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PubMed1983

Visual problem solving in the absence of lexical semantics: evidence from dementia.

A case of dissociation between visual and praxic skills and linguistic, referential, and semantic skills is described in a case of dementia. Testing revealed that her visual processing was intact and that she was capable of a wide range of visually based tasks. These included copying geometric figures and block designs; matching items on the basis of number, color, or relative size, matching upper and lower case letters, sequencing numerals, orienting pictures, coins, and letters appropriately, and solving problems based on the identification of geometric patterns or on the identification of logical sequences based on pattern alternation, number, and size increases. In contrast, she was completely incapable of matching pictures on the basis of their semantic or referential meaning; similarly she had lost all comprehension of language. It was argued that this case demonstrates the capacities of a visual system cut off from all symbolic and semantic processes. When examined with other case studies this case provides information about the nature of and interaction between modality-specific perceptual processing and semantic processing, and information about the various cognitive factors involved in visual gnosis. The CT scan indicated frontal atrophy and marked anterior temporal atrophy, a pattern that is consistent with Pick's disease. The case was discussed in terms of the correlation between the pattern of observed strengths and deficits and the neurological pattern of atrophy.

Dementia↗
PubMed1988

A highly conserved mouse gene with a propensity to form pseudogenes in mammals.

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

Animals↗
PubMed2002

Redundancy gains and costs in cognitive processing: effects of short stimulus onset asynchronies.

In 7 experiments, the influence of varying stimulus onset asynchronies (SOAs) on the processing of redundant information about words and pseudowords was investigated. All stimuli were visually presented once or twice with 2 copies of the same item flashed either simultaneously or with short SOAs between presentations. The experiments revealed a redundancy gain for words that was absent for pseudowords. Furthermore, the redundancy gain disappeared at an SOA of 50 ms, and there was a gradual performance decline at longer SOAs. However, probing SOAs of 150 and 300 ms revealed that, compared with presentation of 1 target stimulus alone, words were processed significantly faster when target and redundantcopy appeared with a 150-ms lag. The results are tentatively explained in a neurocognitive framework.

Adult↗
PubMed1992

Write now: a guide to publishing.

1. With the elimination of time constraints, fear of rejection, and lack of understanding of the publication process, effective communication and sharing of experiences and expertise among nurses can be enhanced through writing for publication. 2. Important author responsibilities, such as being aware of the number of manuscript copies to be sent with the original, typing and reference format of the journal, letters of transmittal, and acknowledgements, can be found in the journal's guidelines. 3. Understanding the publication process and learning how to use the components can lead to positive results for prospective authors.

Nursing Research↗
PubMed2001

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Amino Acid Sequence↗
PubMed1992

Quantitative PCR with internal controls.

We examine the use of internal controls for estimating the expected initial copy number of the target in a polymerase chain reaction (PCR). We base our investigation on an extended branching-process model. In terms of that model, we delineate the necessary assumptions for this methodology to yield approximately unbiased answers, and we provide means for testing some of those assumptions. We show how to design a series of PCRs to attain optimal precision of the estimate. We provide an algorithm for conducting the statistical analysis of the data, including a formula for a confidence interval for the unknown expected initial copy number.

Algorithms↗
PubMed1983

The mechanics of assembling an international drug dossier.

An international registration dossier is a comprehensive scientific document used to obtain worldwide licensing approval of a drug by diverse health authorities. Its creation, processing, compilation, and dispatch to the field by a regulatory affairs department of a major, US-based, multinational pharmaceutical company is dependent upon many interrelated activities beginning years prior to its physical assembly. The following activities are briefly discussed to show the magnitude of the task and the type of activities encountered: planning and scheduling; creating, organizing, and formatting; typing; copying and collating; working with extramural suppliers and services; and finally, shipping the document to the field. Suggestions for making the process more efficient are offered.

Drug Information Services↗
PubMed1993

Somatic hypermutation of an immunoglobulin mu heavy chain transgene.

