bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Human Papillomavirus Type 16 E7 Phosphorylation by the S100 MRP-8/14 Protein Complex
Authors: Tugizov Sharof , Berline Jennifer , Herrera Rossana , Penaranda Maria Elena , Nakagawa Mayumi , Palefsky Joel
Published: December 2004

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G
1
phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.
Roles of Carboxyl-Terminal and Farnesylated Residues in the Functions of the Large Hepatitis Delta Antigen
Authors: O'Malley Brendan , Lazinski David W.
Published: December 2004

The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.
Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance
Authors: Chen Hongying , Gill Andrew , Dove Brian K. , Emmett Stevan R. , Kemp C. Fred , Ritchie Mark A. , Dee Michael , Hiscox Julian A.
Published: December 2004

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.
Loss and Gain of Elicitor Function of
Soybean Mosaic Virus
G7 Provoking
Rsv1
-Mediated Lethal Systemic Hypersensitive Response Maps to P3
Authors: Hajimorad M. R. , Eggenberger A. L. , Hill J. H.
Published: December 2004

Rsv1
, a single dominant resistance gene in soybean PI 96983 (
Rsv1
), confers extreme resistance against all known American strains of
Soybean mosaic virus
(SMV), except G7 and G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. To identify the elicitor of
Rsv1
-mediated LSHR, chimeras were constructed by exchanging fragments between the molecularly cloned SMV-G7 (pSMV-G7) and SMV-G7d (pSMV-G7d), and their elicitor functions were assessed on PI 96983 (
Rsv1
). pSMV-G7-derived chimeras containing only P3 of SMV-G7d lost the elicitor function, while the reciprocal chimera of pSMV-G7d gained the function. The P3 regions of the two viruses differ by six nucleotides, of which two are translationally silent. The four amino acid differences are located at positions 823, 915, 953, and 1112 of the precursor polypeptide. Analyses of the site-directed point mutants of both the viruses revealed that nucleotide substitutions leading to translationally silent mutations as well as reciprocal amino acid substitution at position 915 did not influence the loss or gain of the elicitor function. pSMV-G7-derived mutants with amino acid substitutions at any of the other three positions lost the ability to provoke LSHR but induced SHR instead. Two concomitant amino acid substitutions at positions 823 (V to M) and 953 (K to E) abolished pSMV-G7 elicitor function, provoking
Rsv1
-mediated SHR. Conversely, pSMV-G7d gained the elicitor function of
Rsv1
-mediated LSHR by a single amino acid substitution at position 823 (M to V), and mutants with amino acid substitutions at position 953 or 1112 induced SHR instead of mosaic. Taken together, the data suggest that strain-specific P3 of SMV is the elicitor of
Rsv1
-mediated LSHR.
Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function
Authors: Davido David J. , von Zagorski William F. , Lane William S. , Schaffer Priscilla A.
Published: December 2004

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol.
76:
1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.
Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp
Authors: Cevallos Randal C. , Sarnow Peter
Published: December 2004

The Taura syndrome virus (TSV), a member of the
Dicistroviridae
family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNA
met
. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.
A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases
Authors: Seybert Anja , Posthuma Clara C. , van Dinten Leonie C. , Snijder Eric J. , Gorbalenya Alexander E. , Ziebuhr John
Published: December 2004

Nidoviruses (
Coronaviridae
,
Arteriviridae
, and
Roniviridae
) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn
2+
is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in
trans
by wild-type ZBD, suggesting a critical function of the ZBD in
cis
. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.
In Vitro and In Vivo Relevance of Infectious Laryngotracheitis Virus gJ Proteins That Are Expressed from Spliced and Nonspliced mRNAs
Authors: Fuchs Walter , Wiesner Dorothee , Veits Jutta , Teifke Jens P. , Mettenleiter Thomas C.
Published: December 2004

