bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of segregation strength and an external field on the thermal line edge and line width roughness spectra of a diblock copolymer resist
Authors: Bosse August W.
Published: April 2011

The author uses computer simulations to measure the spectra of thermal line edge roughness (LER) and line width roughness (LWR) in a two-dimensional, symmetric, lamellar diblock copolymer melt over a range of segregation strengths. These measurements demonstrate that increased segregation strength results in a significant suppression of periodic LER and LWR with a wavelength of order of the pattern pitch. The author also examines the effects of a chemically selective external field on the LER and LWR spectra and finds that not only does the external field suppress long-wavelength and periodic LER and LWR but also it acts to decouple interface-interface fluctuations, as measured by the interface-interface covariance. These results will prove useful for block copolymer (BCP) resist design as they highlight the ability to tune the LER and LWR spectra via manipulating BCP segregation strength and the properties of a chemically selective external field. Furthermore, these simulations will function as an important baseline model for planned diffraction experiments aimed at measuring the BCP thermal LER and LWR spectra.
Impact of Polar Ozone Depletion on Subtropical Precipitation
Authors: Kang S. M. , Polvani L. M. , Fyfe J. C. , Sigmond M.
Published: April 2011

The Antarctic ozone hole has led to increased summertime precipitation in the subtropics of the Southern Hemisphere.
Molecular Characterization of Copper Resistance Genes from Xanthomonas citri subsp.
citri
and Xanthomonas alfalfae subsp. citrumelonis
Authors: Behlau Franklin , Canteros Blanca I. , Minsavage Gerald V. , Jones Jeffrey B. , Graham James H.
Published: April 2011

Copper sprays have been widely used for control of endemic citrus canker caused by
Xanthomonas citri
subsp.
citri
in citrus-growing areas for more than 2 decades.
Xanthomonas alfalfae
subsp.
citrumelonis
populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu
r
) strains in both
X. citri
subsp.
citri
and
X. alfalfae
subsp.
citrumelonis
, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in
X. citri
subsp.
citri
and
X. alfalfae
subsp.
citrumelonis
and to compare these organisms to other Cu
r
bacteria. Copper resistance determinants from
X. citri
subsp.
citri
strain A44(pXccCu2) from Argentina and
X. alfalfae
subsp.
citrumelonis
strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes
copL
,
copA
,
copB
,
copM
,
copG
,
copC
,
copD
, and
copF
were identified in
X. citri
subsp.
citri
A44. The same ORFs, except
copC
and
copD
, were also present in
X. alfalfae
subsp.
citrumelonis
1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in
X. citri
subsp.
citri
strain A44 is mostly due to
copL
,
copA
, and
copB
, which are the genes in the cloned cluster with the highest nucleotide homology (≥92%) among different Cu
r
bacteria.
CXCR3-Dependent Plasma Blast Migration to the Central Nervous System during Viral Encephalomyelitis
Authors: Marques Cristina P. , Kapil Parul , Hinton David R. , Hindinger Claudia , Nutt Stephen L. , Ransohoff Richard M. , Phares Timothy W. , Stohlman Stephen A. , Bergmann Cornelia C.
Published: April 2011

Immunoglobulin in cerebral spinal fluid and antibody secreting cells (ASC) within the central nervous system (CNS) parenchyma are common hallmarks of microbial infections and autoimmune disorders. However, the signals directing ASC migration into the inflamed CNS are poorly characterized. This study demonstrates that CXCR3 mediates CNS accumulation of ASC during neurotropic coronavirus-induced encephalomyelitis. Expansion of CXCR3-expressing ASC in draining lymph nodes prior to accumulation within the CNS was consistent with their recruitment by sustained expression of CXCR3 ligands during viral persistence. Both total and virus-specific ASC were reduced greater than 80% in the CNS of infected CXCR3−/−mice. Similar T cell CNS recruitment and local T cell-dependent antiviral activity further indicated that the ASC migration defect was T cell independent. Furthermore, in contrast to the reduction of ASC in the CNS, neither virus-specific ASC trafficking to bone marrow nor antiviral serum antibody was reduced relative to levels in control mice. Impaired ASC recruitment into the CNS of infected CXCR3−/−mice coincided with elevated levels of persisting viral RNA, sustained infectious virus, increased clinical disease, and mortality. These results demonstrate that CXCR3 ligands are indispensable for recruitment of activated ASC into the inflamed CNS and highlight their local protective role during persistent infection.
Analysis of HIV-1 Expression Level and Sense of Transcription by High-Throughput Sequencing of the Infected Cell
Authors: Lefebvre Gregory , Desfarges Sébastien , Uyttebroeck Frédéric , Muñoz Miguel , Beerenwinkel Niko , Rougemont Jacques , Telenti Amalio , Ciuffi Angela
Published: April 2011

