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At least 1,621 records · Page 90Linked to original sources
PubMed2000

The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways.

Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions. Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components. The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation. Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole. In either case, the mechanism of vesicle formation is not known. Autophagic uptake displays a greater capacity for cytosolic protein sequestration. This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell. We have analyzed the biosynthesis of Aut7p, a protein required for both pathways. We find Aut7p expression is induced by nitrogen starvation. Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components. Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown. Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion. These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.

Autophagy-Related Protein 8 Family↗
PubMed2001

Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae.

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Autophagy↗
PubMed2003

Haematological characteristics predicting susceptibility for ascites. 1. High carbon dioxide tensions in juvenile chickens.

1. Male broilers of two different genetic stocks, a pure broiler sire line (A) and commercially available Ross broilers (B), were used to study the effect of haematological characteristics in juvenile chickens on the development of clinical ascitic signs. Production performance (body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR)) from 448 birds per stock was measured from 2 to 5 weeks of age. Mortality was recorded from 2 to 6 weeks of age. The birds were housed at a low ambient temperature to stimulate the incidence of ascites. 2. From each stock, 32 birds with the highest (high risk: HRc) and 32 birds with the lowest (low risk: LRc) carbon dioxide tensions (pCO2) in venous blood were selected at 11 d of age. These birds were marked for future blood sampling to determine changes in pCO2 with age to relate these values to ascites susceptibility. 3. At 2 weeks of age all birds (including HRc and LRc birds) were allotted to 32 floor pens (one HRc and one LRc in each pen) per stock. Venous blood samples were collected weekly from HRc and LRc birds for blood gas analysis and haematocrit, and at week 5 also for thyroid hormone (T3, T4) concentrations in plasma. At 5 weeks of age all HRc and LRc birds were examined post-mortem, relative heart, lung, and liver weights and arterial pressure index (API) values were recorded. 4. Birds from stock A showed a lower BWG and FCR and notably higher ascites mortality compared with stock B. An effect of pCO2 tensions at d 11 was found on the incidence of ascitic signs in selected birds of both stocks up to week 5. From the HRc groups 30% of the birds showed ascitic signs, whereas this was only 8% in the LRc group. LRc birds of stock B in particular showed constant low API values (20 +/- 3%) and none of these birds showed signs of ascites. 5. Our results suggest that the ascites problem in Ross birds can be eliminated by selection for low pCO2 tensions in venous blood. Stock effects on API, liver weight, lung weight, and plasma thyroid hormone independent of pCO2 showed a more complex picture of the ascitic signs in stock A compared with B. 6. We concluded that in this experiment a high pCO2 tension in venous blood measured at d 11 was a reliable predictor for ascites susceptibility observed at 5 weeks of age. A low pCO2 tension provides an appropriate criterion for genetic selection, whereas a high pCO2 tension emphasises the necessity for intensive management in poultry houses.

Animals↗
PubMedDate not supplied

Disability status differentials across fifteen Asian and Pacific Islander groups and the effect of nativity and duration of residence in the U.S.

This study examines disparities in disability status across 15 Asian and Pacific Islander American (API) subpopulations and how nativity and duration in the U.S. influence these differences. Employing three disability questions (work limitations, mobility limitations, and self-care limitations) from the 1990 PUMS, the authors find substantial heterogeneity in disability status across API subgroups: while Japanese American adults have the most favorable outcomes, Other Southeast Asian adults (Laotians, Hmong, and Cambodians), followed by Vietnamese and Pacific Islander adults, suffer from a high risk of disabilities. Many of the disparities in disability status across API subpopulation adults are attributable to differentials in demographic characteristics and SES. The inclusion of an interaction term of age and nativity/duration of residence in the U.S. in multivariate regression analyses demonstrates that the effect of nativity/duration plays a different role across age, net of demographic, and SES risk factors. The overall findings are also consistent with previous studies on the relationship between immigrant health and nativity/duration. That is, immigrants with short duration in the U.S. have superior health status, measured by risk of disability, than longer-term immigrants and their U.S.-born counterparts.

Activities of Daily Living↗
PubMed1979

Theoretic and experimental analysis of the ability of a commercially prepared system to detect mixtures of bacteria.