The positional homologue in the infectious laryngotracheitis virus (ILTV) genome of the glycoprotein gJ gene of herpes simplex virus and the gp2 gene of equine herpesvirus 1 is expressed into four proteins of 85, 115, 160, and 200 kDa (J. Veits, B. Köllner, J. P. Teifke, H. Granzow, T. C. Mettenleiter, and W. Fuchs, Avian Dis.
47:
330-342, 2003). RNA analyses revealed that these proteins are expressed from two different late (γ2) transcripts, an unspliced 5.5-kb and a spliced 4.3-kb mRNA that are translated into proteins of 985 and 611 amino acids, respectively. ILTV gJ is incorporated into virions and is modified by N- and O-linked glycosylation. After cotransfection of chicken cells with genomic DNA of a pathogenic ILTV strain and transfer plasmids, gJ-negative ILTV mutants could be isolated. In vitro growth studies demonstrated that deletion of the gJ gene has only minor effects on direct cell-to-cell spread as measured by plaque size. However, progeny virus titers of ILTV-ΔgJ were significantly reduced in comparison to those of the parental virus and a gJ rescue mutant. After experimental infection of chickens the gJ rescue mutant, like wild-type ILTV, caused severe disease and considerable mortality, whereas ILTV-ΔgJ was significantly attenuated. All immunized animals were protected against subsequent challenge infection with virulent ILTV. In sera collected after immunization with the gJ-rescue mutant or with wild-type ILTV, gJ-specific antibodies were detectable by immunofluorescence on cells that had been transfected with a gJ expression plasmid. As expected, no gJ-specific antibodies were found in sera obtained from chickens immunized with ILTV-ΔgJ. Thus, gJ deletion mutants of ILTV might be usable as attenuated live-virus vaccines. Furthermore, the gJ gene might constitute a reliable marker for serological discrimination between vaccinated and field virus-infected chickens.
Studies of Ebola Virus Glycoprotein-Mediated Entry and Fusion by Using Pseudotyped Human Immunodeficiency Virus Type 1 Virions: Involvement of Cytoskeletal Proteins and Enhancement by Tumor Necrosis Factor Alpha
Authors: Yonezawa Akihito , Cavrois Marielle , Greene Warner C.
Published: December 2004

The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by β-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.
Transformation of Madin-Darby Canine Kidney Epithelial Cells by Sheep Retrovirus Envelope Proteins
Authors: Liu Shan-Lu , Miller A. Dusty
Published: December 2004

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) induce epithelial tumors in the airways of sheep and goats. In both of these simple retroviruses, the envelope (Env) protein is the active oncogene. Furthermore, JSRV Env can transform cultured cells by two distinct mechanisms. In rat and mouse fibroblasts, the cytoplasmic tail of JSRV Env is essential for transformation, which involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the virus receptor hyaluronidase 2 (Hyal2) is not involved. In contrast, in the BEAS-2B human bronchial epithelial cell line, transformation is mediated by JSRV Env binding to Hyal2 followed by Hyal2 degradation and activation of the receptor tyrosine kinase RON, the activity of which is normally suppressed by Hyal2. Here we show that JSRV and ENTV Env proteins can also transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism similar to that observed in fibroblast cell lines. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 appears uninvolved. These results show that the JSRV and ENTV Env proteins can transform epithelial cells besides BEAS-2B cells and argue against a model for Env transformation involving different pathways that are uniquely active in fibroblasts or epithelial cells.
Biochemical Fingerprints of Prion Infection: Accumulations of Aberrant Full-Length and N-Terminally Truncated PrP Species Are Common Features in Mouse Prion Disease
Authors: Pan Tao , Wong Poki , Chang Binggong , Li Chaoyang , Li Ruliang , Kang Shin-Chung , Wisniewski Thomas , Sy Man-Sun
Published: December 2004