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.
Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease
Authors: Romano Keith P. , Laine Jennifer M. , Deveau Laura M. , Cao Hong , Massi Francesca , Schiffer Celia A.
Published: April 2011

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.
Virulence and Genetic Compatibility of Polymerase Reassortant Viruses Derived from the Pandemic (H1N1) 2009 Influenza Virus and Circulating Influenza A Viruses
Authors: Song Min-Suk , Pascua Philippe Noriel Q. , Lee Jun Han , Baek Yun Hee , Park Kuk Jin , Kwon Hyeok-il , Park Su-Jin , Kim Chul-Joong , Kim Hyunggee , Webby Richard J. , Webster Robert G. , Choi Young Ki
Published: April 2011

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.
T4-Like Genome Organization of the Escherichia coli O157:H7 Lytic Phage AR1
Authors: Liao Wei-Chao , Ng Wailap Victor , Lin I-Hsuan , Syu Wan-Jr , Liu Tze-Tze , Chang Chuan-Hsiung
Published: April 2011

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of
Escherichia coli
O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate
Podoviridae
and
Siphoviridae
families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced
E. coli
O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain.
Penaeus monodon TATA Box-Binding Protein Interacts with the White Spot Syndrome Virus Transactivator IE1 and Promotes Its Transcriptional Activity
Authors: Liu Wang-Jing , Chang Yun-Shiang , Huang Wei-Tung , Chen I-Tung , Wang K. C. Han-Ching , Kou Guang-Hsiung , Lo Chu-Fang
Published: April 2011

We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the
Penaeus monodon
TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both
in vitro
and
in vivo
. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused “squelching” of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV
ie1
and
PmTBP
by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of
ie1
and
PmTBP
expression also significantly reduced WSSV replication and the expression of the viral early gene
dnapol
(DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1's transactivation activity that can enhance viral gene expression and help in virus replication.
Subversion of Cellular Autophagy Machinery by Hepatitis B Virus for Viral Envelopment
Authors: Li Jianhua , Liu Yinghui , Wang Zekun , Liu Kuancheng , Wang Yaohui , Liu Jiangxia , Ding Huanping , Yuan Zhenghong
Published: April 2011

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.
Proprioceptive deficits after ACL injury: are they clinically relevant?
Authors: Gokeler Alli , Benjaminse Anne , Hewett Timothy E , Lephart Scott M , Engebretsen Lars , Ageberg Eva , Engelhardt Martin , Arnold Markus P , Postema Klaas , Otten Egbert , Dijkstra Pieter U
Published: April 2011

Objective

To establish the clinical relevance of proprioceptive deficits reported after anterior cruciate ligament (ACL) injury.

Material and methods

A literature search was done in electronic databases from January 1990 to June 2009. Inclusion criteria for studies were ACL deficient (ACL-D) and ACL reconstruction (ACL-R) articles written in English, Dutch or German and calculation of correlation(s) between proprioception tests and clinical outcome measures. Clinical outcome measures were muscle strength, laxity, hop test, balance, patient-reported outcome, objective knee score rating, patient satisfaction or return to sports. Studies included in the review were assessed on their methodological quality.

Results

In total 1161 studies were identified of which 24 met the inclusion criteria. Pooling of all data was not possible due to substantial differences in measurement techniques and data analysis. Most studies failed to perform reliability measurements of the test device used. In general, the correlation between proprioception and laxity, balance, hop tests and patient outcome was low. Four studies reported a moderate correlation between proprioception, strength, balance or hop test.

