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PubMed2000

Pleomorphic carcinoma of the breast: clinicopathological analysis of 26 cases of an unusual high-grade phenotype of ductal carcinoma.

AIMS: Pleomorphic carcinoma is a poorly described entity whose phenotype is not well recognized as within the morphological spectrum of breast carcinoma. The purpose of this report is to describe the clinicopathological features of this tumour, and to promote its recognition as an unusual high-grade morphological variant of mammary ductal carcinoma. METHODS AND RESULTS: Histological slides of breast carcinomas (N = 64) coded between 1978 and 1995 as having pleomorphic or anaplastic features were reviewed. Pleomorphic carcinoma (N = 26) was diagnosed when > or = 50% of the tumour manifested a pleomorphic cell population (> sixfold variation in nuclear size). Tumours of lobular origin were excluded. All neoplasms occurred in women with a mean age of 53 years. Patients underwent biopsy and/or mastectomy (n = 24) or lumpectomy (n = 2). The tumours' mean size was 54 mm. All were high-grade neoplasms. The pleomorphic cell population comprised 50-100% of the tumour; 31% had a prominent spindled morphology. Fifty-eight per cent of the tumours were initially misclassified by referring pathologists as sarcomas or carcinomas, possibly metastatic. Adjacent DCIS or a transition to classic ductal carcinoma was present in 73%. Five (19%) patients were stage I and three (12%) had stage IV disease. Axillary dissections yielded > or = 3 (mean 7.2) positive lymph nodes in 52%. Most (68%) tumours were aneuploid; a high S-phase (> 10%) was present in 63%. All neoplasms were ER negative and all but one were PR negative. p53 expression was present in 71%; none expressed bcl-2. c-erbB-2 was detected in four (19%) node-positive and in 0 (0%) node-negative cases (P = 0.01). Of 16 patients with follow-up, 6 (38%) were disease-free (mean, 74 months), four (25%) alive with disease (mean, 33 months) and six (38%) dead of disease at a mean of 22 months. CONCLUSIONS: Pleomorphic carcinoma is a prognostically unfavourable lesion and represents the extreme end of the morphological spectrum of grade III infiltrating ductal carcinoma.

Adult↗

PubMed2003

The impact of drugs against dementia on cognition in aging and mild cognitive impairment.

OBJECTIVE: To define the actual knowledge of the impact of drugs for the treatment of dementia on the improvement of cognition in aging and mild cognitive impairment. METHOD: We conducted a Medline search for studies with the parameters drug and cognition. Only drugs that had demonstrated cognition improvements in dementia according to actual methodological standards were included. Drugs had to be approved in at least one American or European nation. RESULTS: Donepezil, galantamine, Ginkgo biloba EGb 761 (definition see editorial), memantine and rivastigmine fulfilled these criteria. There were no systematic investigations on the effects of these drugs on cognition in healthy adults or patients with mild cognitive impairment (MCI). Regarding Ginkgo biloba, two studies on MCI patients could be identified. In healthy adults, we could only include one study investigating donepezil and seven investigating Ginkgo biloba EGb 761. According to the findings, donepezil significantly increased the percentage of REM sleep and REM density, whereas REM latency was reduced. This was interpreted as a sign of improved cognition. Ginkgo biloba EGb 761 improved cognition in healthy controls according to Delayed Free Recall and Delayed Recognition of the WAIS-III and the Color Naming of the Stroop Test as well as in MCI in Digit Copying, Dual Coding Task and speed of response on a computerized version of a classification task. The highest effects were observed after 6 weeks of treatment with 180 mg/d. CONCLUSIONS: Due to the lack of standard procedures for investigating cognition improvement in healthy aging, results have to be interpreted cautiously. In healthy adults as well as in individuals categorized as having MCI, Ginkgo biloba EGb 761 improved cognition in some but not all neuropsychological tests. The single positive result with donepezil raises hope that other drugs may also contribute to cognitive improvement, even in healthy adults. However, the data do not allow any conclusions regarding an improvement following a specific "neuropsychological cluster." Studies on prevention have not been completed so far.

Adult↗

PubMed1983

Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.

Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor.

Amino Acid Sequence↗

PubMed1995

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes o-diphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and Thr-177.

Amino Acid Sequence↗

PubMed2005

Exploring cellular activity of locked nucleic acid-modified triplex-forming oligonucleotides and defining its molecular basis.

Triplex-forming oligonucleotides (TFOs), as DNA-binding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine.oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect.

Base Sequence↗

PubMed2006

Molecular dynamics simulations show that bound Mg2+ contributes to amino acid and aminoacyl adenylate binding specificity in aspartyl-tRNA synthetase through long range electrostatic interactions.

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.

Adenosine Monophosphate↗

PubMed1996

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.