Infection with any one of three strains of mouse scrapie prion (PrP
Sc
), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrP
Sc
strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrP
Sc
are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrP
Sc
aggregates, indicating that they are physically associated with PrP
Sc
. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrP
Sc
infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.
Development of a Rotavirus-Shedding Model in Rhesus Macaques, Using a Homologous Wild-Type Rotavirus of a New P Genotype
Authors: McNeal Monica M. , Sestak Karol , Choi Anthony H.-C. , Basu Mitali , Cole Michael J. , Aye Pyone P. , Bohm Rudolf P. , Ward Richard L.
Published: December 2004

Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.
Novel Adeno-Associated Virus Vector Vaccine Restricts Replication of Simian Immunodeficiency Virus in Macaques
Authors: Johnson Philip R. , Schnepp Bruce C. , Connell Mary J. , Rohne Daniela , Robinson Suzanne , Krivulka Georgia R. , Lord Carol I. , Zinn Rebekah , Montefiori David C. , Letvin Norman L. , Clark K. Reed
Published: December 2004

Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.
Assessing investment in manual handling risk controls: a scoring system for use in observational studies: Table 1
Authors: Smedley J , Poole J , Waclawski E , Stevens A , Harrison J , Buckle P , Coggon D
Published: December 2004

Most UK hospitals now have manual handling policies, but few studies have assessed their impact. To facilitate such research, a system for ranking the investment in manual handling risk controls was devised and applied to 109 acute hospitals in the UK. High scoring hospitals performed well on all aspects of manual handling risk management. Low scoring hospitals had a manual handling policy and recorded accidents and sickness absence, but had limited resource for expert manpower and equipment.
Microarray analysis for identification ofPlasmodium-refractoriness candidate genes in mosquitoes
Authors: Chen Haifeng , Wang Jianxin , Liang Ping , Karsay-Klein Monica , James Anthony A , Brazeau Daniel , Yan Guiyun
Published: December 2004

The identification and cloning of genes conferring mosquito refractoriness to the malaria parasite is critical for understanding malaria transmission mechanisms and holds great promise for developing novel approaches to malaria control. The mosquito midgut is the first major site of interaction between the parasite and the mosquito. Failure of the parasite to negotiate this environment can be a barrier for development and is likely the main cause of mosquito refractoriness. This paper reports a study on Aedes aegypti midgut expressed sequence tag (EST) identification and the determination of genes differentially expressed in mosquito populations susceptible and refractory to the avian malaria parasite Plasmodium gallinaceum. We sequenced a total of 1200 cDNA clones and obtained 1183 high-quality mosquito midgut ESTs that were computationally collapsed into 105 contigs and 251 singlets. All 1200 midgut cDNA clones, together with an additional 102 genetically or physically mapped Ae. aegypti clones, were spotted on single arrays with 12 replicates. Of those interrogated microarray elements, 28 (2.3%) were differentially expressed between the susceptible and refractory mosquito populations. Twenty-seven elements showed at least a two-fold increase in expression in the susceptible population level relative to the refractory population and one clone showed reduced expression. Sequence analysis of these differentially expressed genes revealed that 10 showed no significant similarity to any known genes, 6 clones had matches with unannotated genes of Anopheles gambiae, and 12 clones exhibited significant similarity to known genes. Real-time quantitative RT-PCR of selected clones confirmed the mRNA expression profiles from the microarray analysis.Key words: microarray, vector competence, real-time PCR, EST.
Polymorphism and differentiation of rainbow trout Y chromosomes
Authors: Felip Alicia , Fujiwara Atushi , Young William P , Wheeler Paul A , Noakes Marc , Phillips Ruth B , Thorgaard Gary H
Published: December 2004

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.Key words: sex chromosomes, sex markers, cytogenetics, rainbow trout, fish.
Hypervariable plastid locus variation and intron evolution in theAnacamptis palustrislineage
Authors: Cozzolino Salvatore , Cafasso Donata , Pellegrino Giuseppe , Musacchio Aldo , Widmer Alex
Published: December 2004