Conclusion

There is limited evidence that proprioceptive deficits as detected by commonly used tests adversely affect function in ACL-D and ACL-R patients. Development of new tests to determine the relevant role of the sensorimotor system is needed. These tests should ideally be used as screening tests for primary and secondary prevention of ACL injury.
Can shared decision making increase the uptake of evidence in clinical practice?
Authors: Légaré France , Shemilt Michèle , Stacey Dawn
Published: April 2011

Despite copious research and clear policies in many healthcare systems, evidence based practice has yet to be widely adopted. Part of the problem is insufficient consideration of the patient–clinician consultation, which lies at the heart of clinical practice and is where most decisions are made. Shared decision making (SDM)—the interactive process in which patients and clinicians decide on healthcare together—capitalises on the consultation to better translate the best evidence into clinical decisions while taking the patient's values and preferences into account. This paper takes stock of interventions that seek to embed SDM in clinical practice, such as patient decision aids that target both patients and clinicians. It also presents challenges that remain: among others, the paucity of evidence on effective implementation strategies and the lack of consideration of how SDM works when care is delivered by interprofessional teams. The paper then reviews current initiatives to improve and disseminate SDM across the healthcare continuum, and discusses why SDM should be encouraged as a means to leverage evidence based practice. The evidence suggests that finding ways to overcome the challenges and promote SDM will accelerate the uptake of evidence in gastroenterology and hepatology clinical practice.
Possible people, complaints, and the distinction between genetic planning and genetic engineering
Authors: Delaney James J
Published: April 2011

Advances in the understanding of genetics have led to the belief that it may become possible to use genetic engineering to manipulate the DNA of humans at the embryonic stage to produce certain desirable traits. Although this currently cannot be done on a large scale, many people nevertheless object in principle to such practices. Most often, they argue that genetic enhancements would harm the children who were engineered, cause societal harms, or that the risks of perfecting the procedures are too high to proceed. However, many of these same people do not have serious objections to what is called ‘genetic planning’ procedures (such as the selection of sperm donors with desirable traits) that essentially have the same ends. The author calls the view that genetic engineering enhancements are impermissible while genetic planning enhancements are permissible the ‘popular view’, and argues that the typical reasons people give for the popular view fail to distinguish the two practices. This paper provides a principle that can salvage the popular view, which stresses that offspring from genetic engineering practices have grounds for complaint because they are identical to the pre-enhanced embryo, whereas offspring who are the result of genetic planning have no such grounds.
Impact of a care pathway in acute pancreatitis
Authors: McCallum Iain J D , Hicks Gareth J , Attwood Stephen , Seymour Keith
Published: April 2011

Background

Previous studies have shown that accurate process of care predicts quality of care. Few examples currently exist for process of care for the acute surgical patient. A recent region wide audit had identified good outcomes for patients with acute pancreatitis at our institution but aspects of care that could be improved.

Methods

For this re-audit, a simple written care pathway for the management of those presenting with acute pancreatitis was introduced in our institution from February to July 2009. The audit standards were set against the British Society of Gastroenterology (BSG) guidelines for management of acute pancreatitis and were compared with the previous region wide audit.

Results

Marked improvements were noted in the rates of abdominal imaging achieved within 24 h of diagnosis (35.2% vs 47.7%), severity stratification within 48 h of diagnosis (28.7% vs 75%), critical care admission for those classified as severe (39.3% vs 63.6%) and definitive treatment during index admission (22.2% vs 38.5%). Survival rates were 100% for this audit cycle and 95% for all patients within the region wide audit. Despite these improvements, care still does not reach the standards set out by BSG.

