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PubMed1985

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides.

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.

Amino Acid Sequence↗
PubMed2001

The evolution of prestige: freely conferred deference as a mechanism for enhancing the benefits of cultural transmission.

This paper advances an "information goods" theory that explains prestige processes as an emergent product of psychological adaptations that evolved to improve the quality of information acquired via cultural transmission. Natural selection favored social learners who could evaluate potential models and copy the most successful among them. In order to improve the fidelity and comprehensiveness of such ranked-biased copying, social learners further evolved dispositions to sycophantically ingratiate themselves with their chosen models, so as to gain close proximity to, and prolonged interaction with, these models. Once common, these dispositions created, at the group level, distributions of deference that new entrants may adaptively exploit to decide who to begin copying. This generated a preference for models who seem generally "popular." Building on social exchange theories, we argue that a wider range of phenomena associated with prestige processes can more plausibly be explained by this simple theory than by others, and we test its predictions with data from throughout the social sciences. In addition, we distinguish carefully between dominance (force or force threat) and prestige (freely conferred deference).

Journal Article↗
PubMed2004

Objective method of comparing DNA microarray image analysis systems.

Many image analysis systems are available for processing the images produced by laser scanning of DNA microarrays. The image processing system takes pixel-level intensity data and converts it to a set of gene-level expression or copy number summaries that will be used in further analyses. Image analysis systems currently in use differ with regard to the specific algorithms they implement, ease of use, and cost. Thus, it would be desirable to have an objective means of comparing systems. Here we describe a systematic method of comparing image processing results produced by different image analysis systems using a series of replicate microarray experiments. We demonstrate the method with a comparison of cDNA microarray data generated by the UCSF Spot and the GenePix image processing systems.

Algorithms↗
PubMed1999

Getting it right: the teaching of philosophical health care ethics.

This article seeks to show one way in which moral philosophy, considered by the authors to be essential to the nursing and midwifery curricula, can be presented to achieve an optimal learning experience for nurses and midwives. It demonstrates that what might be considered a standard approach, that is, one that begins with ethical principles concerned with rights and duties and then often follows a linear pattern of teaching, may be in danger of promoting a focus on standardized outcomes. Such use of philosophy could therefore actually detract from the process of care. Moral philosophy underpinning health care ethics is commonly misperceived as a method of problem solving when there is an obvious dilemma regarding appropriate care and/or treatment. However, it is readily recognized that key principles within philosophy, for example, deontology and utilitarianism, despite their approach to a standard or criterion of right action, are both deficient in terms of providing ready-made right decisions. This is because their main virtue is to expose the difficulty rather than to solve the problem. Given these difficulties, any subsequent principles such as respect, beneficence, nonmaleficence and justice, incur the same deficiencies a fortiori. It can be argued that the complexity of the environment in which nurses and midwives now practice requires them to develop a capability that begins with the philosophical construction of an issue. This can subsequently enable a recognition of the essential nature of their own involvement as a nurse or midwife. By so doing, nurses and midwives can then bring issues into a nursing or midwifery paradigm and ensure that this perspective informs debate. Ultimately the focus is on the process by which care decisions are made. The intent therefore, is not simply for nurses or midwives to learn moral philosophy or to copy what is considered by others to be right action, but to recognize that a number of right actions are possible and, in so doing, develop their ability either to choose or influence a final action through a valid process. This article proposes and demonstrates by case example that what is often considered as a chance effect for nurses and midwives learning moral philosophy should be seen as the main effect and intended outcome.

Bioethical Issues↗
PubMed1998

In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing.

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400-600 copies of HPV 16, HeLa 229 cells with 10-50 copies of HPV 18, and SiHa cells with 1-2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.

3,3'-Diaminobenzidine↗
PubMed2006

Genome-wide loss of heterozygosity and copy number alteration in esophageal squamous cell carcinoma using the Affymetrix GeneChip Mapping 10 K array.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide. Comprehensive genomic characterization of ESCC will further our understanding of the carcinogenesis process in this disease. RESULTS: Genome-wide detection of chromosomal changes was performed using the Affymetrix GeneChip 10 K single nucleotide polymorphism (SNP) array, including loss of heterozygosity (LOH) and copy number alterations (CNA), for 26 pairs of matched germ-line and micro-dissected tumor DNA samples. LOH regions were identified by two methods--using Affymetrix's genotype call software and using Affymetrix's copy number alteration tool (CNAT) software--and both approaches yielded similar results. Non-random LOH regions were found on 10 chromosomal arms (in decreasing order of frequency: 17p, 9p, 9q, 13q, 17q, 4q, 4p, 3p, 15q, and 5q), including 20 novel LOH regions (10 kb to 4.26 Mb). Fifteen CNA-loss regions (200 kb to 4.3 Mb) and 36 CNA-gain regions (200 kb to 9.3 Mb) were also identified. CONCLUSION: These studies demonstrate that the Affymetrix 10 K SNP chip is a valid platform to integrate analyses of LOH and CNA. The comprehensive knowledge gained from this analysis will enable improved strategies to prevent, diagnose, and treat ESCC.