Data on the organization of a hypervariable chloroplast locus in the Anacamptis palustris (Orchidaceae) lineage are provided and used to infer the pattern of molecular evolution in this group. A large survey of sequence variation in A. palustris and allied taxa reveals several repeat types differing in number and organization that occur in the same plastid region. The resulting repeat type network suggests that at least seven different minisatellite loci evolved near each other in the tRNALEUintron and indicates the presence of at least three main phyletic lines in the A. palustris lineage.Key words: Anacamptis palustris, intron evolution, plastid DNA, tandem repeat, VNTRs.
Genetic relationship among cultivated and wild grapevine accessions from Tunisia
Authors: Snoussi H , Slimane M Harbi Ben , Ruiz-García L , Martínez-Zapater J M , Arroyo-García R
Published: December 2004

We have used nuclear and chloroplast molecular markers to genotype cultivated and wild accessions of Vitis vinifera L. from Tunisia and assess their genetic relationships. Fifty-five distinct genotypes were identified among 80 cultivated accessions, including 18 genotypic groups containing between 2 and 5 accessions per group. They could represent a total of 60 distinct cultivars owing to berry colour variation found within identical genotype groups. Most of the 55 genotypes represent unique table grape genotypes except for one of them that was found identical to the genotype of table grape cultivar Rosseti. Hybridization among cultivars as well as self pollinations seems to have played an important role in their origin since several groups of closely related cultivars were observed. Furthermore, a parentage analysis showed a high probability for a parent hybrid relationship within two groups of three cultivars. No strong genetic similarities were found between cultivated and wild samples indicating that the cultivated accessions do not derive from local Vitis vinifera L. populations but could have been introduced from other regions in historic times.Key words: Vitis vinifera, Tunisian grapevine varieties, wild accessions, table grapes.
AFLP targeting of the 1-cM region conferring thevrs1gene for six-rowed spike in barley,Hordeum vulgareL.
Authors: He Congfen , Sayed-Tabatabaei Badraldin Ebrahim , Komatsuda Takao
Published: December 2004

Spike morphology is a key characteristic in the study of barley domestication, yield, and use. Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley. We developed five amplified fragment length polymorphism (AFLP) markers tightly linked to the vrs1 locus using well-characterized near-isogenic lines as plant materials. The AFLP markers were integrated into three different maps, in which 'Azumamugi' was used as the maternal parent. Of the three maps, Hordeum vulgare L. 'Azumamugi' × H. vulgare 'Golden Promise' showed recombination of the AFLP markers and the vrs1 locus (closest, 0.05 cM), providing the best mapping population for positional cloning of alleles at the vrs1 locus. Conversion of AFLP bands into polymorphic sequence-tagged sites (STSs) is necessary for further high-throughput genotype scoring and for bacterial artificial chromosome (BAC) library screening. We cloned and sequenced the five AFLP bands and synthesized primer pairs. PCR amplification generated DNAs of the same size from all four parental lines for each marker. Restriction endonuclease treatment of e40m36-1110/AccIII, e34m13-260/Psp1406I, e52m32-270/FokI, and e31m26-520/MnlI revealed fragment length polymorphisms between 'Azumamugi' and all the two-rowed parents. Allelism between the AFLPs and corresponding STS markers was confirmed genetically, indicating the usefulness of the STSs as genetic markers.Key words: positional cloning, codominance, near-isogenic lines, high-resolution maps, STSs.
One hundred fifty-four genetic markers for the turkey (Meleagris gallopavo)
Authors: Knutson Todd P , Chaves Lee D , Hall Majken K , Reed Kent M
Published: December 2004