Conclusion

Predefined processes of care may help to recognise those developing or likely to develop severe pancreatitis, ensure accurate documentation of severity, expedite critical care review and/or admission, and help to encourage the timely management of those with a treatable underlying cause of their pancreatitis.
Effect of a Sports Food Bar on Fat Utilisation and Exercise Duration
Authors: Kolkhorst Fred W. , MacTaggart Jason N. , Hansen Matt R.
Published: April 2011

The manufacturer claims that using the Access Fat Conversion Activity Bar increases fat utilisation, which would have a glycogen-sparing effect and delay the onset of fatigue from endurance exercise. This claim was tested using seven trained distance runners who performed two trials of treadmill running at 73% of VO2 max to exhaustion. In a counterbalanced design, subjects ingested either one Access Bar with water or water only prior to treadmill running. Times to exhaustion for the control and Access treatment trials were 104.6 ± 24.9 min and 93.9 ± 21.4 min, respectively, and were not signiflcantly different (p > .05). Differences between trials were not observed for the respiratory exchange ratio, blood lactate or glucose concentrations, plasma glycerol concentration, or perceived exertion. Based on results from this study, it was concluded that the Access Bar does not affect fat or carbohydrate utilisation and does not improve exercise endurance. Key words: ergogenic aid, fatigue, glycerol, lipolysis, respiratory exchange ratio
Metabolic and Hormonal Responses to Exercise in Partially Hepatectomised Rats
Authors: Lavoie Jean-Marc , Warren Claude , Arcelin Katia , Latour Martin G. , Désy François , Shinoda Motoo , Éthier Chantal , Gascon-Barré Marielle
Published: April 2011

To characterise how the liver affects metabolic and hormonal exercise responses, hepatectomised (70%; HX) rats were submitted to a 30- or 50-min treadmill exercise (26 m/min, 0% slope) 48 hr or 7 days after surgery (reduced or normal liver mass, respectively). To determine whether metabolic effects of liver mass reduction during exercise were caused by reduced capacity of the liver to produce glucose, metabolic and hormonal responses to the same exercise protocol were measured in 48-hr HX rats. Euglycemia, maintained by exogenous glucose infusion, produced attenuated lactate, insulin, and glucagon values in 48-hr HX rats but did not affect FFA, glycerol, and plasma catecholamine responses. Results indicate that metabolic and hormonal exercise responses are amplified in 48-hr HX rats. Maintaining euglycemia in 48-hr HX rats during exercise does not reduce all responses. Intrahepatic events, similar to those in a short-term (48-hr) HX liver, may influence metabolic and hormonal exercise responses. Key words: insulin, glucagon, catecholamines, glycemia, hepatectomy, hepatic receptors
Induction of p53-independent p21 during ceramide-induced G1 arrest in human hepatocarcinoma cells
Authors: Kim Won Ho , Kang Kyung Hwa , Kim Mie Young , Choi Kyung Hee
Published: April 2011

Ceramide is known to induce pRb (retinoblastoma gene product) dephosphorylation through the activation of ceramide-activated protein phosphatase (CAPP) during G1 arrest, but other molecular mechanisms linked to regulation of pRb dephosphorylation during ceramide-induced G1 arrest are poorly understood. In this paper, we investigated whether p21, a cdk (cyclin-dependent kinase) inhibitor, is involved in the induction of pRb dephosphorylation during ceramide-induced G1 arrest. In SK-Hep-1 cells, the addition of ceramide resulted in pRb dephosphorylation and G1 arrest. The activity of cdk2 was inhibited in response to ceramide during this process. p21 protein and mRNA were remarkably induced, while the protein level of p53, known as a transcriptional activator of p21, was not elevated at the same condition. p21 induction was also observed in the Hep3B cells lacking a functional p53 after exposure to ceramide. Although p21 is induced in ceramide-treated Hep3B cells, Hep3B cells do not induce G1 arrest, because Hep3B cells are deficient in a functional pRb protein. To confirm that pRb is a critical target for the induction of G1 arrest by inhibiting cdk2 activity through p53-independent p21, pRb-expressing vector was transfected into Hep3B cells. After treatment with ceramide, pRb-expressing cells (pRb+/+), but not pRb-/-cells, were arrested in G1 phase. In pRb+/+ cells, ceramide-mediated G1 arrest was accompanied by the accumulation of hypophosphorylated pRb and p21 associated with cdk2. Together, these results suggest that p21, induced through p53-independent pathway, participates in the induction of pRb dephosphorylation by inhibiting cdk2 activity during ceramide-mediated G1 arrest in hepatocarcinoma cells.

