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PubMed1993

Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.

Profilaggrin is an intermediate filament-associated protein of cornified epithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisional activation of multiply charged precursor ions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-termini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino- and carboxyl-termini were "ragged", suggesting that processing may involve exopeptidase action after an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of an intronless mouse S-adenosylmethionine decarboxylase gene coding for a functional enzyme strongly expressed in the liver.

A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library. Surprisingly, the gene proved to be intronless. With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA. Moreover, the gene contained a poly(dA) tract at the 3' end and was flanked by 13-base pair direct repeats. Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome. By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional. Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver. When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme. Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit. Importantly, the protein encoded by the intronless gene was functional, i.e. it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure.

Adenosylmethionine Decarboxylase↗
PubMed2002

Length distribution of long interspersed nucleotide elements (LINEs) and processed pseudogenes of human endogenous retroviruses: implications for retrotransposition and pseudogene detection.

Deciphering the human genome includes reliable identification and structural characterization of individual retrotransposon elements. The most active group of autonomous transposable elements, the long interspersed nuclear elements (LINE), transpose themselves as well as other RNAs, including those of human endogenous retroviruses (HERV). During this transposition, however, the LINE-encoded reverse transcriptase (RT) often abortively dissociates from the RNA template, leaving a prematurely terminated, 5' truncated copy. We have analyzed the length distributions of LINEs and of processed pseudogenes derived from HERV-W. As expected, we have found that the majority of 5' truncated LINEs and HERV-W processed pseudogenes show a prevalence of very short elements terminated close to the 3' end. On the other hand, the number of complete elements is far above the expectation. The characteristic distribution in both cases indicates two important conclusions: (i) dissociation of LINE RT from the template cannot be fully explained by low processivity of RT modelled as a stochastic, Poisson-type process. (ii) Currently cited numbers of pseudogenes within the human genome are underestimated, since a large percentage of pseudogenes are terminated in the 3' untranslated region and remain undetectable in translated homology searches of protein databases against the human genome.

Endogenous Retroviruses↗
PubMed1997

A promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation.

X-chromosome inactivation is the process by which a cell recognizes the presence of two copies of an X chromosome early in the development of XX embryos and chooses one to be active and one to be inactive. Although it is commonly believed that the initiation of X inactivation is random, with an equal probability (50:50) that either X chromosome will be the inactive X in a given cell, significant variation in the proportion of cells with either X inactive is observed both in mice heterozygous for alleles at the Xce locus and among normal human females in the population. Families in which multiple females demonstrate extremely skewed inactivation patterns that are otherwise quite rare in the general population are thought to reflect possible genetic influences on the X-inactivation process. Here we report a rare cytosine to guanine mutation in the XIST minimal promoter that underlies both epigenetic and functional differences between the two X chromosomes in nine females from two unrelated families. All females demonstrate preferential inactivation of the X chromosome carrying the mutation, suggesting that there is an association between alterations in the regulation of XIST expression and X-chromosome inactivation.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2002

How DNA lesions are turned into mutations within cells?

Genomes of all living organisms are constantly injured by endogenous and exogenous agents that modify the chemical integrity of DNA and in turn challenge its informational content. Despite the efficient action of numerous repair systems that remove lesions in DNA in an error-free manner, some lesions, that escape these repair mechanisms, are present when DNA is being replicated. Although replicative DNA polymerases are usually unable to copy past such lesions, it was recently discovered that cells are equipped with specialized DNA polymerases that will assist the replicative polymerase during the process of Translesion Synthesis (TLS). These TLS polymerases exhibit relaxed fidelity that allows them to copy past lesions in DNA with an inherent risk of generating mutations at high frequency. We present recent aspects related to the genetics and biochemistry of TLS and highlight some of the remaining hot topics of this field.

Animals↗
PubMed1985

The Rey-Osterrieth Complex as a diagnostic measure of neuropsychological outcome of brain injury.

The Rey-Osterrieth Complex was devised to investigate perceptual organization and visual memory in brain-damaged patients. Osterrieth analyzed the patient's method of procedure as well as his specific copying errors, but the test is most commonly regarded as a measure of visual memory. In our work with traumatically brain-injured patients we found the Rey-Osterrieth Complex useful as a high load examiner of disorders of cognitive process. In our application of the test the drawing quality in the copy session is seen as being related to the kind of disorder, while the neuropsychological deficit is emphasized by the drawing quality in the memory session. The present paper is concerned with different thinking disorders as they are expressed in both the copy and memory parts of the Rey-Osterrieth Complex.

Adult↗
PubMedDate not supplied

The C3b/C4b receptor (CR1, CD35) on erythrocytes: methods for study of the polymorphisms.

This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.

Animals↗
PubMed1999

Difficulty in writing Japanese semantic characters in a 9-year-old boy with Williams syndrome.