We have analyzed somatic hypermutation of an immunoglobulin (Ig) heavy chain transgene. Hybridomas expressing the transgene were produced from immunized transgenic mice and transgene copies were sequenced to assay for mutation. In two IgM-producing hybridomas, as well as in several IgG-producing hybridomas, mutations were found in the VDJ region of the transgene. In the IgM-producing hybridomas, both mutated and unmutated transgene copies were present and expressed as mRNA. Several mutated transgene copies were present in a single cell and these showed different patterns of mutation. Two IgG-producing hybridomas isolated from a single animal also showed a hierarchical pattern of mutation indicating that transgene mutations can accumulate during B cell proliferation, similar to the mutational process for endogenous antibody genes. Among hybridomas that expressed both IgG and IgM molecules derived from the transgene, the isotype-switched gamma transgene copy exhibited a higher level of mutation than the mu transgene copies. Our results indicate that the 15-kb ARSmu transgene contains all the sequence information required to target the Ig-specific hypermutational machinery, and raise the possibility that sequences associated with the endogenous CH locus might enhance somatic mutation.

Amino Acid Sequence↗
PubMed2006

Inducible Escherichia coli fermentation for increased plasmid DNA production.

Bacterial plasmids are the vectors of choice for DNA vaccines and short-term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100-250 mg of plasmid DNA/l of culture medium, using standard high-copy pUC origin-containing plasmids. In order to address this initial and yield-limiting upstream step, we identified novel fermentation control parameters for fed-batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed-batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid-mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5-10-fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities.

Bioreactors↗
PubMed1990

Multidimensional digital image representations using generalized Kaiser-Bessel window functions.

Inverse problems that require the solution of integral equations are inherent in a number of indirect imaging applications, such as computerized tomography. Numerical solutions based on discretization of the mathematical model of the imaging process, or on discretization of analytic formulas for iterative inversion of the integral equations, require a discrete representation of an underlying continuous image. This paper describes discrete image representations, in n-dimensional space, that are constructed by the superposition of shifted copies of a rotationally symmetric basis function. The basis function is constructed using a generalization of the Kaiser-Bessel window function of digital signal processing. The generalization of the window function involves going from one dimension to a rotationally symmetric function in n dimensions and going from the zero-order modified Bessel function of the standard window to a function involving the modified Bessel function of order m. Three methods are given for the construction, in n-dimensional space, of basis functions having a specified (finite) number of continuous derivatives, and formulas are derived for the Fourier transform, the x-ray transform, the gradient, and the Laplacian of these basis functions. Properties of the new image representations using these basis functions are discussed, primarily in the context of two-dimensional and three-dimensional image reconstruction from line-integral data by iterative inversion of the x-ray transform. Potential applications to three-dimensional image display are also mentioned.

Fourier Analysis↗
PubMed1992

Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa.

The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities.

Chromosome Mapping↗
PubMed2027

A Restriction-Free Cloning Approach for Molecular Engineering of Plasmids.

Molecular cloning by PCR amplification using a highly processive, high-fidelity DNA polymerase represents a robust and versatile technique for the precise manipulation of nucleic acid sequences. This approach enables the insertion, replacement, or modification of specific DNA fragments within a cloning vector, thereby generating an accurate copy of a gene or viral segment for downstream applications, such as protein expression, site-directed mutagenesis, and structural or functional analyses. The use of processive, high-fidelity polymerases significantly reduces the occurrence of base substitution errors, ensuring sequence integrity throughout the amplification process. Traditionally, restriction enzymes have been employed to facilitate directional cloning; however, alternative methods allow for mutagenesis without the need for unique and specific restriction sites and can be applied to virtually any cloning or seamless DNA assembly strategy. In this chapter, we describe a restriction enzyme-free and ligation-free PCR-based protocol widely applicable to any circular vector. This method enables targeted mutagenesis of the chikungunya virus (CHIKV) genome, offering a fast, efficient, and reliable strategy for generating mutant constructs suitable for virological and molecular studies.

Cloning, Molecular↗
PubMed1999

Recent amplification of the human FRG1 gene during primate evolution.

