bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial oxidative phosphorylation in autosomal dominant optic atrophy
Authors: Mayorov Vladimir I , Lowrey Angela J , Biousse Valerie , Newman Nancy J , Cline Susan D , Brown Michael D
Published: September 2008

Background

Autosomal dominant optic atrophy (ADOA), a form of progressive bilateral blindness due to loss of retinal ganglion cells and optic nerve deterioration, arises predominantly from mutations in the nuclear gene for the mitochondrial GTPase, OPA1. OPA1 localizes to mitochondrial cristae in the inner membrane where electron transport chain complexes are enriched. While OPA1 has been characterized for its role in mitochondrial cristae structure and organelle fusion, possible effects of OPA1 on mitochondrial function have not been determined.

Results

Mitochondria from six ADOA patients bearing OPA1 mutations and ten ADOA patients with unidentified gene mutations were studied for respiratory capacity and electron transport complex function. Results suggest that the nuclear DNA mutations that give rise to ADOA in our patient population do not alter mitochondrial electron transport.

Conclusion

We conclude that the pathophysiology of ADOA likely stems from the role of OPA1 in mitochondrial structure or fusion and not from OPA1 support of oxidative phosphorylation.
Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera
Authors: Tooming-Klunderud Ave , Fewer David P , Rohrlack Thomas , Jokela Jouni , Rouhiainen Leo , Sivonen Kaarina , Kristensen Tom , Jakobsen Kjetill S
Published: September 2008

Background

Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.

Results

A comparative analysis of the adenylation domains from the first module of McyB (McyB1) and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants.

Conclusion

We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may influence the ability of a particular strain to interact with its environment.
Accelerated evolutionary rates in tropical and oceanic parmelioid lichens (Ascomycota)
Authors: Lumbsch H Thorsten , Hipp Andrew L , Divakar Pradeep K , Blanco Oscar , Crespo Ana
Published: September 2008

Background

The rate of nucleotide substitutions is not constant across the Tree of Life, and departures from a molecular clock have been commonly reported. Within parmelioid lichens, the largest group of macrolichens, large discrepancies in branch lengths between clades were found in previous studies. Using an extended taxon sampling, we test for presence of significant rate discrepancies within and between these clades and test our a priori hypothesis that such rate discrepancies may be explained by shifts in moisture regime or other environmental conditions.

Results

In this paper, the first statistical evidence for accelerated evolutionary rate in lichenized ascomycetes is presented. Our results give clear evidence for a faster rate of evolution in two Hypotrachyna clades that includes species occurring in tropical and oceanic habitats in comparison with clades consisting of species occurring in semi-arid and temperate habitats. Further we explore potential links between evolutionary rates and shifts in habitat by comparing alternative Ornstein-Uhlenbeck models.

Conclusion

Although there was only weak support for a shift at the base of a second tropical clade, where the observed nucleotide substitution rate is high, overall support for a shift in environmental conditions at cladogenesis is very strong. This suggests that speciation in some lichen clades has proceeded by dispersal into a novel environment, followed by radiation within that environment. We found moderate support for a shift in moisture regime at the base of one tropical clade and a clade occurring in semi-arid regions and a shift in minimum temperature at the base of a boreal-temperate clade.
Chromosomal evolution of the PKD1 gene family in primates
Authors: Kirsch Stefan , Pasantes Juanjo , Wolf Andreas , Bogdanova Nadia , Münch Claudia , Pennekamp Petra , Krawczak Michael , Dworniczak Bernd , Schempp Werner
Published: September 2008

Background

The autosomal dominant polycystic kidney disease (ADPKD) is mostly caused by mutations in the PKD1 (polycystic kidney disease 1) gene located in 16p13.3. Moreover, there are six pseudogenes of PKD1 that are located proximal to the master gene in 16p13.1. In contrast, no pseudogene could be detected in the mouse genome, only a single copy gene on chromosome 17. The question arises how the human situation originated phylogenetically. To address this question we applied comparative FISH-mapping of a human PKD1-containing genomic BAC clone and a PKD1-cDNA clone to chromosomes of a variety of primate species and the dog as a non-primate outgroup species.