Identifying and selectively breeding for improved traits is one of the ultimate goals of genetic research in agriculturally important species. Genome characterization and analysis are important first steps in this process. Genetic linkage maps based on the linear order of polymorphic DNA markers are typically developed through statistical analysis of inheritance patterns in pedigreed families. To develop microsatellite markers for further improvement of the turkey genetic linkage map, small-insert genomic libraries were screened for tandem repeats. Oligonuclotide primers were designed to amplify 164 microsatellite-containing fragments from genomic DNA. Genetic polymorphisms at 154 markers were determined by genotyping the F1 individuals of two resource populations. Markers determined as segregating in the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) reference population were used to genotype F2 individuals and a two-point linkage analysis was performed.Key words: turkey, Meleagris gallopavo, microsatellites, genetic linkage.
Genetic mapping of three alleles at thePm3locus conferring powdery mildew resistance in common wheat (TriticumaestivumL.)
Authors: Huang Xiu-Qiang , Hsam Sai L.K , Mohler Volker , Röder Marion S , Zeller Friedrich J
Published: December 2004

A set of differential isolates of Blumeria graminis f.sp. tritici was used to identify 10 alleles at the Pm3 locus on the short arm of chromosome 1A. Three F3populations were used to map Pm3h in Abessi, Pm3i in line N324, and Pm3j alleles in GUS 122 relative to microsatellite markers. In total, 13 marker loci were mapped on chromosome 1AS and 1 marker on 1AL. The order of marker loci in the 3 mapping populations is consistent with previously published maps. All 3 alleles were mapped in the distal region of chromosome 1AS. The present study indicated that microsatellite markers are an ideal marker system for comparative mapping of alleles at the same gene locus in different mapping populations. The linkage distances of the closest microsatellite marker, Xgwm905–1A, to Pm3h, Pm3i, and Pm3j were 3.7 cM, 7.2 cM, and 1.2 cM, respectively. The microsatellite marker Xgwm905–1A cannot be used to distinguish between Pm3 alleles. The development of specific markers for individual Pm3 alleles is discussed on the basis of the recently cloned Pm3b allele.Key words: genetic mapping, marker-assisted selection, microsatellite markers, Pm3 locus, powdery mildew resistance, Triticum aestivum.
'fuzzy ProjectManager' — FRAMEWORK FOR SOFTWARE PROJECT MANAGEMENT USING FUZZY LOGIC
Authors: SIWANI IMRAN , CAPRETZ MIRIAM
Published: December 2004

Project Management is the application of knowledge, skills, tools and techniques to project activities in order to meet project requirements. The success of any project relies heavily on the initial estimation of all project parameters. The absence of reliable estimations leads to ineffective project planning, over- or under-commitment of resources and therefore an increased likelihood of a software project failure. Fuzzy Logic is a soft-computing technique used to effectively solve uncertainties due to imprecise inputs to generate linguistic or quantitative outputs. This paper presents a novel framework for project management incorporating fuzzy logic known as 'fuzzy ProjectManager'. Furthermore, this paper demonstrates the application of fuzzy logic as a feasible technique for improved estimation accuracy of all software project estimations to ensure higher software project success rates.
PRODUCTION MANAGEMENT — SIMPLE OR COMPLEX
Authors: HALEVI GIDEON
Published: December 2004

Theoretical production planning and scheduling is actually very simple task: The plant gets orders which defines the product, the quantity and delivery dates. The resources of the plants are known, the product bill of material is known and the task of production scheduling is to make sure that the orders will be ready on time, that's all.

It seems strange that in order to meet this simple task, over 100 complex production planning methods were proposed. Some of the outstanding ones are: PICS; MRP; ERP; GT; TOC; FMS; IMS; CIM; CE; JIT; Kanaban; TQM; Agent…, AGILE etc. Yet the search for "THE" method is carried on.

In this paper an attempt to analyze why production planning is regarded as a complex task, and why the search for "THE" production planning method is still an open topic for researchers. Furthermore, to demonstrate how introduction of flexibility will restore the simplicity of production planning.
CDC/AHA Workshop on Markers of Inflammation and Cardiovascular Disease
Authors: Biasucci Luigi M.
Published: December 2004