Amino Acid Sequence↗

PubMed1988

Recognition of similar epitopes on varicella-zoster virus gpI and gpIV by monoclonal antibodies.

Two monoclonal antibodies, MAb43.2 and MAb79.0, prepared against varicella-zoster virus (VZV) proteins were selected to analyze VZV gpIV and gpI, respectively. MAb43.2 reacted only with cytoplasmic antigens, whereas MAb79.0 recognized both cytoplasmic and membrane antigens in VZV-infected cells. Immunoprecipitation of in vitro translation products with MAb43.2 revealed only proteins encoded by the gpIV gene, whereas MAb79.0 precipitated proteins encoded by the gpIV and gpI genes. Pulse-chase analysis followed by immunoprecipitation of VZV-infected cells indicated reactivity of MAb43.2 with three phosphorylated precursor species of gpIV and reactivity of MAb79.0 with the precursor and mature forms of gpI and gpIV. These results indicated that (i) MAb43.2 and MAb79.0 recognize different epitopes on VZV gpIV, (ii) glycosylation of gpIV ablates recognition by MAb43.2, and (iii) gpIV is phosphorylated. To map the binding site of MAb79.0 on gpI, the pGEM transcription vector, containing the coding region of the gpI gene, was linearized, and three truncated gpI DNA fragments were generated. RNA was transcribed from each truncated fragment by using SP6 RNA polymerase, translated in vitro in a rabbit reticulocyte lysate, and immunoprecipitated with MAb79.0 and human sera. The results revealed the existence of an antibody-binding site within 14 amino acid residues located between residues 109 to 123 on the predicted amino acid sequences of gpI. From the predicted amino acid sequences, 14 residues on gpI (residues 107 to 121) displayed a degree of similarity (36%) to two regions (residues 55 to 69 and 245 to 259) of gp IV. Such similarities may account for the binding of MAb79.0 to both VZV gpI and gpIV.

Amino Acid Sequence↗

PubMed2004

Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.

A recognition site for the cAMP-dependent protein kinase was introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make a variant of MAb-chCC49 containing a highly stable phosphate. To design this monoclonal antibody (MAb) without changing its immunoreactivity or biological properties, molecular modeling was used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. We selected one position to mutate on the heavy chain based on molecular dynamics study of the solvated antibody. A vector expressing the mutant was constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5. MAb-WW5 contained the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity, and retained the phosphate stably. Compared with MAb-chCC49K1, another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5 showed much improved stability: about a 10-fold increase in resistance to hydrolysis. MAb-WW5 exhibited the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells as we observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.

Adenocarcinoma↗

PubMed1991

Chimeric gamma-delta signal joints. Implications for the mechanism and regulation of T cell receptor gene rearrangement.

Rearrangement of Ag receptor genes requires recognition by the lymphocyte recombinase of heptamer-nonamer signal sequences followed by two endonucleolytic cleavages and two DNA ligations to form the coding and signal joints. The phenomenon of trans-rearrangement, in which Ag receptor gene segments located on different chromosomes recombine to yield chimeric products, provides an in vivo system in which to investigate the ability of the recombinase to carry out each of these functions in trans. Trans-rearrangements between TCRG and TCRD loci, similar in structure and frequency to those observed previously in human lymphoid tissues, were demonstrated in normal mouse thymus by PCR with crossed V gamma/J delta and V delta/J gamma primer pairs. A simple mechanistic model for trans-rearrangement was then tested. This model posits an ability of the recombinase to catalyze the formation of both coding and signal joints in trans and therefore predicts that trans-rearrangements will generate chimeric signal joints. In adult thymus, chimeric D delta 2-J gamma 1 and D delta 2-J gamma 2 signal joints, containing fused heptamer-nonamer sequences, could be detected by PCR and were each present at frequencies sufficient to account for a large proportion of the corresponding TCRG/TCRD trans-rearrangements. In agreement with the predictions of the model, chimeric signal joints were found as both linear chromosomal and circular episomal DNA. The results provide a framework for understanding the formation of chromosomal translocations in normal and neoplastic lymphoid cells and support the possibility of a looping mechanism for standard gene rearrangement. To test the form of regulation of TCRG rearrangement, the frequencies of specific signal joints from standard and trans-rearrangements were compared. Although J gamma 1 and J gamma 2 segments participated with equal frequency in trans-rearrangement with D delta 2, only the J gamma 1 segment participated in standard rearrangement with V gamma 5. The results suggest that V-J recombination in the TCRG locus is regulated directly at the DNA level by cis-acting constraints which do not affect the accessibility of individual TCRG gene segments to recombination in trans.

Animals↗

PubMed1991

Identification of novel rhodopsin mutations associated with retinitis pigmentosa by GC-clamped denaturing gradient gel electrophoresis.