Results

Comparative FISH with the PKD1-cDNA clone clearly shows that in all primate species studied distinct single signals map in subtelomeric chromosomal positions orthologous to the short arm of human chromosome 16 harbouring the master PKD1 gene. Only in human and African great apes, but not in orangutan, FISH with both BAC and cDNA clones reveals additional signal clusters located proximal of and clearly separated from the PKD1 master genes indicating the chromosomal position of PKD1 pseudogenes in 16p of these species, respectively. Indeed, this is in accordance with sequencing data in human, chimpanzee and orangutan. Apart from the master PKD1 gene, six pseudogenes are identified in both, human and chimpanzee, while only a single-copy gene is present in the whole-genome sequence of orangutan. The phylogenetic reconstruction of the PKD1-tree reveals that all human pseudogenes are closely related to the human PKD1 gene, and all chimpanzee pseudogenes are closely related to the chimpanzee PKD1 gene. However, our statistical analyses provide strong indication that gene conversion events may have occurred within the PKD1 family members of human and chimpanzee, respectively.

Conclusion

PKD1 must have undergone amplification very recently in hominid evolution. Duplicative transposition of the PKD1 gene and further amplification and evolution of the PKD1 pseudogenes may have arisen in a common ancestor of Homo, Pan and Gorilla ~8 MYA. Reticulate evolutionary processes such as gene conversion and non-allelic homologous recombination (NAHR) may have resulted in concerted evolution of PKD1 family members in human and chimpanzee and, thus, simulate an independent evolution of the PKD1 pseudogenes from their master PKD1 genes in human and chimpanzee.
Heat stress-responsive transcriptome analysis in heat susceptible and tolerant wheat (Triticum aestivum L.) by using Wheat Genome Array
Authors: Qin Dandan , Wu Haiyan , Peng Huiru , Yao Yingyin , Ni Zhongfu , Li Zhenxing , Zhou Chunlei , Sun Qixin
Published: September 2008

Background

Wheat is a major crop in the world, and the high temperature stress can reduce the yield of wheat by as much as 15%. The molecular changes in response to heat stress are poorly understood. Using GeneChip®Wheat Genome Array, we analyzed genome-wide gene expression profiles in the leaves of two wheat genotypes, namely, heat susceptible 'Chinese Spring' (CS) and heat tolerant 'TAM107' (TAM).

Results

A total of 6560 (~10.7%) probe sets displayed 2-fold or more changes in expression in at least one heat treatment (falsediscoveryrate, FDR, α = 0.001). Except for heat shock protein (HSP) and heat shock factor (HSF) genes, these putative heat responsive genes encode transcription factors and proteins involved in phytohormone biosynthesis/signaling, calcium and sugar signal pathways, RNA metabolism, ribosomal proteins, primary and secondary metabolisms, as well as proteins related to other stresses. A total of 313 probe sets were differentially expressed between the two genotypes, which could be responsible for the difference in heat tolerance of the two genotypes. Moreover, 1314 were differentially expressed between the heat treatments with and without pre-acclimation, and 4533 were differentially expressed between short and prolonged heat treatments.

Conclusion

The differences in heat tolerance in different wheat genotypes may be associated with multiple processes and mechanisms involving HSPs, transcription factors, and other stress related genes. Heat acclimation has little effects on gene expression under prolonged treatments but affects gene expression in wheat under short-term heat stress. The heat stress responsive genes identified in this study will facilitate our understanding of molecular basis for heat tolerance in different wheat genotypes and future improvement of heat tolerance in wheat and other cereals.
Genome sequencing and analysis reveals possible determinants of Staphylococcus aureus nasal carriage
Authors: Sivaraman Karthikeyan , Venkataraman Nitya , Tsai Jennifer , Dewell Scott , Cole Alexander M
Published: September 2008

Background

Nasal carriage of Staphylococcus aureus is a major risk factor in clinical and community settings due to the range of etiologies caused by the organism. We have identified unique immunological and ultrastructural properties associated with nasal carriage isolates denoting a role for bacterial factors in nasal carriage. However, despite extensive molecular level characterizations by several groups suggesting factors necessary for colonization on nasal epithelium, genetic determinants of nasal carriage are unknown. Herein, we have set a genomic foundation for unraveling the bacterial determinants of nasal carriage in S. aureus.

