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Results for “process mapping”

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At least 973 records · Page 54Linked to original sources
PubMed1993

Evaluation of product equivalence during process optimization for manufacture of a human IgM monoclonal antibody.

We have developed a battery of tests to characterize monoclonal antibodies and assess the effect of potential manufacturing process changes. Tryptic peptide mapping, molecular weight determination by HPLC and classical light scattering, isoelectric focussing, oligosaccharide mapping by HPAE-PAD chromatography, circular dichroism spectra and differential scanning calorimetry were applied as sensitive assays of antibody structure. Biological activity was assessed by measurement of specific antigen binding activity, binding spectrum and opsonic activity. Pharmacokinetics was assessed by clearance rate studies in rats. The sensitivity of this battery of assays was demonstrated by the ability to readily detect differences between a human monoclonal antibody (IgM-2) produced by in vitro cell culture versus ascites culture. These same tests support equivalence of a second monoclonal antibody (IgM-1) produced before and after in vitro cell culture process improvements which resulted in a twofold increase in product titer.

Animals↗
PubMed1986

Low temperature causes accumulation of unspliced fibroin mRNA precursor molecules in silkworm larvae.

Silk fibroin premessenger mRNA is a large (17 kb) molecule containing a single intron. Previous S1 mapping studies yielded evidence of processing cleavages at sites within the intron, in addition to the major cleavages at the intron/exon boundaries. We have performed S1 mapping experiments using RNA from animals which had been incubated at low temperature. These experiments show a marked accumulation of full-length fibroin mRNA precursor relative to controls, and a reduction in the abundance of molecules lacking portions of the intron. These results demonstrate a partial uncoupling of transcription and splicing in vivo.

Animals↗
PubMed1997

Gene trapping and functional genomics.

Classical genetics depends upon investigation of function by random destruction with little information on structure. Modern mapping using random polymorphisms, cloning and sequencing investigates structure without function. The genome projects with their rapid gene discovery are, however, redefining classical genetic approaches. The efficient translation of this wealth of new information into insights in biological function at molecular, cellular and organismal levels requires large-scale approaches to the generation of mutants. Gene trapping in embryonic stem (ES) cells allows an efficient approach to the functional analysis of the murine genome. The usually separate processes of gene discovery, mapping, the observation of the expression pattern and the mutant phenotype in vivo, can be integrated by the use of an indexed library of insertionally mutated ES cell clones. It should be possible to generate mutants for a large proportion of the genes of the mammalian genome.

Animals↗
PubMed2002

Mechanism of the process formation; podocytes vs. neurons.

In this review article we discuss the common mechanism for cellular process formation. Besides the podocyte, the mechanism of process formation, including cytoskeletal organization and signal transduction, etc., has been studied using neurons and glias as model systems. There has been an accumulation of data showing common cell biological features of the podocyte and the neuron: 1) Both cells possess long and short cell processes equipped with highly organized cytoskeletal systems; 2) Both show cytoskeletal segregation; microtubules (MTs) and intermediate filaments (IFs) in podocyte primary processes and in neurites, while actin filaments (AFs) are abundant in podocyte foot processes in neuronal synaptic regions; 3) In both cells, process formation is mechanically dependent on MTs, whose assembly is regulated by various microtubule- associated proteins (MAPs); 4) In both cells, process formation is positively regulated by PP2A, a Ser/Thr protein phosphatase; 5) In both cells, process formation is accelerated by laminin, an extracellular matrix protein. In addition, recent data from our and other laboratories have shown that podocyte processes share many features with neuronal dendrites: 1) Podocyte processes and neuronal dendrites possess MTs with mixed polarity, namely, plus-end-distal and minus-end-distal MTs coexist in these processes; 2) To establish the mixed polarity of MTs, both express CHO1/MKLP1, a kinesin-related motor protein, and when its expression is inhibited formation of both podocyte processes and neuronal dendrites is abolished; 3) The elongation of both podocyte processes and neuronal dendrites is supported by rab8-regulated basolateral-type membrane transport; 4) Both podocyte processes and neuronal dendrites express synaptopodin, an actin-associated protein, in a development-dependent manner; interestingly, in both cells, synaptopodin is localized not in the main shaft of processes but in thin short projections from the main shaft. We propose that the podocyte process and the neuronal dendrite share many features, while the neuronal axon should be thought of as an exceptionally differentiated cellular process.