Aged↗
PubMed2005

Comparative genomic hybridization.

Altering DNA copy number is one of the many ways that gene expression and function may be modified. Some variations are found among normal individuals ( 14, 35, 103 ), others occur in the course of normal processes in some species ( 33 ), and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur prior to or shortly after fertilization, whereas DNA dosage alterations that occur in somatic cells are frequent contributors to cancer. Detecting these aberrations, and interpreting them within the context of broader knowledge, facilitates identification of critical genes and pathways involved in biological processes and diseases, and provides clinically relevant information. Over the past several years array comparative genomic hybridization (array CGH) has demonstrated its value for analyzing DNA copy number variations. In this review we discuss the state of the art of array CGH and its applications in medical genetics and cancer, emphasizing general concepts rather than specific results.

Chromosome Aberrations↗
PubMed1998

A partial hprt gene duplication generated by non-homologous recombination in V79 Chinese hamster cells is eliminated by homologous recombination.

Here, the sequence in the hprt gene of the duplication mutant SPD8 originating from V79 Chinese hamster cells was determined. The duplication arose after non-homologous recombination between exon 6 and intron 7, resulting in an extra copy of the 3' portion of exon 6, of exon 7 and of flanking intron regions. Only a duplication of exon 7 is present in the mRNA, since the duplicated exon 6 lacks its 5' splice site and is removed during RNA processing. The findings in this study suggest that the non-homologous recombination mechanism which occurred here may have been initiated by endonucleases, rather than by a spontaneous double strand break. Subsequently, 14 spontaneous SPD8 revertants with a functional hprt gene were isolated and characterized using PCR and sequencing. The data revealed that although the SPD8 cell line arose by non-homologous recombination, it reverts spontaneously by homologous recombination. Interestingly, the downstream copy of exon 7 was restored by this process. This was indicated by the presence of a specific mutation, a T-to-G transversion, close to the breakpoint, a characteristic unique to the SPD8 clone. Our results suggest that the spontaneous reversion of this cell line by homologous recombination may involve an exchange, rather than a conversion mechanism.

Animals↗
PubMed1996

A functional dominant mutation in Schizosaccharomyces pombe RNase MRP RNA affects nuclear RNA processing and requires the mitochondrial-associated nuclear mutation ptp1-1 for viability.

The essential gene for RNase MRP RNA, mrp1, was identified previously in Schizosaccharomyces pombe by homology to mammalian RNase MRP RNAs. Here we describe distinct site-specific mutations in RNase MRP RNA that support a conserved role for this ribonucleoprotein in nucleolar 5.8S rRNA processing. One characterized mutation, mrp1-ND90, displays dominance and results in accumulation of unspliced precursor RNAs of dimeric tRNA(Ser)-tRNA(Met)i, suggesting a novel nuclear role for RNase MRP in tRNA processing. Cells carrying the mrp1-ND90 mutation, in the absence of a wild-type copy of mrp1, additionally require the mitochondrially associated nuclear mutation ptp1-1 for viability. Analysis of this mrp1 mutation reinforces previous biochemical evidence suggesting a role for RNase MRP in mitochondrial DNA replication. Several mutations in mrp1 result in unusual cellular morphology, including alterated nuclear organization, and are consistent with a broader nuclear role for RNase MRP in regulating a nuclear signal for septation; these results are a further indication of the multifunctional nature of this ribonucleoprotein.

Base Sequence↗
PubMed2006

Short blocks from the noncoding parts of the human genome have instances within nearly all known genes and relate to biological processes.

Using an unsupervised pattern-discovery method, we processed the human intergenic and intronic regions and catalogued all variable-length patterns with identically conserved copies and multiplicities above what is expected by chance. Among the millions of discovered patterns, we found a subset of 127,998 patterns, termed pyknons, which have additional nonoverlapping instances in the untranslated and protein-coding regions of 30,675 transcripts from 20,059 human genes. The pyknons arrange combinatorially in the untranslated and coding regions of numerous human genes where they form mosaics. Consecutive instances of pyknons in these regions show a strong bias in their relative placement, favoring distances of approximately 22 nucleotides. We also found pyknons to be enriched in a statistically significant manner in genes involved in specific processes, e.g., cell communication, transcription, regulation of transcription, signaling, transport, etc. For approximately 1/3 of the pyknons, the intergenic/intronic instances of their reverse complement lie within 380,084 nonoverlapping regions, typically 60-80 nucleotides long, which are predicted to form double-stranded, energetically stable, hairpin-shaped RNA secondary structures; additionally, the pyknons subsume approximately 40% of the known microRNA sequences, thus suggesting a possible link with posttranscriptional gene silencing and RNA interference. Cross-genome comparisons reveal that many of the pyknons have instances in the 3' UTRs of genes from other vertebrates and invertebrates where they are overrepresented in similar biological processes, as in the human genome. These unexpected findings suggest potential unique functional connections between the coding and noncoding parts of the human genome.