Results

MLST analysis revealed no lineage specific differences between carrier and non-carrier strains suggesting a role for mobile genetic elements. We completely sequenced a model carrier isolate (D30) and a model non-carrier strain (930918-3) to identify differential gene content. Comparison revealed the presence of 84 genes unique to the carrier strain and strongly suggests a role for Type VII secretion systems in nasal carriage. These genes, along with a putative pathogenicity island (SaPIBov) present uniquely in the carrier strains are likely important in affecting carriage. Further, PCR-based genotyping of other clinical isolates for a specific subset of these 84 genes raise the possibility of nasal carriage being caused by multiple gene sets.

Conclusion

Our data suggest that carriage is likely a heterogeneic phenotypic trait and implies a role for nucleotide level polymorphism in carriage. Complete genome level analyses of multiple carriage strains of S. aureus will be important in clarifying molecular determinants of S. aureus nasal carriage.
Activity of the Bacillus anthracis20 kDa protective antigen component
Authors: Hammamieh Rasha , Ribot Wilson J , Abshire Terry G , Jett Marti , Ezzell John
Published: September 2008

Background

Anthrax is caused byBacillus anthracisthat produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63and PA20). PA63forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.

Both proteolytically cleaved protective antigens (PA63and PA20fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63fragment has been thoroughly studied while little is known about the PA20.

Methods

In this study we examined the role of PA20using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83to obtain PA20using limited digestion of this recombinant PA83with trypsin.

Results

Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of ~40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20orB. anthracisspores/vegetative cells.

Conclusion

This study shows that the PA20has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.
Determinants of health-related quality of life in elderly in Tehran, Iran
Authors: Tajvar Maryam , Arab Mohammad , Montazeri Ali
Published: September 2008

Background

As Iran started to experience population ageing, it is important to consider and address the elderly people's needs and concerns, which might have direct impacts on their well-being and quality of life. There have been only a few researches into different aspects of life of the elderly population in Iran including their health-related quality of life. The purpose of this study was to measure health-related quality of life (HRQoL) of elderly Iranians and to identify its some determinant factors.

Methods

This was a cross-sectional survey of a random sample of community residents of Tehran aged 65 years old and over. HRQoL was measured using the Short From Health Survey (SF-36). The study participants were interviewed at their homes. Uni-variate analysis was performed for group comparison and logistic regression analysis conducted to predict quality of life determinants.

Results

In all, 400 elderly Iranian were interviewed. The majority of the participants were men (56.5%) and almost half of the participants were illiterate (n = 199, 49.8%). Eighty-five percent of the elderly were living with their family or relatives and about 70% were married. Only 12% of participants evaluated their economic status as being good and most of people had moderate or poor economic status. The mean scores for the SF-36 subscales ranged from 70.0 (SD = 25.9) for physical functioning to 53.5 (SD = 29.1) for bodily pain and in general, the respondents significantly showed better condition on mental component of the SF-36 than its physical component (mean scores 63.8 versus 55.0). Performing uni-variate analysis we found that women reported significantly poorer HRQoL. Multiple logistic regression analysis showed that for the physical component summary score of the SF-36, age, gender, education and economic status were significant determinants of poorer physical health-related quality of life; while for the mental component summary score only gender and economic status were significant determinants of poorer mental health-related quality of life. The analysis suggested that the elderly people's economic status was the most significant predictor of their HRQoL.

Conclusion

The study findings, although with a small number of participants, indicate that elderly people living in Tehran, Iran suffer from relatively poor HRQoL; particularly elderly women and those with lower education. Indeed to improve quality of life among elderly Iranians much more attention should be paid to all aspects of their life including their health, and economic status.
Anopheles gambiae complex along The Gambia river, with particular reference to the molecular forms of An. gambiae s.s
Authors: Caputo Beniamino , Nwakanma Davis , Jawara Musa , Adiamoh Majidah , Dia Ibrahima , Konate Lassana , Petrarca Vincenzo , Conway David J , della Torre Alessandra
Published: September 2008

Background

The geographic and temporal distribution of M and S molecular forms of the major Afrotropical malaria vector species Anopheles gambiae s.s. at the western extreme of their range of distribution has never been investigated in detail.

