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PubMed2003

Human hippocampal and parahippocampal activity during visual associative recognition memory for spatial and nonspatial stimulus configurations.

Evidence from animal studies points to the importance of the parahippocampal region (PHR) [including entorhinal, perirhinal, and parahippocampal (PHC) cortices] for recognition of visual stimuli. Recent findings in animals suggest that PHR may also be involved in visual associative recognition memory for configurations of stimuli. Thus far, however, such involvement has not been demonstrated in humans. In fact, it has been argued that associative recognition in humans is critically dependent on the hippocampal formation (HF). To better understand the division of function between HF and PHR during recognition memory in humans, we measured the activity of both areas in healthy young adults during an associative recognition memory task using functional magnetic resonance imaging. To more precisely characterize the nature of the associations that might be coded by the HF and PHR during recognition, subjects were required to learn and were later tested for associations based on either the spatial arrangements of two stimuli or the identity of two stimuli (a face and a tool). An area in the PHC was found to be more active for recognized old configurations than new configurations in both the spatial and identity conditions. The HF, on the other hand, was more active for recognition of new configurations than old configurations and also more active in the spatial than the identity condition. These data highlight the involvement of PHR in the long-term coding of associative relationships between stimuli and help to clarify the nature of its functional distinction from the HF.

Adult↗

PubMed2000

The categorical perception of colors and facial expressions: the effect of verbal interference.

A series of five experiments examined the categorical perception previously found for color and facial expressions. Using a two-alternative forced-choice recognition memory paradigm, it was found that verbal interference selectively removed the defining feature of categorical perception. Under verbal interference, there was no longer the greater accuracy normally observed for cross-category judgments relative to within-category judgments. The advantage for cross-category comparisons in memory appeared to derive from verbal coding both at encoding and at storage. It thus appears that while both visual and verbal codes may be employed in the recognition memory for colors and facial expressions, subjects only made use of verbal coding when demonstrating categorical perception.

Adolescent↗

PubMed2005

Principles of microRNA-target recognition.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA-target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families.

3' Untranslated Regions↗

PubMed1987

Immunoglobulin variable region heptamer-nonamer recognition sequence joined to rearranged D-J segment: implications for the immunoglobulin recombinase mechanism.

We have found a novel immunoglobulin gene rearrangement in a murine hybridoma in which a heavy chain variable region (VH) heptamer-nonamer recognition sequence is joined to the diversity segment (D) through head-to-head fusion. The heptamer-nonamer recognition sequence and its adjacent 5' DNA are derived from the downstream flanking region of a germline VH gene. Sequence analysis indicates that this adjacent DNA is homologous to the downstream flank of VH108B, and it has characteristics of RNA processing that may suggest it was derived from an mRNA intermediate; these unusual features indicate that the segment is a processed gene. Because of head-to-head fusion, the recognition sequence and the flanking sequence are in opposite transcriptional polarity to D. The latter is joined correctly at its 3' border to a joining (J) gene segment. A gamma 1 constant region (but not mu) is located further downstream. Thus this fragment has several features common to normal immunoglobulin heavy chain gene rearrangement despite the unusual joining event involving V-D. Linkage of the VH heptamer-nonamer recognition sequence to D has not been observed previously. Although the recognition sequence described is inverted with respect to D and J, the endonucleolytic process that cleaved the recognition sequence at the 5' border of the heptamer before rearranging it to D was accurate. We suggest that of the three functions associated with the recombinase reaction; recognition, cutting, and ligation, only recognition and cutting may be limited to specific structures, and the ligation step may be less restricted because it is not confined to forming coding-to-coding or flank-to-flank joints. This aberrant ligation product suggests that the information leading to normal rearrangements may be found in structures that include more than the recognition sequences or coding regions alone, because the joining described here has spliced the incorrect end of a recognition sequence to a coding region to yield a nonproductive recombination.

Animals↗

PubMed1995

The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B, RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames.

The DNA sequence of a 26,677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3' end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine beta-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases.

Amino Acid Sequence↗

PubMed1989

DNA synthesis in CD4- and CD8-positive cells in synovial fluid of patients with reactive and rheumatoid arthritis.

