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At least 523 records · Page 29Linked to original sources
PubMed2006

Comprehensive thermodynamic analysis of 3' double-nucleotide overhangs neighboring Watson-Crick terminal base pairs.

Thermodynamic parameters are reported for duplex formation of 48 self-complementary RNA duplexes containing Watson-Crick terminal base pairs (GC, AU and UA) with all 16 possible 3' double-nucleotide overhangs; mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest-neighbor analysis, the addition of a second dangling nucleotide to a single 3' dangling nucleotide increases stability of duplex formation up to 0.8 kcal/mol in a sequence dependent manner. Results from this study in conjunction with data from a previous study [A. S. O'Toole, S. Miller and M. J. Serra (2005) RNA, 11, 512.] allows for the development of a refined nearest-neighbor model to predict the influence of 3' double-nucleotide overhangs on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against five oligomers with various core duplex sequences. Phylogenetic analysis of naturally occurring miRNAs was performed to support our results. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent upon the identity of the 3' double-nucleotide overhang. Thermodynamic parameters for 3' single terminal overhangs adjacent to a UA pair are also presented.

Adenine↗
PubMed2006

Comprehensive immunohistological analysis of the endothelin system in human kidney grafts.

BACKGROUND: In experimental models of renal transplantation, upregulation of the endothelin (ET) system and amelioration of renal injury by ET-receptor blockers have been documented. In contrast, little information is available on the expression of the ET system in human kidney allografts. It was the purpose of the present study to analyse by immunohistology the expression of ET-1 as well as of the two ET receptors (ET-RA and ET-RB) in the different cells and compartments of kidney grafts and control kidneys. METHODS: Fifty-five graft biopsies were taken from 55 kidney allograft recipients (mean age: 32+/-2.8 years) who were all on a calcineurin inhibitor. The indication for biopsies was delayed graft function or suspected rejection. The underlying diagnoses were acute allograft rejection (n = 14), chronic allograft nephropathy (n = 14), cyclosporin A (CSA) toxicity (n = 10), post-operative acute tubular necrosis (ATN) (n = 11) and recurrent primary disease (n = 6). As control, tissues of non-grafted kidneys with ATN (mean age: 35+/-24 years), of primary glomerulonephritis (mean age: 69+/-10 years) and of non-tumour-bearing parts of eight tumour nephrectomy specimens (mean age: 67+/-5 years) were assessed. The biopsies were scored using the 1997 Banff criteria. Expression of ET-1, ET-RA and ET-RB as well as of vascular endothelial growth factor was evaluated by immunohistochemistry and a semi-quantitative scoring system. Interstitial infiltrating cells were characterized using antibodies against T cells, B cells and macrophages (CD3, CD20 and CD68). RESULTS: Control cases showed only faint expression of ET-1 in glomeruli (in podocytes and endothelial cells), whereas marked expression was seen in distal, but less in proximal tubular cells. The interstitium was completely negative. ET-1 expression was seen in vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC). Only faint expression of ET-RA and ET-RB was found in glomeruli and tubuli (distal more than proximal). Marked ET-RA and ET-RB expression was seen in VEC and VSMC. In all transplanted kidneys, irrespective of the underlying diagnosis, expression of ET-1, ET-RA and ET-RB was markedly higher compared with control kidneys. ET-1 was strikingly upregulated in glomeruli and tubuli, but surprisingly not in the vasculature of grafts with CSA toxicity. Expression of ET-RB was markedly increased in CSA toxicity in glomeruli, tubuli and vessels. In grafts with ATN and acute rejection, pronounced expression of ET-RA was noted. There was a strong correlation between proteinuria and expression of ET-1 in glomeruli and proximal tubuli and of ET-RB in proximal tubuli. CONCLUSIONS: The above data in human kidney allograft biopsies are consistent with an important role of the ET system in different types of renal allograft damage. This finding extends and clarifies the somewhat contradictory results in animal models.

Acute Disease↗
PubMed2001

A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes allowed us to predict cis-regulatory regions involved in cell-wall construction in specific organs of Arabidopsis.