Materials and methods

Collections of indoor-resting An. gambiae s.l. females were carried out along a ca. 400 km west to east transect following the River Gambia from the western coastal region of The Gambia to south-eastern Senegal during 2005 end of rainy season/early dry season and the 2006 rainy season. Specimens were identified to species and molecular forms by PCR-RFLP and the origin of blood-meal of fed females was determined by ELISA test.

Results

Over 4,000 An. gambiae s.l. adult females were collected and identified, 1,041 and 3,038 in 2005 and 2006, respectively. M-form was mainly found in sympatry with Anopheles melas and S-form in the western part of the transect, and with Anopheles arabiensis in the central part. S-form was found to prevail in rural Sudan-Guinean savannah areas of Eastern Senegal, in sympatry with An. arabiensis. Anopheles melas and An. arabiensis relative frequencies were generally lower in the rainy season samples, when An. gambiae s.s. was prevailing. No large seasonal fluctuations were observed for M and S-forms. In areas where both M and S were recorded, the frequency of hybrids between them ranged from to 0.6% to 7%.

Discussion

The observed pattern of taxa distribution supports the hypothesis of a better adaptation of M-form to areas characterized by water-retaining alluvial deposits along the Gambia River, characterized by marshy vegetation, mangrove woods and rice cultivations. In contrast, the S-form seems to be better adapted to free-draining soil, covered with open woodland savannah or farmland, rich in temporary larval breeding sites characterizing mainly the eastern part of the transect, where the environmental impact of the Gambia River is much less profound and agricultural activities are mainly rain-dependent. Very interestingly, the observed frequency of hybridization between the molecular forms along the whole transect was much higher than has been reported so far for other areas.

Conclusion

The results support a bionomic divergence between the M and S-forms, and suggest that the western extreme of An. gambiae s.s. geographical distribution may represent an area of higher-than-expected hybridization between the two molecular forms.
Augmentation of Pulmonary Epithelial Cell IL-8 Expression and Permeability by Pre-B-cell Colony Enhancing Factor
Authors: Li Hailong , Liu Peng , Cepeda Javier , Fang Deyu , Easley R Blaine , Simon Brett A , Zhang Li Qin , Ye Shui Qing
Published: September 2008

Background

Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability.

Methods

Human pulmonary alveolar epithelial cells (cell line and primary cells) were transfected with human PBEF cDNA or PBEF siRNA and then cultured in the presence or absence of TNFα. PBEF and IL-8 expression were analyzed by RT-PCR and Western blotting. In addition, changes in pulmonary alveolar epithelial and artery endothelial cell barrier regulation with altered PBEF expression was evaluated by an in vitro cell permeability assay.

Results

Our results demonstrated that, in human pulmonary alveolar epithelial cells, the overexpression of PBEF significantly augmented basal and TNFα-stimulated IL-8 secretion by more than 5 to 10-fold and increased cell permeability by >30%; the knockdown of PBEF expression with siRNA significantly inhibited basal and TNFα-stimulated IL-8 secretion by 70% and IL-8 mRNA levels by 74%. Further, the knockdown of PBEF expression also significantly attenuated TNFα-induced cell permeability by 43%. Similar result was observed in human pulmonary artery endothelial cells.

Conclusion

These results suggest that PBEF may play a vital role in basal and TNFα-mediated pulmonary inflammation and pulmonary epithelial barrier dysfunction via its regulation of other inflammatory cytokines such as IL-8, which could in part explain the role of PBEF in the susceptibility and pathogenesis of ALI. These results lend further support to the potential of PBEF to serve as a diagnostic and therapeutic target to ALI.
Identification of symptom domains in ulcerative colitis that occur frequently during flares and are responsive to changes in disease activity
Authors: Joyce Joel C , Waljee Akbar K , Khan Tahira , Wren Patricia A , Dave Maneesh , Zimmermann Ellen M , Wang Sijian , Zhu Ji , Higgins Peter DR
Published: September 2008

Background

Ulcerative colitis disease activity is determined by measuring symptoms and signs. Our aim was to determine which symptom domains are frequent and responsive to change in the evaluation of disease activity, which are those defined by three criteria: 1) they occur frequently during flares; 2) they improve during effective therapy for ulcerative colitis; and 3) they resolve during remission.