Inflammation is a recognized key component of acute coronary syndromes. Such pathogenetic achievement has led to the use of inflammatory cells and proteins as prognostic markers in these syndromes. A number of markers have been proposed, including proinflammatory cytokines such as interleukin-6, interleukin-1RA, and tumor necrosis factor-α, adhesion molecules such as intracellular adhesion molecule-1 and vascular adhesion molecule-1 and markers of cell activation. Although all are of scientific interest, the clinical use of these markers is limited by their high cost, low availability, and unfavorable biological profile. Conversely, common markers of inflammation such as C-reactive protein (CRP), the prototypic acute phase protein, and to a lesser extent fibrinogen, have been proven to be reliable and important markers of risk in ischemic heart disease. CRP, in particular, has been found to be associated with short- and long-term prognosis in acute coronary syndromes, including ST-elevation myocardial infarction, and in stable angina, and to predict the risk of restenosis and major events, including death, after revascularization procedures. CRP has been consistently found to be independent from other risk factors and to have an incremental value beyond the common risk factors and biochemical markers of risk, including troponin. Whether CRP also should be used as a guide to therapy is still a matter of discussion that deserves further, properly designed studies.
Predictors of Quality-of-Life Benefit After Percutaneous Coronary Intervention
Authors: Spertus John A. , Salisbury Adam C. , Jones Philip G. , Conaway Darcy Green , Thompson Randall C.
Published: December 2004

Background—

Improving patients’ quality of life is a primary indication for percutaneous coronary intervention (PCI), yet little is known about patient characteristics associated with greater quality-of-life improvement from the procedure. This study was conducted to identify patient characteristics associated with quality-of-life benefit after PCI.

Methods and Results—

A consecutive series of 1518 patients undergoing PCI in nonacute myocardial infarction settings were prospectively enrolled into an observational study documenting their postprocedural health status. We examined univariate and multivariable associations between baseline patient characteristics and quality of life 1 year after the procedure using the disease-specific Seattle Angina Questionnaire (SAQ) to quantify the impact of patients’ coronary disease on their quality of life. Baseline angina frequency and physical function were the strongest predictors of quality-of-life improvement 1 year after PCI. In comparing patients without angina to those experiencing monthly, weekly, and daily angina, the quality-of-life improvements (mean±SEM) were 21.4±2.1, 30.7±2.2, and 34.6±2.6 points greater (
P
<0.001). Patients with mild, moderate, and severe physical limitation improved 13.8±1.9, 20.0±2.1, and 13.5±3.5 points more than those with minimal baseline physical limitation (
P
<0.001). These findings were maintained in multivariable models correcting for baseline differences in demographic, clinical, disease-severity, and health-status variables.

Conclusions—

Preprocedural angina frequency is the most important prognostic indicator of quality-of-life improvement after PCI. Although substantial quality-of-life benefits are attained in most patients with preprocedural angina, more careful consideration of the potential benefits and risks of the procedure are needed in asymptomatic patients.
Both Cell Fusion and Transdifferentiation Account for the Transformation of Human Peripheral Blood CD34-Positive Cells Into Cardiomyocytes In Vivo
Authors: Zhang Sui , Wang Dachun , Estrov Zeev , Raj Sean , Willerson James T. , Yeh Edward T.H.
Published: December 2004

Background—

Adult human peripheral blood CD34-positive (CD34
+
) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients.

Methods and Results—

We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34
+
cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34
+
cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA
+
and troponin T
+
or Nkx2.5
+
cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA
−
, troponin T
+
cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34
+
cells contained human X chromosome only.

Conclusions—

Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34
+
cells into cardiomyocytes in vivo.
Transgenic Mouse Model of Ventricular Preexcitation and Atrioventricular Reentrant Tachycardia Induced by an AMP-Activated Protein Kinase Loss-of-Function Mutation Responsible for Wolff-Parkinson-White Syndrome
Authors: Sidhu Jasvinder S. , Rajawat Yadavendra S. , Rami Tapan G. , Gollob Michael H. , Wang Zhinong , Yuan Ruiyong , Marian A.J. , DeMayo Francesco J. , Weilbacher Donald , Taffet George E. , Davies Joanna K. , Carling David , Khoury Dirar S. , Roberts Robert
Published: December 2004

Background—

We identified a gene (
PRKAG2
) that encodes the γ-2 regulatory subunit of AMP-activated protein kinase (AMPK) with a mutation (Arg302Gln) responsible for familial Wolff-Parkinson-White (WPW) syndrome. The human phenotype consists of ventricular preexcitation, conduction abnormalities, and cardiac hypertrophy.