The Arabidopsis thaliana genome sequencing project has revealed that multigene families, such as those generated by genome duplications, are more abundant among plant genomes than among animal genomes. To gain insight into the evolutionary implications of the multigene families in higher plants, we examined the XTH gene family, a group of genes encoding xyloglucan endotransglucosylase/hydrolase, which are responsible for cell-wall construction in plants. Expression analysis of all members (33 genes) of this family, using quantitative real-time RT-PCR, revealed that most members exhibit distinct expression profiles in terms of tissue specificity and responses to hormonal signals, with some members exhibiting similar expression patterns. By comparing the flanking sequences of individual genes, we identified four sets of large-segment duplications and two sets of solitary gene duplications. In each set of gene duplicates, long nucleotide sequences, ranging from one to two hundred base pairs, are conserved. Furthermore, gene duplicates exhibit similar organ-specific expression profiles. These facts allowed us to predict putative cis-regulatory regions, particularly those responsible for cell-wall construction, and hence for morphogenesis, that are specific for certain organs or tissues in plants.

Arabidopsis↗
PubMed2003

Sentinel lymph node biopsy has no benefit for patients with primary cutaneous melanoma metastatic to a lymph node: an assertion based on comprehensive, critical analysis: part I.

The thesis is set forth in this treatise that there is no place in the routine practice of medicine for the procedure for melanoma known conventionally and universally as sentinel node biopsy. Our assertion is based on assessment of the extensive body of literature devoted to the subject of treatment of melanoma before any metastasis has manifested itself clinically and of that dedicated to therapy for overt metastatic melanoma by a variety of modalities, chief among those addressed here being elective lymph node dissection and sentinel lymph node biopsy. In this era of sentinel lymph node biopsy, elective lymph node dissection has been modified to include only patients with metastasis of melanoma to lymph nodes, a procedure now termed "selective complete lymph node dissection." Among adjuvant medical therapies, the most popular today is interferon alpha-2B. Critical, incisive scrutiny of the literature leads to the conclusion, incontrovertibly, that elective lymph node dissection has no benefit for a patient and that all modifications of it also are devoid of value. The reason, logically, for the lack of utility of elective lymph node dissection becomes apparent by virtue of the route taken by cells of melanoma as they metastasize; those cells proceed in the same fashion as does lymph, bacteria, foreign material (including vital dyes and radioactive tracers), and other kinds of cells, to wit, by passing rapidly through nodes, including the sentinel one, and even bypassing entirely the nodes. In reality, cells of metastatic melanoma are not held up in nodes for any significant period of time, contrary to what is asserted repeatedly, but without any basis in fact, by many students of the subject. Moreover, not a single adjuvant medical therapy available currently is effective against metastatic melanoma and, therefore, none of them should be invoked to justify performance of sentinel node biopsy. Even if the sentinel node is found to house cells of melanoma, which, as a rule, conveys a grim message regarding the future, the finding in an individual patient is meaningless; a particular patient may live in harmony with metastases of melanoma for more than 30 years and even die of an unrelated malady. In short, no surgeon, pathologist, or oncologist is a seer, diviner, or prophet when it comes to predicting accurately the outcome for a patient with metastasis of melanoma; the end could come in weeks, months, or decades. If, however, a sentinel node is found to contain nary a cell of metastatic melanoma, it, too, means nothing for an individual patient because the existence of metastases widely is not excluded by that finding. In short, sentinel node biopsy cannot be considered the standard of care in the daily practice of medicine; it is woefully substandard because it is without benefit. There is no justification, whatsoever, for the procedure, scientifically or practically, and for that reason it should be abandoned, without delay, now.

Female↗
PubMed2002

Comprehensive genotypic analysis of leukemia: clinical and therapeutic implications.

Over the past several years, the application of a spectrum of cytogenetic and molecular diagnostic techniques has dramatically improved our understanding of the pathophysiology of leukemia. These techniques include chromosomal translocations visualized by G-banding techniques, fluorescence in-situ hybridization, spectral karyotyping, comparative genomic hybridization, loss of heterozygosity analysis, and characterization of point mutations by DNA sequence analysis. We will review the application of these techniques, update novel findings utilizing these techniques over the past year as they apply to specific leukemias, and review the clinical and therapeutic implications of these findings.

Genotype↗
PubMed1998

A functional MRI analysis of comprehension processes of Japanese sentences.