The validity of the biochemical identification of bacteria presumes that the inoculum consists of a pure growth of bacteria. The detection of mixtures of bacteria with the Analytab Enteric (API 20E) system was tested. Using a computer to predict the response of the API 20E system to mixed cultures, the presence of a mixture was not detected in 15% of all pairwise combinations of 324 common organisms of the Enterobacteriaceae family and in 19% of all pairwise combinations of Enterobacteriaceae with four gram-positive cocci. These theoretic predictions were confirmed experimentally for selected mixtures in which the bacteria were combined in equal concentrations. These results indicate that a purity subculture plate must be used to insure the detection of mixtures of bacteria, because the API 20E system fails to detect a large number of mixtures and may erroneously identify the organisms in such mixtures.

Bacteriological Techniques↗
PubMed1982

Comparison of two methods for same-day identification of Enterobacteriaceae.

Two commercial methods, API 20E (as modified for same-day enterobacterial identification) and Micro-ID, were evaluated for ability to provide useful same-day information of 368 clinically isolated Enterobacteriaceae. Organisms included Escherichia coli (54), Shigella (7), Edwardsiella tarda (1), Salmonella enteritidis (10), Citrobacter (30), Klebsiella (55), Enterobacter (68), Hafnia alvei (2), Serratia (33), Proteus (64), Morganella morganii (24), Providencia (18), and Yersinia enterocolitica (2). Methods were those of manufacturers without supplemental tests. API at five hours identified 78.5% of strains to species, 9.5% to genus only, 10.1% as part of a spectrum of identifications (SI), and 1.9% incorrect. Micro-ID at four hours yielded 90.0% correct identification to species and 3.3% to genus only, 4.0% SI, and 2.7% incorrect. API identification of many Serratia, Citrobacter, Providencia strains was to genus only; most incorrect results occurred in Serratia marcescens. Micro-ID identified most organisms to species; incorrect identifications were mainly S. marcescens and Klebsiella pneumoniae. Both systems provided excellent identification of E. coli. Both methods sacrifice a degree of accuracy that varies with the species tested, as compared to overnight systems, but both provide rapid information of potential clinical value.

Culture Media↗
PubMed1985

Microcomputer assisted identification of Bacillus species.

A microcomputer based system for the identification of unknown isolates of Bacillus species is described. The identification matrix includes 78 test probabilities for 38 recognised species and other groups in the genus Bacillus and it is based on the work of Logan and Berkeley (1984). Morphological characters together with the results of tests using API 20E and API 50CHB, read after 24 and 48 h incubation, are used to obtain a probabilistic identification of an unknown aerobic endospore forming rod. Any differences between the observed and expected results for any identified organism are listed. Identification can be attempted on the basis of a limited set of test results, although this is rarely if ever done with this largely API based system, and if the unknown cannot be successfully identified then a set of additional tests can be selected which should permit identification. The computer system can store and recall test results entered for any isolate. This feature allows the accumulation of data on isolates which could be used to update the identification matrix in future taxonomic studies.

Bacillus↗
PubMed2003

GeneComber: combining outputs of gene prediction programs for improved results.

UNLABELLED: We recently demonstrated that combining the output from Genscan and HMMgene can provide increased accuracy of gene predictions. We have created a robust software system that runs algorithms previously described on DNA sequences and provides a public web interface to the system for use by the biological community worldwide. The GeneComber system performs ab initio gene prediction by first taking a user inputted DNA sequence and running Genscan and HMMgene. The outputs of Genscan and HMMgene are then integrated using the EUI, GI and EUI_frame algorithms. All results are then stored into a relational database management system (RDBMS) and can then be retrieved through a web interface. The web interface provides a unified view of the GeneComber predictions by graphically overlaying outputs from Genscan, HMMgene, EUI, GI and EUI_frame. Outputs can also be retrieved in general feature format (GFF) or FASTA format. The software is written in the Perl programming language and is both dependent on and interoperable with the Bioperl toolkit. It includes high-level application programming interfaces (APIs) to run Genscan, HMMgene and a database API to insert prediction results into an RDBMS. The APIs are assembled into the genecomber script which is executed by the web interface or can be run directly from the Unix command line. The web interface is written in PHP and is structured so as to be easily modified for viewing data from any database that stores gene structures. AVAILABILITY: The GeneComber public web interface and supplementary information is located at http://bioinformatics.ubc.ca/genecomber The source code is released under the GNU General Public License and is available at ftp://ftp.bioinformatics.ubc.ca/pub/genecomber/software.

Algorithms↗
PubMed2005

caBIONet--A .NET wrapper to access and process genomic data stored at the National Cancer Institute's Center for Bioinformatics databases.