Methods and Results—

To elucidate the molecular basis for the phenotype, transgenic mice were generated by cardiac-restricted expression of the wild-type (TG
WT
) and mutant(TG
R302Q
)
PRKAG2
gene with the cardiac-specific promoter α-myosin heavy chain. ECG recordings and intracardiac electrophysiology studies demonstrated the TG
R302Q
mice to have ventricular preexcitation (PR interval 10±2 versus 33±5 ms in TG
WT
,
P
<0.05) and a prolonged QRS (20±5 versus 10±1 ms in TG
WT
,
P
<0.05). A distinct AV accessory pathway was confirmed by electrical and pharmacological stimulation and substantiated by induction of orthodromic AV reentrant tachycardia. Enzymatic activity of AMPK in the mutant heart was significantly reduced (0.009±0.003 versus 0.025±0.001 nmol · min
−1
· g
−1
in nontransgenic mice), presumably owing to the mutation disrupting the AMP binding site. Excessive cardiac glycogen was observed. Hypertrophy was confirmed by increases in heart weight (296 versus 140 mg in TG
WT
) and ventricular wall thickness.

Conclusions—

We have developed a genetic animal model of WPW that expresses a mutation responsible for a familial form of WPW syndrome with a phenotype identical to that of the human, including induction of supraventricular arrhythmia. The defect is due to loss of function of AMPK. Elucidation of the molecular basis should provide insight into development of the cardiac conduction system and accessory pathways.
Aldosteronism and a Proinflammatory Vascular Phenotype
Authors: Ahokas Robert A. , Sun Yao , Bhattacharya Syamal K. , Gerling Ivan C. , Weber Karl T.
Published: December 2004

Background—

Chronic, inappropriate (relative to dietary Na
+
) elevations in circulating aldosterone, such as occur in congestive heart failure, are accompanied by a proinflammatory vascular phenotype involving the coronary and systemic vasculature. An immunostimulatory state with activated peripheral blood mononuclear cells (PBMCs) precedes this phenotype and is induced by a fall in cytosolic free [Mg
2+
]
i
and subsequent Ca
2+
loading of these cells and transduced by oxidative/nitrosative stress.

Methods and Results—

We sought to further validate this hypothesis in rats with aldosterone/1%NaCl treatment (ALDOST) by using several interventions as cotreatment: a Mg
2+
-supplemented diet; amlodipine, a CCB; and
N
-acetylcysteine, an antioxidant. Blood samples were obtained at weeks 1 to 4 of ALDOST to monitor [Mg
2+
]
i
, [Ca
2+
]
I
, and H
2
O
2
production in PBMCs. Coronal ventricular sections were examined for invading inflammatory cells and 3-nitrotyrosine labeling, a marker of oxidative/nitrosative stress. In response to ALDOST and compared with untreated controls, we found an early and persistent reduction in [Mg
2+
]
i
with a subsequent rise in [Ca
2+
]
i
and H
2
O
2
production, each of which was either attenuated or abrogated by the Mg
2+
-supplemented diet and by
N
-acetylcysteine, whereas amlodipine prevented Ca
2+
loading and an altered redox state. Cotreatment with these interventions either markedly attenuated or prevented the appearance of the proinflammatory coronary vascular phenotype and the presence of 3-nitrotyrosine in invading inflammatory cells.

Conclusions—

We suggest that the immunostimulatory state that appears during aldosteronism and leads to a proinflammatory coronary vascular phenotype is induced by a fall in [Mg
2+
]
i
with Ca
2+
loading of PBMCs and is transduced by H
2
O
2
production in these cells.