Methods

Twenty-eight symptom domains, 16 from standard indices and 12 novel domains identified by ulcerative colitis patient focus groups, were evaluated. Sixty subjects with ulcerative colitis were surveyed, rating each symptom on the three criteria with a 100 mm Visual Analogue Scale. Frequent and responsive symptoms were defined a priori as those whose median Visual Analogue Scale rating for all 3 criteria was significantly greater than 50.

Results

Thirteen of the 28 symptom domains were identified as both frequent in ulcerative colitis flares and responsive to changes in disease activity. Seven of these 13 symptom domains were novel symptoms derived from ulcerative colitis patient focus groups including stool mucus, tenesmus, fatigue, rapid postprandial bowel movements, and inability to differentiate liquid or gas from solid stool when rectal urgency occurs. Ten of the 16 symptom domains from standard indices were either infrequent or unresponsive to changes in disease activity.

Conclusion

Only some of the symptoms of ulcerative colitis that are important to patients are included in standard indices, and several symptoms currently measured are not frequent or responsive to change in ulcerative colitis patients. Development of survey measures of these symptom domains could significantly improve the assessment of disease activity in ulcerative colitis.
Apigenin and luteolin modulate microglial activation via inhibition of STAT1-induced CD40 expression
Authors: Rezai-Zadeh Kavon , Ehrhart Jared , Bai Yun , Sanberg Paul R , Bickford Paula , Tan Jun , Shytle R Douglas
Published: September 2008

Background

It is well known that most neurodegenerative diseases are associated with microglia-mediated inflammation. Our previous research demonstrates that the CD40 signaling is critically involved in microglia-related immune responses in the brain. For example, it is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (IFN-γ)-induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN-γ and amyloid-β (Aβ) peptide. Recent studies have shown that certain flavonoids possess anti-inflammatory and neuroprotective properties distinct from their well-known anti-oxidant effects. In particular, flavonoids, apigenin and luteolin have been found to be effective CD40 immunomodulators.

Methods

Cultured microglia, both N9 and primary derived lines, were treated with flavonoids in the presence of IFN-γ and/or CD40 ligation to assess any anti-inflammatory effects and/or mechanisms. CD40 expression on microglia was analyzed by fluorescence activated cell sorting (FACS). Anti-inflammatory effects and mechanisms were confirmed by ELISA for interlekin-6 (IL-6) and TNF-α, lactate dehydrogenase (LDH) assay, and STAT1 Western blotting.

Results

Apigenin and luteolin concentration-dependently suppressed IFN-γ-induced CD40 expression. Apigenin and luteolin also suppressed microglial TNF-α and IL-6 production stimulated by IFN-gamma challenge in the presence of CD40 ligation. In addition, apigenin and luteolin markedly inhibited IFN-γ-induced phosphorylation of STAT1 with little impact on cell survival.

Conclusion

Our findings provide further support for apigenin and luteolin's anti-inflammatory effects and suggest that these flavonoids may have neuroprotective/disease-modifying properties in various neurodegenerative disorders, including Alzheimer's disease (AD).
Human T-cell leukemia virus type I infects human lung epithelial cells and induces gene expression of cytokines, chemokines and cell adhesion molecules
Authors: Teruya Hiromitsu , Tomita Mariko , Senba Masachika , Ishikawa Chie , Tamayose Maki , Miyazato Akiko , Yara Satomi , Tanaka Yuetsu , Iwakura Yoichiro , Fujita Jiro , Mori Naoki
Published: September 2008

Background

Human T-cell leukemia virus type I (HTLV-I) is associated with pulmonary diseases, characterized by bronchoalveolar lymphocytosis, which correlates with HTLV-I proviral DNA in carriers. HTLV-I Tax seems to be involved in the development of such pulmonary diseases through the local production of inflammatory cytokines and chemokines in T cells. However, little is known about induction of these genes by HTLV-I infection in lung epithelial cells.