Key words: C6-ceramide, G1 arrest, p21, hepatocarcinoma cells.
Activity-dependent morphological synaptic plasticity in an adult neurosecretory system: magnocellular oxytocin neurons of the hypothalamus
Authors: Majdoubi Mohammed El , Poulain Dominique A , Theodosis Dionysia T
Published: April 2011

Oxytocin and vasopressin neurons, located in the supraoptic and paraventricular nuclei of the hypothalamus, send their axons to the neurohypophysis where the neurohormones are released directly into the general circulation. Hormone release depends on the electrical activity of the neurons, which in turn is regulated by different afferent inputs. During conditions that enhance oxytocin secretion (parturition, lactation, and dehydration), these afferents undergo morphological remodelling which results in an increased number of synapses contacting oxytocin neurons. The synaptic changes are reversible with cessation of stimulation. Using quantitative analyses on immunolabelled preparations, we have established that this morphological synaptic plasticity affects both inhibitory and excitatory afferent inputs to oxytocin neurons. This review describes such synaptic modifications, their functional significance, and the cellular mechanisms that may be responsible.Key words: oxytocin, vasopressin, GABA, glutamate, noradrenaline, hypothalamo-neurohypophysial system, lactation.
Flammulin: A novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes
Authors: Wang H X , Ng T B
Published: April 2011

A protein with a molecular weight of 40 kDa, capable of inhibiting cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 0.25 nM, was isolated from fruiting bodies of the mushroom Flammulina velutipes. The protein, designated flammulin, was devoid of ribonuclease activity. Flammulin was unadsorbed on DEAE-cellulose at neutral pH and low ionic strength and adsorbed on CM-Sepharose and Affi-gel blue gel under similar conditions. Its N-terminal sequence demonstrates sites of similarity to those of plant ribosome-inactivating proteins (RIPs).Key words: mushroom, ribosome-inactivating protein, fruiting body.
Chromatin: Mysteries solved?
Authors: Peterson Craig L
Published: April 2011

Over the past few years we have seen enormous progress in uncovering the critical roles that chromatin structure has on the control of gene expression, the regulation of developmental processes, and the control of cell cycle checkpoints. No longer is chromatin research the "last bastion of scoundrels." The recent intensity of chromatin research, however, might lead a young scientist to conclude that the field is saturated or that all the big mysteries have been solved. This view could not be further from the truth! Here I briefly outline four areas of chromatin research where new paradigms and mysteries are still waiting to be discovered.Key words: chromatin, DNA repair, SWI/SNF.
Crowbars and ratchets: Hsp100 chaperones as tools in reversing protein aggregation
Authors: Glover John R , Tkach John M
Published: April 2011

Molecular chaperones have the capacity to prevent inappropriate interactions between aggregation-prone folding or unfolding intermediates created in the cell during protein synthesis or in response to physical and chemical stress. What happens when surveillance by molecular chaperones is evaded or overwhelmed and aggregates accumulate? Recent progress in the elucidation of Hsp100/Clp function suggests that intracellular aggregates or stable complexes can be progressively dissolved by the action of chaperones that act as molecular crowbars or ratchets. These insights set the stage for new progress in the understanding and treatment of diseases of protein folding.Key words: molecular chaperone, Hsp100, aggregation, amyloid.
Localization of Merkel cells at hairless and hairy human skin sites using keratin 18
Authors: Fradette Julie , Godbout Marie-Josée , Michel Martine , Germain Lucie
Published: April 2011