Animals↗
PubMed2005

Suitability of the vegetation types in Mexico's Tamaulipas state for the siting of hazardous waste treatment plants.

A land suitability study was carried out by applying a multiple-criteria technique to 12 different vegetation types in Mexico's Tamaulipas state to help select potentially suitable sites for hazardous waste treatment plants. Species richness, spatial distribution, and uniqueness were selected as the criteria for estimating a vegetation type's suitability. Using the analytical hierarchy process, we ranked and mapped vegetation types, then compared the results with rankings of the same vegetation types based only on their number of endemic species. The suitabilities of the various vegetation types were ordered in more or less the same way by both methods, except in two cases for which the results were very different. The method proved to be a useful tool despite the availability of only partial (mostly qualitative) information; under such circumstances, expert experience can be incorporated in the evaluation process to a limited degree. The technique described in this paper has a high potential to aid decisions when many opinions and options must be considered simultaneously.

Ecosystem↗
PubMed1992

Silanized silica bound trypsin as analytical probe.

Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Stimulation of tubulin polymerization by MAP-2. Control by protein kinase C-mediated phosphorylation at specific sites in the microtubule-binding region.

Microtubule-associated protein-2 (MAP-2) is extensively phosphorylated on serine and threonine residues, and such modifications affect various cellular processes, including microtubule dynamics. Although MAP-2 phosphorylation has been studied both in vitro and in vivo, nothing is known about the exact location of phosphorylated sites influencing the strength of MAP-2 binding to microtubules. Because the microtubule-binding region (MTBR) retains virtually all of the binding affinity of intact MAP-2 for microtubules, we focused on understanding the effect of protein kinase C phosphorylation on MTBR binding to taxol-stabilized microtubules. We used bacterially expressed MAP-2 MTBR, containing newly introduced CNBr-cleavable methionyl residues, as well as mass spectrometry and site-specific mutagenesis to locate and confirm assignments of critical phosphorylation sites. We report on the localization and role of phosphoryls on two specific residues (Ser-1703 and Ser-1711) in terms of kinase-mediated control of MTBR-stimulated tubulin polymerization. Ser-1703 is situated in the so-called first inter-repeat, and Ser-1711 is located in the second nonidentical repeat. Upon more extensive protein kinase C action, microtubule binding can be abolished by phosphorylation at Ser-1728, but this effect is conditionally dependent on the phosphorylation state at Ser-1703 and/or Ser-1711. Our results suggest that in vivo binding of MAP-2 to microtubules may be controlled by protein kinase C action at these residues, and the strategy presented in this report may facilitate future studies with other kinases.

Amino Acid Sequence↗
PubMed2003

Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping.

Histone acetylation and methylation are processes that are generally considered to play crucial roles in the chromatin-based regulatory mechanism. Characterization of the histones as well as their modification sites has become increasingly important. In this paper, the use of LC-MS and peptide mapping methods to analyze chicken core histones and identify the modification sites is reported. Microbore C(4) HPLC separated the core histones into H2A, H2B, H3 and H4 using HFBA as the ion-pairing agent. The four subclasses of histones and their putative acetylated or methylated isoforms were identified by LC-MS simultaneously. MALDI-TOF and tandem mass spectrometry provided peptide mapping of the modification sites of the histones through trypsin digestion of the HPLC eluents. This approach is straightforward and prospective for further application in the field of chromatin research.

Chromatography, High Pressure Liquid↗
PubMed2003

The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification.

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.

Amino Acid Sequence↗
PubMed1996

The development of a data system for a combination of liquid chromatography or capillary electrophoresis with an ion trap storage/reflectron time-of-flight mass detector.

A data system based upon a 200 MHz transient recorder interface card in a Pentium PC computer is demonstrated for on-line analysis of microbore high-performance liquid chromatography (HPLC), capillary HPLC and capillary electrophoresis (CE) separations using a fast and sensitive ion-trap storage/reflectron time-of-flight mass spectrometric detector (IT-reTOFMS). Under the control of a user-written program, the system is capable of conducting the data acquisition and storage for a minimum of 30 min, at rates exceeding 10 Hz, of individual mass spectra containing 16,000 data points having 10 nsec resolution. The capability is mainly attributed to the use of a data reduction scheme in which only mass intensities higher than a preset threshold are saved as indexed flight-time/intensity pairs. This produces a typical reduction ratio of 30:1 in data set size, yielding faster storage with smaller file size, and permits the complete set of mass spectra to be held in the computer's memory. In addition, the data system is capable of displaying, for real-time evaluation of the analysis, each individual mass spectrum and the total-ion chromatogram. Further, the selected-ion chromatograms of given masses and a 3-dimensional topographic map describing a separation process can be rapidly generated from the collected data for the unambiguous and high fidelity identification of target analytes in a complex mixture.