Results

We tested infection of lung epithelial cells by HTLV-I by coculture studies in which A549 alveolar and NCI-H292 tracheal epithelial cell lines were cocultured with MT-2, an HTLV-I-infected T-cell line. Changes in the expression of several cellular genes were assessed by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and flow cytometry. Coculture with MT-2 cells resulted in infection of lung epithelial cells as confirmed by detection of proviral DNA, HTLV-I Tax expression and HTLV-I p19 in the latter cells. Infection was associated with induction of mRNA expression of various cytokines, chemokines and cell adhesion molecule. NF-κB and AP-1 were also activated in HTLV-I-infected lung epithelial cells.In vivostudies showed Tax protein in lung epithelial cells of mice bearing Tax and patients with HTLV-I-related pulmonary diseases.

Conclusion

Our results suggest that HTLV-I infects lung epithelial cells, with subsequent production of cytokines, chemokines and cell adhesion molecules through induction of NF-κB and AP-1. These changes can contribute to the clinical features of HTLV-I-related pulmonary diseases.
Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging
Authors: Fritz Joëlle V , Didier Pascal , Clamme Jean-Pierre , Schaub Emmanuel , Muriaux Delphine , Cabanne Charlotte , Morellet Nelly , Bouaziz Serge , Darlix Jean-Luc , Mély Yves , de Rocquigny Hugues
Published: September 2008

Background

The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

Results

Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.

Conclusion

We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.
High-throughput Agrobacterium-mediated barley transformation
Authors: Bartlett Joanne G , Alves Sílvia C , Smedley Mark , Snape John W , Harwood Wendy A
Published: September 2008

Background

Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.

Results

A robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos withAgrobacteriumstrain AGL1, carrying vectors from the pBract series that contain thehptgene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising theluc(firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of thelucgene.

Conclusion

This protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.
Frequencies of polymorphisms associated with BSE resistance differ significantly between Bos taurus, Bos indicus, and composite cattle
Authors: Brunelle Brian W , Greenlee Justin J , Seabury Christopher M , Brown Charles E , Nicholson Eric M
Published: September 2008

Background

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect several mammalian species. At least three factors related to the host prion protein are known to modulate susceptibility or resistance to a TSE: amino acid sequence, atypical number of octapeptide repeats, and expression level. These factors have been extensively studied in breeds of Bos taurus cattle in relation to classical bovine spongiform encephalopathy (BSE). However, little is currently known about these factors in Bos indicus purebred or B. indicus × B. taurus composite cattle. The goal of our study was to establish the frequency of markers associated with enhanced susceptibility or resistance to classical BSE in B. indicus purebred and composite cattle.

Results

No novel or TSE-associated PRNP-encoded amino acid polymorphisms were observed for B. indicus purebred and composite cattle, and all had the typical number of octapeptide repeats. However, differences were observed in the frequencies of the 23-bp and 12-bp insertion/deletion (indel) polymorphisms associated with two bovine PRNP transcription regulatory sites. Compared to B. taurus, B. indicus purebred and composite cattle had a significantly lower frequency of 23-bp insertion alleles and homozygous genotypes. Conversely, B. indicus purebred cattle had a significantly higher frequency of 12-bp insertion alleles and homozygous genotypes in relation to both B. taurus and composite cattle. The origin of these disparities can be attributed to a significantly different haplotype structure within each species.

Conclusion

The frequencies of the 23-bp and 12-bp indels were significantly different between B. indicus and B. taurus cattle. No other known or potential risk factors were detected for the B. indicus purebred and composite cattle. To date, no consensus exists regarding which bovine PRNP indel region is more influential with respect to classical BSE. Should one particular indel region and associated genotypes prove more influential with respect to the incidence of classical BSE, differences regarding overall susceptibility and resistance for B. indicus and B. taurus cattle may be elucidated.
A chemical-genetic strategy reveals distinct temporal requirements for SAD-1 kinase in neuronal polarization and synapse formation
Authors: Kim Joanne SM , Lilley Brendan N , Zhang Chao , Shokat Kevan M , Sanes Joshua R , Zhen Mei
Published: September 2008

Background

Neurons assemble into a functional network through a sequence of developmental processes including neuronal polarization and synapse formation. In Caenorhabditis elegans, the serine/threonine SAD-1 kinase is essential for proper neuronal polarity and synaptic organization. To determine if SAD-1 activity regulates the establishment or maintenance of these neuronal structures, we examined its temporal requirements using a chemical-genetic method that allows for selective and reversible inactivation of its kinase activity in vivo.