A 9-year-old boy diagnosed as having Williams syndrome was evaluated using psychological test batteries in order to clarify his ability in language and visual cognition. The subject had difficulty in writing some of the Japanese semantic characters (called Kanji) which he could otherwise read and understand. Although he could write the small components of which the Kanji characters were composed, he could not locate these correctly. This phenomenon is considered to be very similar to the difficulty in copying a figure observed clinically. The Kaufmann Assessment Batteries for Children clearly revealed that the boy had difficulty with the sub-test of spatial memory compared to his average score for simultaneous processing. This result is considered to be closely related to the difficulty in copying figures or writing Kanji characters. On the Illinois Test of Psycholinguistic Abilities, the present authors found that the subject's vocabulary was relatively good, although semantic and pragmatic problems remained. Clarifying the strong and weak points of the abilities of such patients will help to determine the most appropriate mode of education for them.

Apraxia, Ideomotor↗
PubMed1985

The two mechanisms for antigenic variation in Trypanosoma brucei are independent processes.

Antigenic switching in Trypanosoma brucei can occur either by the production of a telomeric copy of a variant surface glycoprotein (VSG) gene through a gene conversion mechanism or by the nonduplicative activation of a telomeric VSG gene. The 5 VSG gene telomeric copy that is expressed in IsTaR 1 variant antigenic type (VAT) 5 is retained in an inactive state following an antigenic switch to VAT A5. This inactive telomeric 5 VSG gene copy is absent following independent single antigenic switches to VATs 1A5 and 11A5. The inactive 5 VSG gene does not appear to have been replaced with the newly expressed VSG gene. Thus, inactive telomeric VSG genes that are capable of being expressed can be lost, presumably through gene conversion to new VSG genes. These results suggest that gene conversion of an inactive VSG gene does not obligately activate the new VSG gene. We conclude that the gene conversion and telomeric activation mechanisms for antigenic switching are separate and independent processes.

Animals↗
PubMed1988

Light and electron microscopic immunocytochemistry of neurons in the blowfly optic lobe reacting with antisera to RFamide and FMRFamide.

Different antisera to the molluscan cardioexcitatory peptide FMRFamide, and its fragment, RFamide (Arg-Phe-NH2), label a distinct population of neurons in the optic lobe of the blowfly, Calliphora erythrocephala. Seven morphological types of RFamide/FMRFamide-like immunoreactive neurons could be distinguished in the optic lobes based on the locations of their cell bodies, their axonal projections and the distribution of their processes. Of these, two types could be resolved in their entire extent, the others were labeled only in their cell bodies and terminal processes or were partly obscured by other immunoreactive processes. The RF-like immunoreactive neurons in the optic lobes are of two main classes: (1) two types of large field projection neurons and (2) five types of local neurons. One type of projection neurons (five in each lobe) connects the entire projected retinal mosaic of the medulla and lobula in the optic lobe with protocerebral centres associated with the mushroom body calyx. The other type (2-3 invading each lobe) has cell bodies in the protocerebrum and contralateral processes invading optic lobes. Of the class of local neurons there are two amacrine RF-like immunoreactive neurons in each medulla. Each of these amacrines supplies the entire mosaic with fine processes. The remaining local RF-like immunoreactive neurons are present in relatively large numbers (one type in more than 2000 copies in each medulla) and-supply the medulla, lobula and lobula plate neuropils with fine varicose processes. In the medulla the RF-like immunoreactive processes are arranged in strict layers whereas in the lobula complex the distribution is diffuse. Electron microscopic immunocytochemistry, using both pre-embedding immuno peroxidase-antiperoxidase and post-embedding protein A-gold labeling, was employed for analysis of cytology and synaptic connections of RF-like immunoreactive neurons in the medulla. The varicosities of the processes of the large field projection neurons were not found to make chemical synapses with other neurons in the medulla. The spines of the RF-like immunoreactive processes of the large medulla amacrines, however, make pre- and postsynaptic contacts with other neural elements. Our findings indicate that an RFamide/FMRFamide-like substance may be used as a neurotransmitter or neuromodulator by optic lobe neurons of different types. The local and projection RF-like immunoreactive pathways probably play different roles in visual processing.

Animals↗
PubMed1998

Radiology report times: impact of picture archiving and communication systems.

OBJECTIVE: We investigated the impact on radiologist reporting time of the change from conventional film to hard-copy computerized radiography and of the subsequent move to soft-copy images on picture archiving and communication system (PACS) workstations. MATERIALS AND METHODS: A controlled before and after research design was undertaken. Data were collected on four occasions: two relating to conventional film, one relating to hard-copy computerized radiography, and one relating to soft-copy PACS images. Data collection was by direct observation of radiology reporting sessions by independent health service researchers. Data were collected on report times, details of images viewed, characteristics of the radiologist, and details of interruptions. To control for potential biases in the before and after comparisons, ordinary least squares multiple regression analysis was used. The principal comparison was between reports with PACS and reports with computerized radiography hard-copy because no change was noted in the organization of the reporting process between these two data collection rounds other than the introduction of the PACS. RESULTS: Data were collected on a total of 5568 report observations. Report time in the PACS data collection period was not significantly different (p = .32) than that in the computerized radiography hard-copy period. Reporting with the PACS was associated with significantly more (p < .01) historical images (i.e., images of the same patient obtained in previous examinations) being viewed. CONCLUSION: Report time was not lengthened by the introduction of the PACS. The finding that more historical images were viewed when the PACS was in use indicates that the PACS brought about a positive change in reporting practice.