There is evidence of multiple copies of the FSHD Region Candidate Gene 1 (FRG1) in humans. Analysis of human FRG1 ESTs showed many of them to be non-processed pseudogenes dispersed throughout the genome. To determine when the amplification of FRG1 occurred, we used a PCR-based approach to identify FRG1 sequences from great apes, chimpanzee, gorilla and orang-utan, and an Old World monkey, Macaca mulatta. In common with humans, multiple copies of FRG1 were detected in the great apes. However, in Macaca mulatta, only two FRG1 loci were identified, one presumed to be the homologue of the human chromosome 4q gene. This is strikingly similar to the distribution of a dispersed 3.3-kb repeat family in primates. A member of this family, D4Z4, maps to the subtelomeric region of 4q, in close proximity to FRG1. We propose that an ancestral duplication of distal 4q included FRG1. This duplication is present in Macaca mulatta whose divergence from hominoids is thought to have occurred at least 33 million years ago. We propose that this telomeric region then underwent further amplification and dispersion events in the great ape lineage, with copies of FRG1 and the 3.3-kb repeats being localized in heterochromatic regions.

Animals↗
PubMed1991

Regulation of polyadenylation in hepatitis B viruses: stimulation by the upstream activating signal PS1 is orientation-dependent, distance-independent, and additive.

Hepatitis B viruses replicate by reverse transcription of a genomic RNA which harbors terminal redundancies. The synthesis of this RNA requires that transcription proceed twice through the polyadenylation (pA) site which, in mammalian strains, is flanked by the variant hexanucleotide UAUAAA and a T-rich downstream domain. These core elements are by themselves virtually defective in 3' end processing and require multiple upstream accessory elements which regulate pA site use. In ground squirrel hepatitis B virus (GSHV), one of these signals (PS1; -215 to -107 relative to UAUAAA) is transcribed only at the 3' end of genomic RNA and as such is analogous to retroviral U3 sequences. PS1 cooperates with other signals to enhance pA site use to very high levels and can be further sub-divided into two regions (A and B) which contribute equally to 3' end processing. Critical residues within PS1B have been localized to a 15 bp A/T-rich stretch which displays homology to other known upstream activating signals. A 15 bp segment within PS1A which has the identical A/T content but a divergent primary sequence plays a diminished role in processing. Furthermore, PS1 can activate GSHV core element usage autonomously. This stimulation has been shown to be additive since multiple copies of PS1 progressively increase polyadenylation, a phenomenon which also demands that PS1 exert its influence from a variety of distances from the hexanucleotide signal.

Base Sequence↗
PubMed2001

The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens.

The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.

Adjuvants, Immunologic↗
PubMed2006

Increased gene copy number of the transcription factor E2F1 in malignant melanoma.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.

Blotting, Western↗
PubMed1987

Processing determinants required for in vitro cleavage of the poliovirus P1 precursor to capsid proteins.

We generated defined alterations in poliovirus protein-processing substrates and assayed the effects of these alterations with an in vitro expression system. A complete cDNA copy of the poliovirus genome was inserted into a bacteriophage T7 transcription vector. Using this expression template, we produced RNA transcripts containing defined regions of the poliovirus capsid precursor polypeptide (P1) and RNA transcripts containing mutations in the P1 and P2 regions. In vitro translation of P1-derived transcripts allowed us to characterize the 3C-mediated cleavage of P1 to capsid proteins. We demonstrated that, for either posttranslational or cotranslational cleavage at any of the Q-G amino acid pairs within P1, almost the entire P1 precursor is required. We also demonstrated that minimal sequences 3' to the 2A coding sequence are required to generate active 2A proteinase in vitro and that two specific four-amino-acid insertions in protein 2C do not alter 2A- or 3C-mediated processing of the poliovirus polyprotein. In addition, we demonstrated that substantial deletion of P1 sequences does not alter 2A-mediated cleavage of the Y-G site at the P1-P2 junction. These results allowed us to compare the P1 sequences required for 2A- versus 3C-mediated processing of the capsid precursor, and we discuss these results in the context of the three-dimensional structure of the capsid proteins.

Amino Acid Sequence↗
PubMed1987

Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion.

An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.

Animals↗