3' Untranslated Regions↗
PubMed1986

The suffix sequence is involved in processing the 3' ends of different mRNAs in Drosophila melanogaster.

We describe here the nucleotide sequences of several genomic and mRNA copies of the suffix, a short dispersed actively transcribed repeat located at the 3' ends of many different genes of Drosophila melanogaster. Only one strand of the suffix is transcribed. The patterns of suffix-containing mRNAs vary during development. The five randomly selected genomic copies of the suffix are 265 bp long and quite conservative in their sequence. The non-transcribed strand is terminated with oligo(A) preceded by AATAAA sequence. No repetitive flanking sequences can be detected. The three other genomic copies selected by hybridization with suffix-containing cDNA clones are less conservative, especially in the 5' part. In particular, they contain short insertions carrying a polyadenylation signal AATAAA at exactly the same position of the suffix. Comparison of genomic and cDNA clones shows that mRNAs are polyadenylated at the last nucleotide of these insertions. The cDNA clones include the same part of the suffix, from the 39th to 112th nucleotide. Thus, a segment of the suffix forms the last exon for both genes. In one case, the beginning of the last intron coincides with the beginning of suffix, creating a very unusual donor splicing site. We conclude that the suffix sequence is directly involved in the formation of the last splicing site and 3'-end maturation of mRNA, at least in the case of the two genes analysed.

Journal Article↗
PubMed1990

Amplification of a long sequence that includes a processed pseudogene for elongation factor 2 in the mouse.

Quantitative Southern blotting analysis has demonstrated that mouse cells contain about 70 copies per haploid genome of a DNA sequence related to the gene for elongation factor 2. The restriction maps of seven cosmids that each carry one copy of the EF2-related sequence (MER) and nucleotide sequences of MERs were highly conserved among the cosmids. Data obtained by such analyses suggest that MERs were produced by the integration of one copy of MER derived from poly(A)+ mRNA for EF2 into a specific site in the mouse genome, with subsequent amplification of MER together with its large flanking sequences during the evolution of the mouse. Furthermore, it appears that the size of each repeating unit is more than 60 kb. Analysis by pulse-field gel electrophoresis suggested that multiple copies of a repeating unit of more than 400 kb (or two units) are clustered at a specific site (or each specific site) in the mouse genome.

Animals↗
PubMed1987

The evolution of visual processing and the construction of seeing systems.

This paper is concerned with the evolution of visual mechanisms and the possibility of copying their principles at different levels of sophistication. It is an old question how the complex interaction between eye and brain evolved when each needs the other as a test-bed for successive improvements. I propose that the primitive mechanism for the separation of stationary objects relies on their relative movement against a background, normally caused by the animal's own movement. Apparently insects and many lower animals use little more than this for negotiating through a three-dimensional world, making adequate responses to individual objects which they 'see' without a cortical system or even without a large brain. In the development of higher animals such as birds or man, additional circuits store memories of the forms of objects that have been frequently inspected from all angles or handled. Simple visual systems, however, are tuned to a feature of the world by which objects separate themselves by movement relative to the eye. In making simple artificial visual systems which 'see', as distinct from merely projecting the image, it is more hopeful to copy the 'ambient' vision of lower animals than the cortical systems of birds or mammals.

Animals↗
PubMedDate not supplied

Lessons from a doctoral thesis.

The production of a doctoral thesis is a time-consuming affair that until recently was done in conjunction with professional publishing services. Advances in computer technology have made many sophisticated desktop publishing techniques available to the microcomputer user. We describe the computer method used, the problems encountered, and the solutions improvised in the production of a doctoral thesis by computer. The Apple Macintosh was selected for its ease of use and intrinsic graphics capabilities. A scanner was used to incorporate text from published papers into a word processing program. The body of the text was updated and supplemented with new sections. Scanned graphics from the published papers were less suitable for publication, and the original data were replotted and modified with a graphics-drawing program. Graphics were imported and incorporated in the text. Final hard copy was produced by a laser printer and bound with both conventional and rapid new binding techniques. Microcomputer-based desktop processing methods provide a rapid and cost-effective means of communicating the written word. We anticipate that this evolving technology will have increased use by physicians in both the private and academic sectors.