The occurrence of MHC class I antigens and microbial antigens derived from the triggering infection of the diseased joints in reactive arthritis (ReA) seems to set the stage for local immune activation. In this report activated lymphocytes are demonstrated by using an avidin-biotin-peroxidase complex (ABC) method combined with autoradiography that identifies DNA synthesis and, thus, activation. Most of the activated T lymphocytes in reactive arthritis were found to belong to the CD8 suppressor/cytotoxic T-lymphocyte subset. In striking contrast, the majority of the activated T lymphocytes detected in rheumatoid arthritis (RA) synovial fluid belonged to the CD4 helper/inducer subset. These findings agree well with the assumption that CD8-positive cells identify the foreign antigen in the context of class I antigens, whereas CD4-positive cells are found to be associated with the recognition of MHC locus II coded HLA antigens.

Adult↗

PubMed1995

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Amino Acid Sequence↗

PubMed1993

Mutagenic effects of restriction enzymes in Chinese hamster cells: evidence for high mutagenicity of Sau3AI at the hprt locus.

CHO cells were exposed to seven different restriction endonucleases by electroporation and their cytotoxicity and mutagenicity measured. Cell killing as determined by a colony formation assay occurred in a concentration-dependent manner for each enzyme. The D0 of the survival curves were: MspI = 24U; AluI = 31U; Sau3AI = 106U; HaeIII = 46U; HinfI = 30U; PvuII = 35U; BamHI = 163U. BamHI and Sau3AI were particularly ineffective in cell killing. For the 6-base recognition sequence enzymes, PvuII (a blunt-ended cutter) was much more cytotoxic per unit electroporated than BamHI (a sticky-ended cutter). Among the 4-base cutters, Sau3AI and HaeIII were generally less cytotoxic than HinfI or PvuII. Cell killing appeared to depend on the nature of the recognition sequence and cutting sites rather than on the cutting frequency. The mutagenic effects of these restriction endonucleases were investigated by measuring the induced frequencies of hprt gene mutations. The mutagenicity of Sau3AI was dramatically higher than the other enzymes, increasing linearly with dose up to 35U. When normalized for survival, the mutagenicity of Sau3AI relative to the other enzymes was even greater. The mutagenic effect of BamHI, which has the same 5' protruding site as Sau3AI, was much lower at similar dose and survival levels. MspI, BamHI, and PvuII which have no recognition sites within the hprt coding sequence were marginally- or non-mutagenic. Based on these results and the distribution of cutting sites within the hprt cDNA for the enzymes studied, the hypothesis is discussed that a region in exon 4 is highly sensitive to the induction of mutants by DNA double-strand breaks.

Animals↗

PubMed2005

Coevolution of protein and RNA structures within a highly conserved ribosomal domain.

The X-ray crystal structure of a ribosomal L11-rRNA complex with chloroplast-like mutations in both protein and rRNA is presented. The global structure is almost identical to that of the wild-type (bacterial) complex, with only a small movement of the protein alpha helix away from the surface of the RNA required to accommodate the altered protein residue. In contrast, the specific hydrogen bonding pattern of the mutated residues is substantially different, and now includes a direct interaction between the protein side chain and an RNA base edge and a water-mediated contact. Comparison of the two structures allows the observations of sequence variation and relative affinities of wild-type and mutant complexes to be clearly rationalized, but reinforces the concept that there is no single simple code for protein-RNA recognition.

Base Sequence↗

PubMed2006

Recognizing DNA.

It has become clear that there is no simple 'code' for protein-DNA recognition and that selecting an optimal binding sequence along the DNA double helix corresponds to more than simply forming a set of specific hydrogen bonds or steric interactions. However, it has been difficult to characterize the so-called indirect components of recognition. While DNA deformation certainly underlies indirect recognition, it is not easy to determine how local fine structure and deformability depend on base sequence or exactly what percentage of recognition should be attributed to such factors. Molecular modelling can help to develop these ideas into a quantitative model, provided the calculations can be carried out fast enough to enable a comprehensive survey of base-sequence effects. I present here some recent results from our group and their consequences for improving our understanding of protein-DNA binding, and their potential for predicting, and eventually modulating, protein-DNA binding.

Base Sequence↗

PubMed2002

Development and validation of a semiautomatic image analysis system for measuring skin desquamation with D-Squames.