Results

We generated a PP1 analog-sensitive variant of SAD-1. Through temporal inhibition of SAD-1 kinase activity we show that its activity is required for the establishment of both neuronal polarity and synaptic organization. However, while SAD-1 activity is needed strictly when neurons are polarizing, the temporal requirement for SAD-1 is less stringent in synaptic organization, which can also be re-established during maintenance.

Conclusion

This study reports the first temporal analysis of a neural kinase activity using the chemical-genetic system. It reveals that neuronal polarity and synaptic organization have distinct temporal requirements for SAD-1.
Salvaging Affymetrix probes after probe-level re-annotation
Authors: de Leeuw Wim C , Rauwerda Han , Jonker Martijs J , Breit Timo M
Published: September 2008

Background

Affymetrix GeneChips can be re-annotated at the probe-level by breaking up the original probe-sets and recomposing new probe-sets based on up-to-date genomic knowledge, such as available in Entrez Gene. This results in custom Chip Description Files (CDF). Using these custom CDFs improves the quality of the data and thus the results of related gene expression studies. However, 44–71% of the probes on a GeneChip are lost in this re-annotation process. Although generally aimed at less known genes, losing these probes obviously means a substantial loss of expensive experiment data. Biologists are therefore very reluctant to adopt this approach.

Findings

We aimed to re-introduce the non-affected Affymetrix probe-sets after these re-annotation procedures. For this, we developed an algorithm (CDF-Merger) and applied it to standard Affymetrix CDFs and custom Brainarray CDFs to obtain Hybrid CDFs. Thus, salvaging lost Affymetrix probes with our CDF-Merger restored probe content up to 94%. Because the salvaged probes (up to 54% of the probe content on the arrays) represent less-reliable probe-sets, we made the origin of all probe-set definitions traceable, so biologists can choose at any time in their analyses, which subset of probe-sets they want to use.

Conclusion

The availability of up-to-date Hybrid CDFs plus R environment allows for easy implementation of our approach.
Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi
Authors: Nacer Adéla , Walker Karen , Hurd Hilary
Published: September 2008

Background

Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of Plasmodium proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of Plasmodium berghei oocysts and in the midgut epithelial cells of Anopheles stephensi was investigated.

Results

An ultrastructural examination of midgut sections from infected and uninfected An. stephensi was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina.

Conclusion

We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear.
Benefits of an educational program for journalists on media coverage of HIV/AIDS in developing countries
Authors: Martinez‐Cajas Jorge L , Invernizzi Cédric F , Ntemgwa Michel , Schader Susan M , Wainberg Mark A
Published: September 2008

Objective

a) To assess the suitability of the curriculum content and didactical quality of information delivered to educate journalists in the J2J program in HIV/AIDS (process evaluation) and b) to explore the effects of such programs on journalists' reporting of HIV/AIDS related information (outcome evaluation).

Design

Descriptive study.

Methods

For the process evaluation, each J2J program curriculum was evaluated for accuracy and pertinence by individuals with high familiarity with HIV/AIDS research. For the outcome evaluation, a survey of J2J attendees and evaluations of the program lectures by attendees were performed in chronological order to determine their perception on usefulness of the program.

Results

Overall, the J2J curriculum is successful in providing journalists with a clear understanding of the current HIV/AIDS medical research objectives and issues with most journalists reporting an increased ability to better investigate and disseminate accurate information on this subject. Furthermore, the journalists surveyed reported positive community responses directly as a result of the J2J training.

Conclusion

The J2J program helps to increase global awareness of pertinent HIV/AIDS concepts. Through this professional development strategy, journalists from around the world may help to amplify efforts to prevent new HIV infections and quench the dissemination of inaccurate information and folklore.
HIV/AIDS, conflict and security in Africa: rethinking relationships
Authors: Becker Joseph U , Theodosis Christian , Kulkarni Rick
Published: September 2008