Bias↗
PubMed2006

PLOTREP: a web tool for defragmentation and visual analysis of dispersed genomic repeats.

Identification of dispersed or interspersed repeats, most of which are derived from transposons, retrotransposons or retrovirus-like elements, is an important step in genome annotation. Software tools that compare genomic sequences with precompiled repeat reference libraries using sensitive similarity-based methods provide reliable means of finding the positions of fragments homologous to known repeats. However, their output is often incomplete and fragmented owing to the mutations (nucleotide substitutions, deletions or insertions) that can result in considerable divergence from the reference sequence. Merging these fragments to identify the whole region that represents an ancient copy of a mobile element is challenging, particularly if the element is large and suffered multiple deletions or insertions. Here we report PLOTREP, a tool designed to post-process results obtained by sequence similarity search and merge fragments belonging to the same copy of a repeat. The software allows rapid visual inspection of the results using a dot-plot like graphical output. The web implementation of PLOTREP is available at http://bioinformatics.abc.hu/PLOTREP/.

Computer Graphics↗
PubMed2004

Quantification of human PPARgamma1 gene expression by competitive PCR using an homologous internal standard.

The polymerase chain reaction (PCR) is useful for amplifying specific mRNAs, particularly those present in low copy numbers. However, due to the exponential nature of the amplification process, PCR cannot readily be used to quantify gene expression. A competitive PCR technique was developed to address this shortcoming. An internal standard that is 100% homologous to, but shorter than, the target gene was constructed. The practicality of the method was demonstrated by determining the expression levels of a human transcription factor, peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) which is normally present in low copy numbers in selected cells. A mock system was used to test the accuracy and sensitivity of the method, which was subsequently used to determine the expression of this receptor in lipopolysaccharide (LPS)-activated monocytes, which are known to express hPPARgamma1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPARgamma1 at the attomole (10(-18)) level in monocytes.

Gene Expression↗
PubMed2005

Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia.

BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.

Amino Acid Sequence↗
PubMed2004

Retroposon mapping in molecular systematics.

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.

Animals↗
PubMed1997

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA.

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAPl-a poly(A)polymerase of Escherichia coli) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.

Bacterial Proteins↗
PubMed1992

Ham-2 corrects the class I antigen-processing defect in RMA-S cells.

The murine major histocompatibility complex (MHC) contains two genes (Ham-1 and Ham-2) that encode members of a super-family of ATP-dependent transport proteins. These genes are believed to mediate the transport of peptide antigen from the cytoplasm into the lumen of the endoplasmic reticulum for binding by MHC class I molecules. Evidence for such a function has come from the rescue of class I surface expression by a cloned copy of the human homologue of Ham-1, PSF-1, in a human cell line that is defective in antigen processing. A mutant murine cell line, RMA-S, has an identical antigen-processing-defective phenotype. Here we show that expression of a cloned copy of the Ham-2 gene in RMA-S cells results in recovery of the ability to process and present class I-restricted antigens to cytotoxic T lymphocytes, and in partial recovery of class I surface expression. Processing defects for classical (H-2 K and D) and non-classical (Qa1 and HMT) class I molecules are corrected by Ham-2. These data indicate that both MHC-linked transporter genes are probably required for class I antigen processing, and that the functional transporter in this pathway may consist of a Ham-1/Ham-2 heterodimer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

The preparation, execution and suppression of copied movements in the human brain.

We used positron emission tomography (PET) to measure movement set-related changes in regional cerebral blood flow (rCBF) when human subjects were asked to copy hand movements. Movement set-related activity in the brain is thought to reflect the processes of movement selection, preparation and inhibition. Four conditions were used. In the first condition, prepare and execute (PE), the hand stimulus to be copied was shown to subjects 3 s before an auditory "go"-cue instructed subjects to execute the movement; a large part of the scanning time was therefore spent in preparing to move. In the immediate execution condition (E), the hand stimulus and the go cue were presented simultaneously. The prepare-only condition (P) was similar to PE, except subjects only prepared to make the movement and did not actually execute any movement when they heard the auditory go-cue. The same stimuli were presented in a baseline condition (B), but the subjects were instructed to neither prepare nor execute movements. There were 5 principle findings: (1) In contrast to a previous study of human set-related activity in which movements were instructed by an arbitrary pattern of LEDs, preparing to make a copied movement causes rCBF changes in area 44 in posterior Broca's area; (2) set-related activity can be recorded in the cerebellar hemispheres and midline; (3) we confirmed that the supramarginal gyrus has a general role in preparing movements - there was more rCBF in the P than the E condition; (4) the cerebellar nuclei and the basal ganglia may be particularly involved in the initiation and execution of a planned movement; these regions were more active in the PE condition than the P condition; (5) the ventrolateral prefrontal cortex and a left anterior cingulate area are part of a distributed system involved in the suppression of a motor response; these areas were significantly more active in the P than the PE condition.

Adult↗