ZnO (AZO and GZO) thin films in a high humidity environment has been investigated for the purpose of finding substitutes for the indium‐tin‐oxide (ITO) thin films used in transparent electrode applications. It was found that the resistivity of polycrystalline AZO and GZO thin films prepared with a thickness in the range from 50 nm to 300 nm at a substrate temperature below 200 °C always increased when long‐term tests were conducted in air at a temperature of 60 °C and a relative humidity of 90%, whereas ITO thin films remained relatively stable in the same environment; however, AZO and GZO thin films with a thickness above approximately 200 nm were sufficiently moisture‐resistant. The resistivity stability of AZO and GZO thin films was considerably related to crystallinity factors such as crystallite size. In addition, the resistivity increase is attributed to carrier transport dominated by grain boundary scattering resulting from the trapping of free electrons due to oxygen adsorption on the grain boundary surface. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Up‐conversion in nanostructured Yb3+–Tm3+ co‐doped sol–gel derived SiO2–LaF3 transparent glass‐ceramics
Authors: Méndez‐Ramos J. , Velázquez J. J. , Yanes A. C. , del‐Castillo J. , Rodríguez V. D.
Published: November 2007

Nanostructured transparent glass‐ceramics with composition of 95SiO2–5LaF3 doped with 0.1 mol% of Tm3+ or co‐doped with 0.1 mol% of Tm3+ and 0.3 mol% of Yb3+ were synthesized by thermal treatment of precursor sol–gel derived glasses. A comparative luminescence analysis in terms of different temperatures of thermal treatments has been carried out. Segregation of LaF3 nanocrystals in the matrix was con‐ firmed by X‐ray diffraction. Blue and red efficient up‐conversion emissions were observed under 980 nm excitation at room temperature. These results indicate partition of a significant fraction of co‐dopants into the precipitated LaF3 nanocrystals. The mechanisms involved in the up‐conversion emissions have been analyzed and can be ascribed to processes with two, three and four photons. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Resistive memory effect in self‐assembled 3‐aminopropyltrimethoxysilane molecular multilayers
Authors: Chauhan A. K. , Aswal D. K. , Koiry S. P. , Padma N. , Saxena V. , Gupta S. K. , Yakhmi J. V.
Published: November 2007

We have observed a pronounced resistive memory effect with an on/off ratio of ∼2 in 3‐aminopropyltrimethoxysilane (APTMS) molecular multilayers trapped with NH+3 ions. The APTMS multilayers were deposited on SiO2/Si(p++) substrates using a self‐assembly process and characterized using various techniques, such as water contact angle, elliposmetery, X‐ray photoelectron microscopy, X‐ray diffraction and atomic force microscopy. The current–voltage characteristics of Hg/APTMS multilayer/Si(p++) devices, in the negative bias region, exhibited a reproducible hysteresis effect along with a negative differential resistance. A plausible explanation of the observed hysteresis in terms of filling and de‐filling of the positive ion traps is proposed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Dislocation imaging for electronics application crystal selection
Authors: Secroun Aurélia , Brinza Ovidiu , Tardieu André , Achard Jocelyn , Silva François , Bonnin Xavier , De Corte Katrien , Anthonis Ans , Newton Mark E. , Ristein Jürgen , Geithner Peter , Gicquel Alix
Published: November 2007

High power electronics are today a real challenge for large band gap materials. Devices as simple as switches have to work at ever higher powers and demand material with exceptional properties: in particular, a high breakdown voltage (at least a few 106 V/cm), a high mobility‐lifetime product (at least 10–3 cm2/V), and a relatively fast response. As far as diamond is concerned, only thick (100 µm) monocrystalline free‐standing films of very good quality are expected to fulfill these requirements. Today's growth techniques still create many defects within the crystal that may deteriorate its electrical properties. It is thus our concern to identify which defects are involved and to find a strategy to select films with properties best suited for our power switching applications. Here, it is shown that nitrogen impurities are one major defect that control electrical properties as soon as 1 ppb is contained in the film and that, for lower values, dislocations seem to play a role. Besides, it is shown that the choice of substrate is involved in the formation of those dislocations within the grown film. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Industrial production of multiwalled carbon nanotubes
Authors: Bierdel M. , Buchholz S. , Michele V. , Mleczko L. , Rudolf R. , Voetz M. , Wolf A.
Published: November 2007