MOTIVATION: The National Cancer Institute's Center for Bioinformatics (NCICB) has developed a Java based data management and information system called caCORE. One component of this software suite is the object oriented API (caBIO) used to access the rich biological datasets collected at the NCI. This API can access the data using native Java classes, SOAP requests or HTTP calls. Non-Java based clients wanting to use this API have to use the SOAP or HTTP interfaces with the data being returned from the NCI servers as an XML data stream. Although the XML can be read and manipulated using DOM or SAX parsers, one loses the convenience and usability of an object oriented programming paradigm. caBIONet is a set of .NET wrapper classes (managers, genes, chromosomes, sequences, etc.) capable of serializing the XML data stream into local .NET objects. The software is able to search NCICB databases and provide local objects representing the data that can be manipulated and used by other .NET programs. The software was written in C# and compiled as a .NET DLL.

Chromosome Mapping↗
PubMed1993

Promotion of preneoplastic lesions and induction of CYP2B by unleaded gasoline vapor in female B6C3F1 mouse liver.

An initiation-promotion protocol was used to test the hypothesis that unleaded gasoline (UG) vapor acts as a liver tumor promoter in female mice under exposure conditions in which UG was hepatocarcinogenic in a cancer bioassay. Twelve day old female B6C3F1 mice were injected with N-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) or vehicle. Starting at 5-7 weeks of age, mice were exposed by inhalation 6 h/day, 5 days/week for 13 weeks to 0 or 2039 p.p.m. of PS-6 blend UG, the same gasoline blend used in the cancer bioassay. Putative preneoplastic lesions in liver, characterized mainly as basophilic foci in H&E-stained liver sections, were found exclusively in mice treated with DEN. While similar numbers of altered hepatic foci were found in DEN-initiated mice treated with 0 or 2039 p.p.m. UG, UG treatment significantly increased both the mean volume (3.2-fold) and the volume fraction (3.6-fold) of the foci. To determine if UG induced CYP2B, a subfamily of cytochrome P450 commonly induced by liver tumor promoters in rodents, pentoxyresorufin-O-dealkylase (PROD) activity was assayed in hepatic microsomes derived from the above livers. UG vapor increased hepatic PROD activity approximately 8-fold, while increasing cytochrome P450 content only approximately 30%. To ascertain if a more recent blend of UG, API 91-1, would have similar biological effects as PS-6, female B6C3F1 mice were gavaged for 3 days with corn oil or 1800 mg/kg/day PS-6 or API 91-1 blend UG. PS-6 and API 91-1 blend UG induced similar increases in relative liver weight (approximately 25%), PROD activity (approximately 9-fold) and hepatocyte labeling index (approximately 8-fold) relative to controls. These data demonstrate that PS-6 blend UG vapor promotes preneoplastic lesions and induces CYP2B in female mouse liver under exposure conditions in which it causes liver tumors, and suggest that a more recent blend of UG may have similar effects.

Animals↗
PubMed2001

Column robustness case study for a liquid chromatographic method validated in compliance with ICH, VICH, and GMP guidelines.

This article presents a case study in dealing with robustness investigations and attempts by our analytical laboratory to address these issues without sacrificing valuable time in revamping the method validation prior to submission. A liquid chromatographic method is developed for the analysis of a novel triazinetrione anticoccidial product. The method effectively separates the active pharmaceutical ingredient (API), impurities, and preservatives in the API and product formulation. For much of the validation, the method holds up to the rigorous guidelines of the International Conference of Harmonization, the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products, and the Good Manufacturing Practices. However, in analyzing a base-degraded sample one of the impurity peaks yields inconsistent retention times (RTs) during a series of injections. When switching the system to another analytical column from the same supplier, this impurity peak elutes at a different retention window and the remaining peaks in the chromatographic profile remain essentially the same. This RT variation of a single peak in the chromatographic profile is observed with additional columns from the same supplier and from different manufacturing lots. This suitability problem is not encountered with the columns used in the method development stage. The method no longer meets the robustness criteria established for pharmaceutical methods. An investigation is commenced and it is discovered that with the addition of tetrabutylammonium hydroxide to the mobile phases, the impurity peak gives a consistent RT in relation to the active peak. The peak shows comparable RTs relative to that of the API peak with columns of different silica lots and bond lots. All peaks, including the aforementioned impurity peak, are well-resolved under the revised high-performance liquid chromatographic conditions. This temporary solution enables continued submission work for FDA, but the robustness of this method is still a concern. After further investigation, it is determined that inhomogeneity of the active sites on the column's stationary phase is the likely culprit. Fortunately, a new column is found to be more suitable for this method and a column qualification study is initiated.

Journal Article↗
PubMed2006

Challenges in the analytical method development and validation for an unstable active pharmaceutical ingredient.