Amino Acid Sequence↗
PubMed1999

Proteolytic processing in infectious bursal disease virus: identification of the polyprotein cleavage sites by site-directed mutagenesis.

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of an immune depressive disease that affects domesticated and wild avian species. The expression strategy of IBDV includes the synthesis of a 110-kDa polyprotein containing the capsid precursor polypeptides. The polyprotein is autocatalitically processed rendering three polypeptides: NH2-VPX-VP4-VP3-COOH. We have carried out a systematic analysis, using a series of plasmids encoding polyproteins containing either deletions or single amino acid substitutions, to identify the processing sites. The results obtained showed the existence of two sites, 511LAA513 and 754MAA756, that are essential for the processing of the VPX-VP4 and VP4-VP3 precursors, respectively. These sequences are highly conserved among IBDV strains form serotypes 1 and 2. A secondary VPX-VP4 processing site was detected in a 19-amino acid stretch located upstream of the 511LAA513 site. Analyses using versions of the 754MAA756 VP4-VP3 processing site containing conservative and nonconservative amino acid substitutions demonstrated that the specificity of the cleavage is dictated by the conserved AA dipeptide.

Amino Acid Sequence↗
PubMed1998

Cyclophosphamide cystitis as a model of visceral pain in rats: a c-fos and Krox-24 study at telencephalic levels, with a note on pituitary adenylate cyclase activating polypeptide (PACAP).

This article is the fifth of a series aimed at mapping brain activities as they result from the development of cyclophosphamide (CP) cystitis in behaving rats using c-fos and Krox-24 expression. The inactive hepatic metabolites of CP are metabolized in the kidney to produce acrolein, which generates cystitis. Data come from animals which were injected once i.p. with either 1 ml saline (sham) or 100 mg/kg CP in 1 ml saline under transient volatile anesthesia and which behaved freely for 1-4 h postinjection, 4 h being the minimum time for cystitis to completely develop. Survival times longer than 4 h were not studied owing to ethical considerations. The first 2 h postinjection cover a period of time over which inputs of multifactorial origin (stress and pain due to the intraperitoneal injection process, possible effects due to the presence of hepatic CP metabolites in blood, cystitis onset) interact in an indistinguishable way; the last 2 h are more cystitis specific as the other effects have vanished. Complete screening of telencephalic levels has been performed. These data complete previously published data at both spinal and subtelencephalic levels. Of all the telencephalic structures, only the bed nucleus of the stria terminalis in the dorsal part of its lateral division (BSTLd) and, to a lesser degree, the nucleus centralis of the amygdala, mostly in its caudal portion (cCeA), appeared to be significantly driven over the most specific cystitis period. Both of these structures had related, but not identical patterns of expression. They both reacted shortly after CP injection, but, while cCeA maintained its activity throughout cystitis development, BSTLd showed a rebound, reaching a peak value when cystitis was fully developed. Both of these areas are the only telencephalic areas to contain high PACAP38 immunoreactivity. This is evidence that, (1) both the BSTLd and cCeA could be the most rostral areas that visceronociceptive inflow would reach when cystitis genesis is under way, and (2) PACAP38 could be one of the neurochemical agents involved in telencephalic visceronociceptive processing. From our complete mapping of brain activities under a fully developed cystitis situation (4 h postinjection), it appears that the activities in BSTLd and cCeA are concomitant with those of both the dorsal vagal complex (DVC), paratrigeminal nucleus (PaT), and the ventrocaudal bulbar reticular formation (vcBRF) at brainstem levels, suggesting they all form the main part of the neural network that subserves the central processing of cystitis-related inputs, comprising pain and associated pseudoaffective responses. Both the DVC and BSTLd, which are the most powerfully driven areas, would be particularly important in such a way. The origin of these activities should be found in both vagal (as sensed through PaT activity) and spinal (pelvic) influences. This network profoundly differs from those reported for painful situations, either somatic or visceral, which controversally accompany positive cardiac inotropism.