Merkel cells are neurosecretory cells of the skin with epithelial features such as desmosomes and expression of keratins 8, 18, 19, and 20. Merkel cells are scarcely distributed in adult human skin. Although they are present in hair follicles, their density is higher at hairless anatomic sites such as palms and soles. These cells are often innervated by sensory nerve fibers and are thought to be specialized mechanosensory skin receptor cells. However, their precise origin and function are not clearly established. The aim of this study was to localize Merkel cells in human hairless and hairy skin by immunohistochemistry with antibodies Ks18.174 and Ks19.1 directed against keratins 18 and 19, respectively. In glabrous skin of palm and sole, Merkel cells have been localized at the bottom of the rete ridges, in the epidermal basal layer. To study Merkel cell distribution at hairy anatomic sites, we have chosen breast skin, a tissue containing small hair follicles typical of those covering most of the body's surface. Merkel cells were present in the interfollicular epidermis. In hair follicles, they have been identified in the isthmus region.Key words: skin, human, Merkel cell, keratin, hair follicle.
Accumulation of microtubule-associated protein 1A (MAP1A) in differentiating P19 embryonal carcinoma cells
Authors: Vaillant A. R. , Brown D. L.
Published: April 2011

We have examined the accumulation of MAP1A in retinoic acid induced P19 embryonal carcinoma (EC) neurons. By immunofluorescent confocal microscopy, MAP1A was detected in the mitotic spindle of undifferentiated cells but was not evident in association with the interphase microtubules in most cells. By day 4 of differentiation, when neurite outgrowth was underway, MAP1A was co-localized with microtubules in all neurites but was absent from growth cones. By day 8, substantial neurite outgrowth had occurred and MAP1A was seen in all processes. At day 12, no further neurite outgrowth was evident and existing neurites were organized into fascicles. Western blotting and ELISA showed that MAP1A protein levels increased during differentiation. Peak accumulation occurred no later than day 8, coinciding with the period of neurite outgrowth, and then decreased after day 8. The results suggest that in differentiating P19 EC cells MAP1A modulates microtubule dynamics during neurite outgrowth.Key words: MAP1A, neuronal cytoskeleton, neurite outgrowth.
Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function
Authors: Fukamizo Tamo , Brzezinski Ryszard
Published: April 2011

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.
RING fingers and B-boxes: zinc-binding protein-protein interaction domains
Authors: Borden Katherine LB
Published: April 2011

The cysteine-rich zinc-binding motifs known as the RING and B-box are found in several unrelated proteins. Structural, biochemical, and biological studies of these motifs reveal that they mediate protein-protein interactions. Several RING-containing proteins are oncoproteins and recent data indicate that proapoptotic activities can be mediated through the RING. 1H NMR methods were used to determine the structures of RINGs and a B-box domain and to monitor the conformational changes these motifs undergo upon zinc ligation. This review discusses in detail the structural features of the RING and B-box domains. Further, possible structure function relationships for these motifs particularly in their role as protein interaction domains are discussed.Key words: RING, B-box, PML, NMR.
Molecular mechanisms of calmodulin's functional versatility
Authors: Zhang Mingjie , Yuan Tao
Published: April 2011

Calmodulin (CaM) is a primary Ca2+-binding protein found in all eukaryotic cells. It couples the intracellular Ca2+ signal to many essential cellular events by binding and regulating the activities of more than 40 different proteins and enzymes in a Ca2+-dependent manner. CaM contains two structurally similar domains connected by a flexible central linker. Each domain of the protein binds two Ca2+ ions with positive cooperativity. The binding of Ca2+ transforms the protein into its active form through a reorientation of the existing helices of the protein. The two helices in each helix-loop-helix Ca2+-binding motif are almost antiparallel in Ca2+-free CaM. The binding of Ca2+ induces concerted helical pair movements and changes the two helices in each Ca2+ binding motif to a nearly perpendicular orientation. These concerted helix pair movements are accompanied by dramatic changes on the molecular surface of the protein. Rather than exhibiting a flat, hydrophilic molecular surface as seen in Ca2+-free CaM, the Ca2+-saturated form of the protein contains a Met-rich, cavity-containing hydrophobic surface in each domain. These hydrophobic surfaces are largely responsible for the binding of CaM to its targets. The unique flexibility and high polarizability of the Met residues located at the entrance of each hydrophobic pocket together with other hydrophobic amino acid residues create adjustable, sticky interaction surface areas that can accommodate CaM's targets, which have various sizes and shapes. Therefore, CaM is able to bind to a large array of targets without obvious sequence homology. Upon binding to its target peptides, the unwinding of the central linker allows the two domains of the protein to engulf the hydrophobic face of target peptides of differing lengths. The binding of Ca2+ reduces the backbone flexibility of CaM. Formation of complexes with its target peptides further decreases the backbone motion of CaM.Key words: calmodulin, NMR, structure and dynamics, peptide targets, ligand specificity.
NMR and CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding
Authors: Kotovych George , Cann John R , Stewart John M , Yamamoto Hitoshi
Published: April 2011