BACKGROUND: D-Squames, have gained wide acceptance for assessing skin desquamation. The amount of corneocytes adhering to D-Squames can be assessed visually by trained observers or by computerized image analysis. Different image analysis algorythms for the evaluation of D-Squames have been published but have not been compared with each other. It was our aim to develop an image analysis system that does not require an expensive image analysis programming tool but should be optimized for routine tasks of analysing large numbers of samples. A second objective of this study was to compare two published image analysis algorythms and visual grading. MATERIAL AND METHODS: The hardware components of the system are a CCD camera connected to a frame grabber card and a light box equipped with fluorescent tubes on two sides that provide a relatively cool, diffuse and even illumination of the sample. The following features were included into the software: generation and identification of bar codes for sample identification; semiautomatic recognition of ROI (region of interest), integration of study design into the analysing process, rapid calculation of desquamation index (DI: integration of the per cent area covered by scales and their thickness distribution) and/or scaling index (SI: distribution of grey values), data storage and export for further analysis. In a first step the system was validated by examining D-Squames covering a wide range of desquamation, by examining different ROI shapes (circle and square), by performing repeat measurements with different positions of the samples and by repeat measurements after re-callibrating the system. In a second step the effect of treatment with different moisturizers was evaluated by the two image analysis parameters DI and SI and compared with hydration measurements (Corneometer). RESULTS: The shape of the ROI showed no influence on the results (variability < 5%). Reproducibility of measurements was satisfactory (COV CDI): 1.7%, COV (SI): 2.6%). There was a good correlation between image analysis results and visual evaluation (means of 3 technicians) (r = 0.986) as well as between the two different image analysis parameters DI and SI (r = 0.971). In the clinical study moisturizer treatment resulted in variable reduction of desquamation that was closely correlated with increase in stratum corneum hydration (r = 0.97). CONCLUSION: Analysing D-Squames with the image analysis system proved to be reproducible, independent of the shape of ROI, cost effective and fast and easy to operate. It has shown to be a suitable and reliable method for the objective determination of desquamation levels.

Emollients↗

PubMed1979

Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy.

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.

Cell Line↗

PubMed1997

[Transcranial color-coded duplex ultrasound in interventional therapy of cerebral aneurysms. A pilot study].

PURPOSE: To assess the diagnostic potential of transcranial colour-coded duplex sonography in the recognition of residual perfusion of coiled cerebral aneurysms. METHOD: 10 patients (7 female, 3 male) with angiographically verified cerebral aneurysms (basilar artery n = 2, mean size 8.5 mm; supraclinoid internal cerebral artery n = 2, mean size 15.5 mm; posterior communicating artery n = 2, mean size 7 mm; anterior communicating artery n = 2, mean size 4.5 mm) were examined by TCCD using a 2.25 MHz probe immediately following embolisation and 12-24 hours prior to angiographic evaluation. RESULTS: 9 patients had a sufficient temporal bone window. Transtemporal localization of the embolised aneurysms was feasible in 7 of the 9 cases. One partially thrombosed aneurysm of the basilar artery (1 mm) and one completely thrombosed aneurysm of the anterior communicating artery could not be visualised. In 7 cases no discrepancies between TCCD and angiography were evident, since rest, perfusion (n = 6) and complete thrombosis were unanimously diagnosed as such. Mean size of the rest perfused lumina was 4.4 mm (minimum 1.5 mm; maximum 8 mm). Platinum coils appeared as hyperechogenic lesions with an echogenicity comparable to the skull. CONCLUSION: With TCCD residual perfusion of coiled aneurysms was feasible in most cases. The method seems to be specific but not sensitive in the detection of incompletely coiled aneurysms. Limitations are insufficient transtemporal bone windows and inaccessible aneurysm location, especially the anterior communicating artery.

Adult↗

PubMed1984

Expression of the sis gene by endothelial cells in culture and in vivo.