We tried to identify the mechanisms directly related to syntactic processing in an fMRI experiment using strictly controlled sets of verbal stimuli. In two conditions, center-embedding and left-branching conditions, the same sets of words were used to construct stimulus sentences, while only the word order reflecting their syntactic structure was different. The subject's task was to understand the relationship among three characters mentioned in the sentences. A difference of activation in Broca's area (BA44 and 45) between the two conditions was found. This result suggests that these areas are involved in syntax-related processing. Furthermore, it was suggested that the posterior part of the frontal lobe (BA6/9) and the inferior parietal area (BA39/40) are involved in the understanding of the relationship among the three characters mentioned in each sentence.

Frontal Lobe↗
PubMed2006

Four novel defective alleles and comprehensive haplotype analysis of CYP2C9 in Japanese.

Genetic variations in cytochrome P450 2C9 (CYP2C9) are known to contribute to interindividual and interethnic variability in response to clinical drugs such as warfarin. In the present study, CYP2C9 from 263 Japanese subjects was resequenced, resulting in the discovery of 62 variations including 32 novel ones. In addition to the two known non-synonymous single nucleotide polymorphisms (SNPs), Ile359Leu (*3; allele frequency=0.030) and Leu90Pro (*13; 0.002), seven novel non-synonymous SNPs, Leu17Ile (0.002), Lys118ArgfsX9 (*25; 0.002), Thr130Arg (*26; 0.002), Arg150Leu (*27; 0.004), Gln214Leu (*28; 0.002), Pro279Thr (*29; 0.002) and Ala477Thr (*30; 0.002), were found. Functional characterization of novel alleles using a mammalian cell expression system in vitro revealed that *25 was a null allele and that *26, *28 and *30 were defective alleles. The *26 product showed a 90% decrease in the Vmax value but little change in the Km value towards diclofenac. Both *28 and *30 products showed two-fold higher Km values and three-fold lower Vmax values than the *1 allele, suggesting the importance of Gln214 and Ala477 for substrate recognition. Linkage disequilibrium and haplotype analyses were performed using the detected variations. Only five haplotypes (frequency >0.02) accounted for most (>87%) of the inferred haplotypes, and they were closely associated with the haplotypes of CYP2C19 in Japanese. Although the haplotype structure of CYP2C9 was rather simple in Japanese, the haplotype distribution was quite different from those previously reported in Caucasians and Africans. Taken together, novel defective alleles and detailed haplotype structures would be useful for determining metabolic phenotypes of CYP2C9 substrate drugs in Japanese and probably Asians.

Alleles↗
PubMed1991

A comprehensive immunological analysis in chronic fatigue syndrome.

A detailed analysis of cell-mediated and antibody-mediated immunity was performed in 20 CDC-defined patients with chronic fatigue syndrome (CFS) and 20 age- and sex-matched healthy controls. CD3+, CD4+, CD8+, and CD20+ lymphocytes were comparable in two groups. Natural killer cells as defined by CD16, CD56 and CD57 antigens were significantly reduced in CFS. A significant increase in the proportions of CD4+ ICAM 1+ T cells was observed in CFS. Monocytes from CFS displayed increased density (as determined by mean fluorescence channel numbers) of intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function associated antigen 1 (LFA-1), but showed decreased enhancing response to recombinant interferon-gamma in vitro. The lymphocyte DNA synthesis in response to phytohaemoglobulin (PHA), Concanavalin A (Con A) and pokeweed mitogen (PWM) was normal but the response to soluble antigens was significantly reduced. Serum IgM, IgG, IgA, and IgG subclasses were normal. In vivo specific antibody response to pneumococcus vaccine was depressed in CFS. Forty percent of patients showed titres of anti-human herpes virus 6 (anti-HHV-6) antibody higher than that in the controls (greater than or equal to 1/80). These data suggest immunological dysfunction in patients with chronic fatigue syndrome. The significance of these observations is discussed.

Adult↗
PubMed2004

Comprehensive EST analysis of tomato and comparative genomics of fruit ripening.

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.

Arabidopsis↗
PubMedDate not supplied

Comprehensive genetic analysis of cancer cells.

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.

Journal Article↗
PubMed2005

Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking.