Most physiological processes are regulated by peptides that perform their functions by interacting with specific receptors on cells. Specific conformations of the peptides are required for correct interactions to take place, and a knowledge of the biologically important conformation is vital for the understanding of biological function. Over the last few years extensive studies using nuclear magnetic resonance and circular dichroism have been carried out on bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and its antagonists with the objective of developing new drugs to combat severe pathologies associated with its production. In the present review, these techniques for the determination of peptide conformation are reviewed and applied to the study of bradykinin and its antagonists. Modeling of these conformational data in the presence of the B2 receptor or an antibody allows the biologically active conformations to be deduced and these are presented in this review.Key words: bradykinin antagonists, conformational analysis, NMR, CD, B2 receptor binding.
Effect of temperature on the kinetics of lactate production and clearance in a rat model of forebrain ischemia
Authors: Lei Hao , Peeling James
Published: April 2011

To investigate the effect of brain temperature on metabolic perturbations during and following forebrain ischemia, localized 1H magnetic resonance spectroscopy was used to measure the kinetics of lactate production and clearance in a rat model of 12- or 20-min forebrain ischemia (two-vessel occlusion with hypotension) at a brain temperature of either 34.5 ± 0.5°C or 37.5 ± 0.5°C. During ischemia, lactate production was modeled with apparent first order kinetics. Hypothermia did not affect the rate or the extent of lactate production during ischemia. Upon reperfusion, a delay in the decrease of the cerebral lactate level was found in the normothermia groups. Such a delay was absent in hypothermia groups, which may reflect faster resumption of cerebral oxidative metabolism upon reperfusion in the hypothermic animals. The rate constant for lactate clearance postischemia was larger for normothermic animals and for the 20-min ischemia groups, perhaps because of increased blood-brain barrier permeability following more severe ischemia.Key words: forebrain ischemia, temperature, hypothermia, lactate, magnetic resonance spectroscopy.
The NMR angle on troponin C
Authors: Gagné Stéphane M , Li Monica X , McKay Ryan T , Sykes Brian D
Published: April 2011

The calcium-induced structural changes in the skeletal muscle regulatory protein troponin C involve a transition from a closed to an open structure with the concomitant exposure of a large hydrophobic interaction site for target proteins. NMR solution structural studies have served to define this conformational change and elucidate the mechanism of the linkage between calcium binding and the induced structural changes. These structural movements are described in terms of interhelical angles in these largely helical proteins. Oddly, the most recent structure of the cardiac system challenges the central paradigm because the calcium-bound structures are not open. The kinetics, energetics, and dynamics of these proteins have also been investigated using NMR.Key words: troponin C, calcium binding protein, NMR, structure, energetics.
Apoptotic signal transduction: emerging pathways
Authors: Wilson Mark R
Published: April 2011