Recognition that the sis gene codes for a protein homologous with at least one of the two chains of platelet-derived growth factor has made it possible to directly assess transcriptional expression of platelet-derived growth factor both in cultured cells and in tissue obtained in vivo. We have found that a 3.7-kilobase RNA homologous to the sis gene is expressed at moderate levels in cultured human and bovine endothelial cells, at low levels in in vivo endothelium from human umbilical vein, and at very low levels in bovine aortic endothelium in vivo. This RNA migrates at the same rate as the previously reported sis band in the HUT 102 human T-cell lymphoma line. This band is not found in RNA extracted from freshly obtained bovine aortic media or from human foreskin fibroblasts or cultured fetal human aortic smooth muscle cells. Our in vitro results suggest that the sis gene is responsible for at least part of the platelet-derived growth factor-like mitogenic activity secreted by cultured endothelial cells and indicate that the sis gene is readily activated in endothelial cells during the transition from in vivo conditions to in vitro growth as a monolayer on plastic. Expression of the sis gene by endothelium in vivo raises the possibility that platelet-derived growth factor has a role in the development of the vascular system in the young animal and in the maintenance of the normal vascular system in the adult.

Animals↗

PubMed1986

A peptide to DNA conversion program.

A modification and extension of the computer program REVCUT (Blumenthal et al, Nucl. Acids Res. 10, 91-101 (1982) is described. The new program searches for restriction endonuclease recognition sites that are not coding DNA sequences of a protein of known aminoacid sequence using bit patterns. The modifications make the program more accurate and extend the range of the restriction endonucleases.

Amino Acid Sequence↗

PubMed1996

Characterization of a binding site for the herpes simplex virus type 1 UL9 origin-binding protein within the UL9 gene.

Gene UL9 of herpes simplex virus type 1 (HSV-1) encodes a sequence-specific origin-binding protein (OBP) that plays a direct and essential role in viral DNA synthesis. A search of the complete HSV-1 genomic sequence for possible OBP binding sites lying outside the known origins of replication revealed the presence of a very close match to the OBP recognition sequence within the UL9 coding region. The ability of OBP to bind to this site (referred to as the 'UL9 box') was confirmed by DNase I footprinting and gel retardation assays, and filter binding experiments demonstrated that the affinity of OBP for the UL9 box was of the same order as for its high affinity sites within the three replication origins. To investigate whether binding of OBP to the UL9 box played a role during viral replication we constructed a mutant virus in which the sequence was altered in such a way as to preserve the encoded amino acid sequence whilst abolishing the ability of OBP to bind. Growth of the virus was indistinguishable from wild-type and no alterations were observed in the accumulation of transcripts from the UL9 region of the genome. In addition, a DNA fragment containing the UL9 box sequence did not exhibit origin activity in a transient assay for viral DNA synthesis. We therefore conclude that binding of OBP to the UL9 box is not essential for virus growth and that expression of the UL9 gene is unlikely to be autoregulated through this site.

Base Sequence↗

PubMed1998

High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha.

Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin mapping at nucleotide resolution of the transcriptionally silent HMLalpha and the active MATalpha shows that unique organized chromatin structure characterizes the silent state of HMLalpha. Precisely positioned nucleosomes abutting the silencers extend over the alpha1 and alpha2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MATalpha, is protected at HMLalpha. Although two precisely positioned nucleosomes incorporate transcription start sites at HMLalpha, the promoter region of the alpha1 and alpha2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus. Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III. At the promoter and the HO site, the structure of HMLalpha in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MATalpha. The discontinuous chromatin structure of HMLalpha contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HMLalpha may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HMLalpha may relate to the combined requirements of transcriptional repression and recombinational competence.

Base Sequence↗

PubMed2005

State-of-the-art modified RNAi compounds for therapeutics.

In recent years, the recognition of non-protein coding RNAs as a functional effector of genetic expression has been highlighted by the discovery of RNA interference (RNAi). RNAi is an intracellular phenomenon that enables the eukaryotic cell to utilize double-stranded RNA molecules to silence gene expression in a sequence-specific manner. The short interfering RNA (siRNA) pathway has been intensively investigated and continues to serve as the basis for the development of potent molecular genetic tools. The power of this technology is most clearly evidenced by the fact that siRNA effector molecules can be chemically synthesized and exogenously delivered to specifically target and silence any gene of choice. This capability enables not only basic research, but also opens the door to a new therapeutic modality. Furthermore, the introduction of certain chemical modifications to siRNA effectors can produce a more robust knockdown of gene expression, hence, optimizing serum stability and increasing target specificity yet limiting the induction of cellular stress response, which are key features for in vivo delivery and successful therapeutics. This article outlines the progress in the development of differentially modified siRNA duplexes and their potential role as human therapeutics.

Animals↗