Arthropod cuticles play an important role as the first barrier against invading pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS/PAGE. Proteins appearing on the gel were designated high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so-called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins designated acidic DE25 and DE29 each contained a Cys-rich domain with sequences similar to those of insect peritrophic matrix proteins and chitinases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin-binding proteins. Alternatively, a low molecular mass chitin-binding fraction was prepared by size exclusion chromatography, and 15 low molecular mass chitin-binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz-type chymotrypsin inhibitor sequence, and P15 contained a Cys-rich motif found in insulin-like growth factor-binding proteins. Interestingly, we observed transglutaminase-dependent polymerization of nearly all high molecular mass chitin-binding proteins, a finding suggests that transglutaminase-dependent cross-linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals.

Animals↗
PubMed2001

Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein.

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

Amino Acid Sequence↗
PubMed2005

Comprehensive bioinformatic analysis of the specificity of human immunodeficiency virus type 1 protease.

Rapidly developing viral resistance to licensed human immunodeficiency virus type 1 (HIV-1) protease inhibitors is an increasing problem in the treatment of HIV-infected individuals and AIDS patients. A rational design of more effective protease inhibitors and discovery of potential biological substrates for the HIV-1 protease require accurate models for protease cleavage specificity. In this study, several popular bioinformatic machine learning methods, including support vector machines and artificial neural networks, were used to analyze the specificity of the HIV-1 protease. A new, extensive data set (746 peptides that have been experimentally tested for cleavage by the HIV-1 protease) was compiled, and the data were used to construct different classifiers that predicted whether the protease would cleave a given peptide substrate or not. The best predictor was a nonlinear predictor using two physicochemical parameters (hydrophobicity, or alternatively polarity, and size) for the amino acids, indicating that these properties are the key features recognized by the HIV-1 protease. The present in silico study provides new and important insights into the workings of the HIV-1 protease at the molecular level, supporting the recent hypothesis that the protease primarily recognizes a conformation rather than a specific amino acid sequence. Furthermore, we demonstrate that the presence of 1 to 2 lysine residues near the cleavage site of octameric peptide substrates seems to prevent cleavage efficiently, suggesting that this positively charged amino acid plays an important role in hindering the activity of the HIV-1 protease.

Algorithms↗
PubMed2006

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis.

The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

Alleles↗
PubMed2006

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases involved in large ribosomal subunit biogenesis.

DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.

Alleles↗
PubMed2025

Comprehensive genomic analysis of antibiotic resistance plasmids in animal-associated Staphylococcus aureus in France.

UNLABELLED: In Staphylococcus aureus, an animal pathogen and zoonotic agent, plasmids play a pivotal role in the acquisition and spread of antibiotic resistance genes (ARGs). This study investigated the plasmid content of 329 S. aureus isolates from livestock and companion animals collected in France between 2010 and 2021. Plasmids (n = 211) were identified from 139 isolates. The major families identified-rep7a, rep20, and rep10-were associated with specific resistance genes (str, cat, blaZ, erm(C)) and exhibited widespread horizontal transfer across different S. aureus sequence types (STs) and animal hosts. In temporal analysis, the rep7a/str and rep7a/cat plasmids circulating in horses were progressively replaced by a rep7a plasmid carrying both str and cat genes. The study also highlighted the presence of mosaic plasmids, which combined elements from different bacterial species/genera, confirming the broad host range of S. aureus plasmids and their ability to acquire ARGs from diverse sources. Moreover, the occurrence of hybrid plasmids (carrying multiple rep genes) underscores the plasticity of these vectors of ARGs. This study emphasizes the need to investigate the mechanisms driving the spread and persistence of antibiotic-resistant plasmids in S. aureus, with a view to developing strategies aimed at combating antibiotic resistance. IMPORTANCE: The spread of antibiotic resistance in Staphylococcus aureus is a growing concern, particularly in animals that can serve as reservoirs for resistant strains. This study highlights the crucial role of plasmids in transmitting resistance genes among different animal hosts and S. aureus lineages. The characterization of 329 isolates collected over 10 years revealed how certain plasmid families are associated with specific resistance genes and how they evolve over time. The occurrence of mosaic and hybrid plasmids further underscores the ability of S. aureus to acquire resistance from diverse bacterial sources. These findings provide key insights into the mechanisms shaping antibiotic resistance in this pathogen and emphasize the fact that understanding plasmid-driven resistance is essential for developing effective interventions to limit the spread of multidrug-resistant S. aureus in both veterinary and human medicine.

Animals↗