Academic Dissertations as Topic↗
PubMed1996

The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation.

To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

Base Sequence↗
PubMed1985

A model for intracellular complexation between gene-5 protein and bacteriophage fd DNA.

A structural model for the helical intracellular complex formed between the gene-5 DNA-binding protein (G 5 BP; approximately 1274 copies) and bacteriophage fd DNA has been derived by an atomic-contact analysis approach. These studies depended in large part on the recently determined high-resolution structure of the G 5 BP dimer and cross-correlations with physical-chemical data available from other techniques. The approach was to systematically scan the full set of helical complexation parameters involved, based upon observed structural and orientational constraints, to determine those compatible with both the structure of the G 5 BP dimer and the overall dimensions of the full complex. This process was monitored throughout by close scrutiny of dimer-dimer contacts and the use of hard-copy and interactive graphics devices. Instead of the wide variety of possibilities that had been expected from such an approach, only one satisfactory assembly of DNA and G 5 BP dimers could be found. The results indicate that phage DNA will be wound to the outside of the helical protein ribbon that forms the core of intracellular complex at a density of five nucleotides per G 5 BP monomer. Bound DNA strands are positioned in two contiguous binding channels, which form as a consequence of the interactions of complexed G 5 BP dimers. These channels run just inside the outer extended beta loops, composed of residue 20-30, and are separated by approximately 3.2 nm. The DNA phosphate backbone is bound at a substantially smaller radial distance (approximately 3.5 nm) than the maximum radius of the intracellular complex as a whole (approximately 4.5 nm) since bound DNA is embedded within these well-defined binding channels. Our studies also indicate that a number of sterically unacceptable contacts, involving residues 38-42, prevent complexation of otherwise complementary dimer surfaces in the absence of nucleic acids. In the process of binding DNA, these residues change conformation thereby allowing self-assembly of dimer units into a helical structure. We propose that these residues act as a two-position stereochemical switch that allows or disallows complex formation in response to the absence or presence of DNA.

Bacteriophages↗
PubMed1997

Major histocompatibility complex gene mapping in the amphibian Xenopus implies a primordial organization.

One of the most provocative recent discoveries in immunology was the description of a genetic linkage in the major histocompatibility complex (MHC) between structurally unrelated genes whose products are involved in processing and presentation of antigens for recognition by T lymphocytes. Genes encoding MHC class I molecules, which bind and present at the cell surface proteolytic fragments of cytosolic proteins, are linked to nonhomologous genes whose products are involved in the production and subsequent transfer of such fragments into the endoplasmic reticulum. In mammals, the class I presentation and processing genes are found in different regions of the MHC. To examine the evolutionary origins of this genetic association, linkage studies were carried out with Xenopus, an amphibian last sharing an ancestor with mammals over 350 million years ago. In contrast to mammals, the single copy Xenopus class I gene is located between the class II and III regions, speculated to be in close linkage with the processing and transport genes. In addition to suggesting a primordial organization of genes involved in class I antigen presentation, these linkage studies further provide insight into the origins of the MHC class III region and the phenomenon of class I gene instability in the mammalian MHC.

Alleles↗
PubMed1998

Effects of propentofylline on adenosine receptor activity in Chinese hamster ovary cell lines transfected with human A1, A2A, or A2B receptors and a luciferase reporter gene.

Propentofylline is neuroprotective in vivo, but its mechanism of action is not completely understood. Previously, propentofylline was shown to block adenosine transport processes, to inhibit three adenosine receptor subtypes, and to inhibit cAMP phosphodiesterase. We tested the effect of propentofylline on adenosine receptor function in Chinese hamster ovary (CHO) cells transfected with human adenosine A1, A2A, or A2B receptors and a luciferase reporter gene under control of a promoter sequence containing several copies of the cAMP response element. We investigated the concentration-dependent inhibitory effects of propentofylline on cAMP phosphodiesterase, adenosine transport processes, and adenosine A1, A2A, and A2B receptors. At concentrations > or = 1 mM, propentofylline increased luciferase activity probably as a result of inhibition of cAMP phosphodiesterase. Inhibition of [3H]adenosine uptake by propentofylline was concentration dependent, with IC50 values of 37-39 microM for the three cell types. Agonist-activated adenosine A1 receptors were antagonized by 100 microM propentofylline, but inhibition of agonist-stimulated A2A or A2B receptors was not observed. In contrast, A1 and A2A receptor mediated effects of adenosine were enhanced by propentofylline at concentrations of 1 and 100 microM, respectively. These data indicate that the net effects of propentofylline in vivo will be dependent on the concentrations of propentofylline and adenosine available and on the subtypes of adenosine receptors, phosphodiesterases, and nucleoside transporters present.

Adenosine↗