The effect of conflict on HIV transmission and regional and global security has been the subject of much recent discussion and debate. Many long held assumptions regarding these relationships are being reconsidered. Conflict has long been assumed to contribute significantly to the spread of HIV infection. However, new research is casting doubt on this assumption. Studies from Africa suggest that conflict does not necessarily predispose to HIV transmission and indeed, there is evidence to suggest that recovery in the “post‐conflict” state is potentially dangerous from the standpoint of HIV transmission. As well, refugee populations have been previously considered as highly infected vectors of HIV transmission. But in light of new investigation this belief is also being reconsidered. There has additionally been concern that high rates of HIV infection among many of the militaries of sub‐Saharan Africa poses a threat to regional security. However, data is lacking on both dramatically elevated prevalence amongst soldiers and a possible negative effect on regional security. Nevertheless, HIV/AIDS remain a serious threat to population health and economic well being in this region. These issues are of vital importance for HIV programming and health sector development in conflict and “post‐conflict” societies and will constitute formidable challenges to the international community. Further research is required to better inform the discussion of HIV, conflict, and security in sub‐Saharan Africa.
Gastrin-Releasing Peptide Messenger Ribonucleic Acid Expression in the Hypothalamic Paraventricular Nucleus Is Altered by Melanocortin Receptor Stimulation and Food Deprivation
Authors: Ladenheim Ellen E. , Behles Robert R. , Bi Sheng , Moran Timothy H.
Published: September 2008

Gastrin-releasing peptide (GRP) is a bombesin-like peptide widely distributed in the gastrointestinal tract and central nervous system. In the brain, GRP mRNA is located in the hypothalamic paraventricular nucleus (PVN), a region that receives neural input from the arcuate nucleus and plays a critical role in food intake and energy balance. Because GRP neurons are localized in the vicinity of projection sites in the PVN for peptides that participate in energy homeostasis, we investigated whether GRP mRNA expression in the PVN may be sensitive to challenges imposed by either 38 h food deprivation or stimulation of the melanocortin system by the melanocortin 3/4 receptor agonist, melanotan II (MTII). We found that food deprivation significantly decreased GRP mRNA expression, whereas lateral ventricular MTII administration increased GRP mRNA expression in ad libitum-fed rats 4 h after administration. Furthermore, administration of MTII at a dose that reduces 24 h food intake and body weight prevented the decrease in GRP mRNA expression observed in animals that were pair fed to the amount of food consumed by those injected with MTII. These results demonstrate that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN, suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance.

Gastrin-releasing peptide gene expression in the hypothalamic paraventricular nucleus is responsive to signals related to energy balance.
A Select Combination of Clinically Relevant Phytoestrogens Enhances Estrogen Receptor β-Binding Selectivity and Neuroprotective Activities in Vitro and in Vivo
Authors: Zhao Liqin , Mao Zisu , Brinton Roberta Diaz
Published: September 2008

We have previously shown that a number of naturally occurring phytoestrogens and derivatives were effective to induce some measures of neuroprotective responses but at a much lower magnitude than those induced by the female gonadal estrogen 17β-estradiol. In the present study, we sought to investigate whether a combination of select phytoestrogens could enhance neural responses without affecting the reproductive system. We performed a range of comparative analyses of the estrogen receptor (ER) α/β binding profile, and in vitro to in vivo estrogenic activities in neural and uterine tissues induced by clinically relevant phytoestrogens: genistein, daidzein, equol, and IBSO03569, when used alone or in combination. Our analyses revealed that both the ERα/β binding profile and neural activities associated with individual phytoestrogens are modifiable when used in combination. Specifically, the combination of genistein plus daidzein plus equol resulted in the greatest binding selectivity for ERβ and an overall improved efficacy/safety profile when compared with single or other combined formulations, including: 1) an approximate 30% increase in ERβ-binding selectivity (83-fold over ERα); 2) a greater effect on neuronal survival against toxic insults in primary neurons; 3) an enhanced activity in promoting neural proactive defense mechanisms against neurodegeneration, including mitochondrial function and β-amyloid degradation; and 4) no effect on uterine growth. These observations suggest that select phytoestrogens in combination have the therapeutic potential of an alternative approach to conventional estrogen therapy for long-term safe use to reduce the increased risk of cognitive decline and neurodegenerative disease associated with menopause in women.

A combination of genistein, daidzein, and equol enhances estrogen receptor β-binding selectivity and estrogenic activities in promoting neuronal survival and brain defense mechanisms without impact on uterine growth.