A sensitive high-performance liquid chromatography (HPLC) impurity profile method for the antibiotic ertapenem is developed and subsequently validated. The method utilizes an Inertsil phenyl column at ambient temperature, gradient elution with aqueous sodium phosphate buffer at pH 8, and acetonitrile as the mobile phase. The linearity, method precision, method ruggedness, limit of quantitation, and limit of detection of the impurity profile HPLC method are found to be satisfactory. The method is determined to be specific, as judged by resolving ertapenem from in-process impurities in crude samples and degradation products that arise from solid state thermal and light stress, acid, base, and oxidative stressed solutions. In addition, evidence is obtained by photodiode array detection studies that no degradate or impurity having a different UV spectrum coeluted with the major component in stressed or unstressed samples. The challenges during the development and validation of the method are discussed. The difficulties of analyzing an unstable active pharmaceutical ingredient (API) are addressed. Several major impurities/degradates of the API have very different UV response factors from the API. These impurities/degradates are synthesized or prepared by controlled degradation and the relative response factors are determined.

Chromatography, High Pressure Liquid↗
PubMed1998

Perceptions of dementia, caregiving, and help seeking among Asian and Pacific Islander Americans.

The United States is becoming more ethnically diverse, and Asian and Pacific Islanders (APIs) are one of the most rapidly growing ethnic minority groups. Of interest to social workers is how the traditional values and practices of these cultures affect their perceptions of disease and use of health services. This article presents information on perceptions of dementia, caregiving, and help seeking for the API group as a whole, with additional information on five of the major API subgroups--Chinese, Japanese, Filipino, Vietnamese, and Hawaiian. Implications for practice, policy, and research are discussed.

Attitude to Health↗
PubMed1999

On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.

Allergens↗
PubMed2002

Contribution of an imidazole-indole stack to high catalytic potency of a lysine-specific serine protease, Achromobacter protease I.

Achromobacter protease I (API), a lysine-specific serine-protease of the trypsin family, has an aromatic-ring stacking Trp 169-His 210 in close proximity to the reactive site. In order to investigate the role of this novel aromatic stacking, several mutants of the two residues were constructed and their kinetic parameters were determined. Three His 210 mutants showed lower activity by one order of magnitude than the wild-type with a peptide substrate of Ala-Ala-Lys-MCA (4-methylcoumaryl-7-amide), but 30-170% activity towards Val-Leu-Lys-MCA, suggesting that His 210 plays a role in keeping high activity toward various substrates by maintaining the active form of the substrate-binding subsite. Kinetic results of eight Trp 169 variants showed a roughly linear relation between k(cat) or K(m) values and the surface area at residue 169. With increasing size of the side-chain, k(cat) values increased, while K(m) values decreased. A systematic kinetic analysis of the activities of Trp 169 mutants toward Lys-MCA, Ala-Lys-MCA, and Ala-Ala-Lys-MCA peptide substrates revealed that large side-chain, rather than aromaticity, plays an important role in retaining the high catalytic activity of API. Due to the presence of the aromatic stacking, API shows one order of magnitude higher activity than bovine trypsin.

Alcaligenes↗
PubMed1996

Characterization of the S1 subsite specificity of aspergillopepsin I by site-directed mutagenesis.

The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1'. However, API accommodates a Lys residue at P1, which leads to activation of trypsinogen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and delta S78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position.

Amino Acid Sequence↗
PubMed1990

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells.

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.

Amino Acid Sequence↗
PubMed1989

The CO-I and CO-II region of honeybee mitochondrial DNA: evidence for variation in insect mitochondrial evolutionary rates.

The sequence of a region of honeybee (Apis mellifera ligustica) mitochondrial DNA, which contains the genes for cytochrome c oxidase subunits I and II (CO-I and CO-II) and inferred genes for tRNA(Asp), tRNA(Leu)UUR, tRNA(Lys), and tRNA(Trp), is presented. The region includes the segment previously identified as incurring a length increase in some other bee strains, including Africanized bees. The sequence information of this study and of that by Vlasak et al. shows that several shifts of tRNA genes have occurred between Apis and Drosophila, but shifts of other kinds of genes have yet to be demonstrated. The CO-I and CO-II gene sequences are both more A+T rich than are the corresponding Drosophila genes. Parsimony analyses using the mouse and Xenopus sequences as outgroups show significantly more amino acid substitutions on the branch to Apis (120) than on that to Drosophila (44), indicating a difference in the long-term evolutionary rates of hymenopteran and dipteran mtDNA.

Amino Acid Sequence↗