Carbon Nanotubes are currently being synthesized by different methods. The methods differ largely by the achievable space‐time yield, amorphous carbon content and carbon purity. High space‐time yields can only be achieved by CVD processes. Though usually low contents of amorphous carbon are observed in CVD processes, C‐purity is generally decreased due to catalyst residues. Bayer Technology Services has developed a high‐yield process for multi‐walled carbon nanotubes (MWNT, trade name Baytubes®) based on a CCVD process starting from light hydrocarbons. The produced Baytubes® are characterized by a very high graphite content and an excellent purity (carbon content >95 wt%). Due to their high purity and quality, Baytubes® can be employed directly in further processing. The Baytubes® were incorporated into different polymers and also into liquids. The primary feature for good mechanical and electrical properties of the polymer composites is a full dispersion of the CNT in the polymer matrix. TEM analysis is an important method to investigate the CNT dispersion on the nanometer scale. But it is also necessary to combine different methods to ensure a good and constant dispersion quality. Thus, different analysis methods were developed by BTS during product development. Process scale‐up is under way and a nominal production capacity of 60 tons per year has already been commissioned. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Electrochemically modified single‐walled carbon nanotubes
Authors: Burghard M. , Maroto A. , Balasubramanian K. , Assmus T. , Forment‐Aliaga A. , Lee E. J. H. , Weitz R. T. , Scolari M. , Nan F. , Mews A. , Kern K.
Published: November 2007

We report on the properties of electrochemically modified SWCNTs bearing a functional organic layer and a low density of noble metal nanoparticles, respectively. First, a novel approach to nanoscale pH sensors is demonstrated which involves the gentle modification of individual metallic tubes via covalent attachment of amino‐substituted phenyl groups. Furthermore, gold particles with sizes of a few tens of nanometres deposited onto individual SWCNTs were found to locally enhance the Raman signals of the underlying nanotube. The enhancement imparted by individual gold particles was estimated to be two orders of magnitude. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
DNA–carbon nanotube interactions and nanostructuring based on DNA
Authors: Vogel Stephanie R. , Müller Katharina , Plutowski Ulrich , Kappes Manfred M. , Richert Clemens
Published: November 2007

The unusual properties of single‐walled carbon nanotubes (SWCNTs) make them attractive for applications in the realm of molecular electronics, but also cause severe experimental difficulties in the purification and handling of these materials. One important step toward routine use of SWCNTs is the development of concentrated, monodisperse suspensions that are suitable for conventional manipulation and chromatography. We have studied the effect of the structure of oligodeoxynucleotides on their ability to generate such suspensions of SWCNTs, as well as the ability to release the nanotubes site‐selectively after (local) heating. Interestingly, short DNA strands were found to be better “surfactants” for the solubilization process than longer ones. The kinetic stability data suggest that the affinity of oligodeoxynucleotides for SWCNTs is greater than that for complementary DNA strands. The implications of this finding for DNA‐based nanostructuring are discussed. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Body emission characteristics from lateral single‐walled carbon nanotube emitters
Authors: Lee W. Y. , Yang F. , Fan L. S. , Tsai C. H.
Published: November 2007

The uniform body emission properties of lateral single‐walled carbon nanotubes (SWNTs) emitters grown by chemical vapor deposition are reported. Lateral SWNTs lying on the substrate were found to possess a turn‐on field of at least 1.67 V/μm and a field enhancement factor of 5223 at most. A flat panel display requiring 1 mA/cm2 was carried out at 4.7 V/μm. The large area uniform luminescence was obtained due to the uniform electrical field intensities throughout the body of SWNTs according to a calculation of electrical field distribution. Good field emission characteristics with a simple process could be more applicable for display applications. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Bond‐curvature effect on burning of single‐wall carbon nanotubes
Authors: Miyata Y. , Kawai T. , Miyamoto Y. , Yanagi K. , Maniwa Y. , Kataura H.
Published: November 2007

Chirality dependent burning of single‐wall carbon nanotubes (SWCNTs) was monitored using optical absorption and photoluminescence spectroscopy for the HiPco and the CoMoCAT SWCNTs. Results have indicated that nanotubes with a higher chiral angle and smaller diameter decomposed more rapidly through thermal oxidation in air. A “local curvature radius” along the C–C bond is found to be a major rate‐determining factor in the combustion process of SWCNTs. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)