Animals↗
PubMed2025

HINN: Hierarchical Input Neural Network identifies multi-omics biomarker for cognitive decline.

Understanding complex diseases requires models that can integrate diverse layers of biological data while yielding insights that are biologically interpretable. Although multi-omics integration with machine learning (ML) has advanced disease prediction and biomarker discovery, most existing approaches overlook the hierarchical and regulatory relationships that connect these molecular layers. Here, we present the Hierarchical Input Neural Network (HINN), a deep learning framework that incorporates known cross-omics relationships directly into its architecture, capturing the flow of information from genomics to epigenomics, transcriptomics, and downstream biological processes. By embedding these relationships, HINN improves both predictive performance and biological interpretability. We applied HINN to blood-derived multi-omics data from individuals with Alzheimer's disease or mild cognitive impairment to predict cognitive scores from standardized assessments. HINN outperformed both baseline and state-of-the-art models and pinpointed multi-omics biomarkers-including SNPs and promoter-region CpG sites in ATP6V1C1 and RCHY1 -that were significantly correlated with plasma p-Tau181 levels. These features map to biologically relevant processes with potential implications for cognitive decline. Our findings demonstrate how combining deep learning with biological knowledge can uncover interpretable, blood-based biomarkers for cognitive decline due to complex diseases such as Alzheimer's. All code and data are openly available at https://github.com/bozdaglab/HINN.

Alzheimer’s disease↗
PubMed2001

Comparative electrophysiological and hemodynamic measures of neural activation during memory-retrieval.

The spatial and temporal characteristics of the brain processes underlying memory retrieval were studied with both event-related potentials (ERP) and positron emission tomography (PET) techniques. Subjects studied lists of 20 words and then performed episodic (old/new judgment) or semantic (living/nonliving decision) retrieval tasks on multiple four-item test lists, each lasting 10 sec. The PET and ERP measurements at test were assessed in relation to both the task (episodic vs. semantic) and the item (old vs. new or living vs. nonliving). Episodic retrieval was associated with increased blood flow in the right frontal lobe (Brodmann Area 10) and a sustained, slowly developing positive ERP shift recorded from the right frontopolar scalp. Semantic retrieval was associated with increased blood flow in the left frontal (Area 45) and temporal (Area 21) lobes but no clear ERP concomitant. The two retrieval tasks also differed from each other in the ERPs to single items in an early (300-500 ms) time window. Item-related comparisons yielded convergent results mainly if the retrieved information was relevant to the given task (e.g., old/new items during episodic retrieval and living/nonliving items during semantic retrieval). Episodically retrieved old items were associated with increased blood flow in the left medial temporal lobe and a transient increase in the amplitude of the late positive component (500-700 ms) of the ERP. Semantically retrieved living items were associated with increased blood flow in the left frontal cortex and anterior cingulate and a transient late frontal slow wave (700-1,500 ms) in the ERPs. These results indicate that the brain regions engaged in memory retrieval are active in either a sustained or transient manner. They map task-related processes to sustained and item-related processes to transient neural activity. But they also suggest that task-related factors can transiently affect early stages of item processing.

Adult↗
PubMed1995

Molecular genetic analysis of the ripening-inhibitor and non-ripening loci of tomato: a first step in genetic map-based cloning of fruit ripening genes.

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato, ripening-inhibitor (rin), and non-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning the rin and nor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning the rin locus is estimated to be 200-300 kb/cM, while the nor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked to rin and nor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains the nor locus.

Base Sequence↗
PubMed2003

MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export.

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.

Calpain↗
PubMed2005

Beta-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin.

Using a yeast two-hybrid system, we screened a human brain cDNA library for possible interacting proteins with the C-terminal cytosolic tail of the beta-secretase beta-amyloid protein converting enzyme (BACE)1. This identified seven potential candidates, including the brain-specific type II membrane protein BRI3. Co-localization and co-immunoprecipitation experiments confirmed that BACE1 and BRI3 co-localize and interact with each other via the cytosolic tail of BACE1. Furthermore, pulse and pulse-chase analyses revealed that the pro-protein convertases furin, and to a lesser extent PC7 and PC5A, process BRI3 into a C-terminal secreted approximately 4-kDa product. Thus, furin efficiently processes both pro-BACE1 and its novel interacting protein pro-BRI3.

Amino Acid Sequence↗
PubMed2004

In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides.

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

Amino Acid Sequence↗