Retinitis pigmentosa (RP) is a group of disorders characterized by progressive degeneration of the outer retina, resulting in night blindness, visual field loss, an abnormal electroretinogram, and characteristic retinal pigmentary changes. An important step in the understanding of RP has been the recognition that some cases of autosomal dominant RP (ADRP) are caused by mutations in the rhodopsin gene. Multiple different point mutations within the coding sequence of the rhodopsin gene have been associated with ADRP. We have developed a GC-clamped denaturing-gradient-gel electrophoresis (DGGE) assay for the coding region of the rhodopsin gene and have used this assay to screen ADRP patients for mutations. The assay consists of amplifying with PCR the five exons of the rhodopsin gene and then analyzing each PCR product by DGGE. We have used this assay to detect three previously unreported rhodopsin base substitutions associated with ADRP. The use of this assay to identify ADRP patients who have various rhodopsin mutations has allowed us to begin studies seeking to correlate molecular genotype with clinical phenotype. Furthermore, GC-clamped DGGE has allowed us to identify families with ADRP not caused by a rhodopsin mutation. Such families will be important in the search for other genes involved in ADRP.

Amino Acid Sequence↗

PubMed1983

[Relations between Ia antigens and the products of Ir genes. A theory of the evolution of the immune response].

To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting an helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

Animals↗

PubMed1983

[Relation between Ia antigens and Ir gene products. A theory on the development of the immune response].

To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting a helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

Animals↗

PubMed1988

A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite.

As a consequence of gene cloning and DNA sequencing several gene families are emerging in the field of cell-cell recognition. These include immunoglobulins, integrins, certain extracellular glycoproteins and a family of functionally unrelated proteins which include factor B. We report here the cloning and sequencing of a gene from Plasmodium falciparum, coding for a protein we call thrombospondin related anonymous protein (TRAP), which shares certain sequence motifs common to other well-characterized proteins. The most significant homology is based around the sequence Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), present in three copies in region I of thrombospondin (TSP), six copies in properdin (P) and one copy in all the circumsporozoite (CS) proteins sequenced so far. TRAP also shares with certain extracellular glycoproteins, including TSP, the cell-recognition signal Arg-Gly-Asp (RGD), which has been shown to be crucial in the interaction of several extracellular glycoproteins with members of the integrin superfamily. Unlike the CS protein, TRAP is expressed during the erythrocytic stage of the parasite life cycle.

Amino Acid Sequence↗

PubMed1990

The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA.

E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the E1A transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription.

Adenovirus Early Proteins↗

PubMed2005

Screening for protease substrate by polyvalent phage display.

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.

Antibody Affinity↗

PubMed2001

Identification and analysis of Arabidopsis expressed sequence tags characteristic of non-coding RNAs.

Sequencing of the Arabidopsis genome has led to the identification of thousands of new putative genes based on the predicted proteins they encode. Genes encoding tRNAs, ribosomal RNAs, and small nucleolar RNAs have also been annotated; however, a potentially important class of genes has largely escaped previous annotation efforts. These genes correspond to RNAs that lack significant open reading frames and encode RNA as their final product. Accumulating evidence indicates that such "non-coding RNAs" (ncRNAs) can play critical roles in a wide range of cellular processes, including chromosomal silencing, transcriptional regulation, developmental control, and responses to stress. Approximately 15 putative Arabidopsis ncRNAs have been reported in the literature or have been annotated. Although several have homologs in other plant species, all appear to be plant specific, with the exception of signal recognition particle RNA. Conversely, none of the ncRNAs reported from yeast or animal systems have homologs in Arabidopsis or other plants. To identify additional genes that are likely to encode ncRNAs, we used computational tools to filter protein-coding genes from genes corresponding to 20,000 expressed sequence tag clones. Using this strategy, we identified 19 clones with characteristics of ncRNAs, nine putative peptide-coding RNAs with open reading frames smaller than 100 amino acids, and 11 that could not be differentiated between the two categories. Again, none of these clones had homologs outside the plant kingdom, suggesting that most Arabidopsis ncRNAs are likely plant specific. These data indicate that ncRNAs represent a significant and underdeveloped aspect of Arabidopsis genomics that deserves further study.

Algorithms↗

PubMed2001

Neuropsychological evidence for case-specific reading: multi-letter units in visual word recognition.

We describe a patient (GK) who shows symptoms associated with Balint's syndrome and attentional dyslexia. GK was able to read words, but not nonwords. He also made many misidentification and mislocation errors when reporting letters in words, suggesting that his word-naming ability did not depend upon preserved position-coded, letter identification. We show that GK was able to read lower-case words better than upper-case words, but upper-case abbreviations better than lower-case abbreviations. Spacing the letters in abbreviations disrupted identification, as did mixing the case of letters within words. These data cannot be explained in terms of letter-based reading or preserved holistic word recognition. We propose that GK was sensitive to the visual familiarity of adjacent letter forms.

Adult↗