Apoptosis is a counterbalance to mechanisms of cell proliferation and is critically important in regulation of the immune system, development, and normal tissue homeostasis. Mammalian signal transduction pathways affecting apoptosis are more complex than their counterparts in the nematode Caenorhabditis elegans, a valuable model system that has provided powerful initial insights into key molecules regulating apoptosis. Despite this complexity, substantial progress has been made in recent years towards defining the nature and detail of signalling pathways bringing about apoptosis in mammalian cells. In particular, the identity and precise substrate specificities of a large family of caspase enzymes, implicated as critical components of the apoptotic machinery, have been defined. In addition, the mechanism by which the cell surface Fas receptor mediates induction of apoptosis, via activation of caspases, has recently been elucidated. A prominent role for mitochondria in cell death pathways has also recently emerged, a clear theme being that mitochondria can trigger degradative events by the release of apoptogenic proteins (e.g., cytochrome c) from the intermembrane space to the cytosol. This review focusses on recent progress in these areas and discusses integration of this knowledge in our overall understanding of the processes that control apoptosis.Key words: caspases, Fas receptor, mitochondria, cytochrome c, AIF.
Noninvasive assessment of pharmaceutical intervention during myocardial ischemia-reperfusion in a canine model using two-dimensional 31P chemical shift imaging
Authors: Campbell Constance M , Wisenberg Gerald , Sykes Jane , Thompson R Terry
Published: April 2011

The metabolic effects during myocardial ischemia and sustained reperfusion of the antianginal agents diltiazem (n = 10) and propranolol (n = 10) were monitored with noninvasive phosphorus nuclear magnetic resonance spectroscopy to establish any correlation between metabolic changes and infarct size. Spectroscopy followed changes in high-energy phosphate concentrations and myocardial intracellular pH during 2 h of left anterior descending coronary artery occlusion and 3 subsequent weeks of reperfusion, in a closed chest canine infarct model. Gadolinium-DTPA enhanced magnetic resonance imaging was used to assess the extent of myocardial injury (infarct size). Microspheres were used to document the zone at risk and the success of reperfusion. Whereas diltiazem appeared to reduce the derangement in high-energy phosphates during coronary occlusion, there was no significant change in infarct size when compared with a previously studied control group. Propranolol, which produced a lesser decline in pH during occlusion and smaller pH changes during early reperfusion, was associated with a significant reduction in the degree of tissue necrosis (compared with controls). There was an inverse correlation (r = -0.51) between the change in myocardial pH (occlusion end to immediate reperfusion) and the recovery index (an index of myocardial salvage). By 1 h into reperfusion, there was a stronger inverse correlation between pH and infarct size (r = -0.75), implying a protective effect of delaying pH recovery during early reperfusion and indicating the potential use of this parameter as a predictor of tissue viability.Key words: diltiazem, propranolol, magnetic resonance spectroscopy, myocardial infarction.
NMR studies of the antimicrobial salivary peptides histatin 3 and histatin 5 in aqueous and nonaqueous solutions
Authors: Brewer Dyanne , Hunter Howard , Lajoie Gilles
Published: April 2011

Conformational studies of the salivary peptides histatin 3 (H3) and histatin 5 (H5) were performed by NMR and circular dichroism (CD) in aqueous and nonaqueous solutions. Histatin 5 has no defined structure in H2O but adopts a more helical conformation in dimethyl sulfoxide and aqueous trifluoroethanol. This is in agreement with the CD analysis, which shows no secondary structure in H2O but increasing helical content in the presence of trifluoroethanol. CD analysis shows that H3 has less propensity to form a helical structure than H5 in similar conditions. The NMR analysis of H3 in H2O at pH 7.4 reveals that its conformational mobility is less than that of H5 as indicated by the observation of backbone cross peaks alphaN (i, i + 1) and NN (i, i + 1) and the slow exchanging amide protons in the C-terminus. However, H3 remains essentially unordered as suggested by the lack of longer range nuclear Overhauser effects (NOEs) in the NOESY spectrum. H3 becomes much more ordered in a mixture of 50:50 H2O - dimethyl sulfoxide as indicated by the numerous NOEs, including several side chain to side chain and side chain to backbone conectivities. Our data suggest that in these conditions H3 contains a turn in the region of K13 to K17 and possibly a 310 helix at the C-terminus. This study demonstrates that H3 and H5 are both conformationally mobile and that each adopt different types of conformations in aqueous and nonaqueous solutions.Key words: histatins, NMR, antimicrobial.
Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells
Authors: Ohan Nicholas W , Tam Ying , Fernando Pasan , Heikkila John J
Published: April 2011

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.Key words: Xenopus laevis, heat shock protein, hsp30, NEPHGE, basic small heat shock proteins, sodium arsenite, herbimycin A.