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At least 1,423 records · Page 79Linked to original sources
PubMed1997

Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization.

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.

Cell Line↗
PubMed1996

The genomic structure of the murine alpha 4 integrin gene.

The VLA-4 (alpha 4 beta 1) integrin is a leukocyte glycoprotein involved in both cell-extracellular matrix and cell-cell interactions. We report here the cloning of the murine alpha 4 gene whose protein product is antigenically related to the human VLA-4 alpha chain. The alpha 4 m gene is about 75 kb long and consists of 28 exons, ranging in size from 46 bp (exon 13) to 437 bp (exon 1). The introns varied from 79 bp (intron 8) to more than 17 kb (intron 2). Three mRNA transcripts from this alpha 4 m gene can be visualized on Northern blot. After cloning the 3' untranslated region (3' UTR), four polyadenylation sites could be identified, presumably responsible for the presence of three to four transcripts of the alpha 4 gene, differing substantially in length.

Animals↗
PubMed1998

[Progress in molecular medicine: "laser capture microdissection"].

BACKGROUND: With the unravelling of the human genome, we now face the challenge of defining the function and clinical relevance of single genes. To do this, we should be able to isolate normal and diseased cells from complex tissue structures to make them accessible to sensitive molecular analyses. Laser Capture Microdissection (LCM) was developed to meet this challenge. METHOD AND APPLICATION: LCM allows the precise dissection of cells with the help of a laser beam under direct visualization in the microscope, and the sterile transfer of these cells into a DNA or RNA isolation buffer. The technique is ideal for investigating cell-cell interactions, for performing mutation analyses, and for the production of high-quality cDNA libraries. Expression studies of known and unknown genes are currently employed successfully to define tissue- and simple cell-specific patterns which help elucidate the etiology and pathogenesis of colon, lung, breast, prostate, adrenal, ovary, and other organ tumors. The LCM system developed at the NIH is, therefore, an important part of the Cancer-Genome Anatomy Project (CGAP), which sequences and publishes the structures of genes that are expressed in human tumors. In combination with the modern cDNA arrays, it will thus be possible to analyze the expression of several thousands of genes in one step and to develop individual therapeutic strategies in the not too distant future. CONCLUSIONS: The LCM is a major advance in molecular medicine, enabling us to combine highly-sensitive gene analysis techniques with conventional histologic and morphologic methods. Applications range from research to diagnosis, and to monitoring disease progression.

Cell Separation↗
PubMed2002

Leber's hereditary optic neuropathy: clinical and molecular genetic results in a patient with a point mutation at np T11253C (isoleucine to threonine) in the ND4 gene and spontaneous recovery.

BACKGROUND: Mitochondrial DNA mutations at nucleotide position (np) 3460 in the ND1 gene, np 11778 in the ND4 gene, and np 14484 in the ND6 gene are commonly considered to be associated with the clinical features of LHON and account for the majority of LHON cases. Here we report the clinical and molecular genetic findings of a LHON patient with a new mitochondrial DNA mutation at np 11253 in the ND4 gene and spontaneous recovery. METHODS: The clinical examination consisted of visual acuity measurements, visual field testing, and ophthalmoscopy over a period of 14 years. Total lymphocyte DNA was analyzed for all common LHON mutations. Because the LHON patient did not harbor any of the common or recently described rare LHON mutations, we performed a sequence analysis of the whole mitochondrial genome. RESULTS: The patient exhibited typical clinical features of LHON. Molecular genetic analysis did not reveal any of the common LHON mutations. Sequence analysis of the mtDNA of the patient and his unaffected sister and niece was performed and showed a T to C missense mutation at np 11253 in the ND4 gene, leading to a replacement of an evolutionary highly conserved isoleucine by a threonine residue. This mutation introduces a polar group into a hydrophobic domain of the protein and induces a significant change in hydrophobicity of the peptide sequence. The mutation was not found among 100 controls. CONCLUSION: The fact that the new mutation at np 11253 is found within a highly conserved region and was not present in any controls implies that this mutation is responsible for LHON in this patient. Interestingly, this point mutation has formerly been reported in the mitochondria of the substantia nigra in an unrelated patient with proven Parkinson's disease.

Adult↗
PubMed1988

Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes.

To investigate the serotypic and genetic diversity of human rotavirus strains, we have tested 513 and 519 fecal rotavirus specimens, respectively, by an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies and by polyacrylamide gel electrophoresis of the segmented RNA genome. Of the 513 specimens, 375 were typed as serotype 1 (47.3%), serotype 2 (2.9%), serotype 3 (2.9%), or serotype 4 (17.7%). In addition, a presumptive new human serotype, tentatively referred to as serotype X in this paper, was found in 1.6% of the specimens tested. The remaining 138 specimens (26.9%) were untypeable. Considerable variation in relative frequency of circulating serotypes was observed with respect to geographic locations and observation periods. Rotavirus RNAs were visualized in 481 of 519 specimens tested. Of these, 415 were typed as 33 electropherotypes, many of which were infrequently detected and were restricted to single epidemics. Analysis of the 291 specimens whose electropherotypes and serotypes were available indicated clearly that a given RNA pattern always corresponded to a particular serotype. Heterogeneity of electropherotypes within a serotype was similarly observed in strains belonging to the four previously established serotypes. The results obtained in this study indicated that antigenic changes on the major neutralization antigen occurred always with concurrent changes of genomic RNA electropherotypes. On the other hand, serotypic changes could not be predicted from the changes in RNA electropherotypes.

Animals↗
PubMed2006

Bigenomic functional regulation of all 13 cytochrome c oxidase subunit transcripts in rat neurons in vitro and in vivo.

Cytochrome c oxidase is a multisubunit, bigenomically encoded inner mitochondrial membrane protein. Its enzymatic activity and amount in the brain vary with metabolic demands, but the precise regulation of all 13 subunits to form a functional holoenzyme in a 1:1 stoichiometry is not well understood. To determine if all 13 subunit transcripts were coordinately regulated by functional alteration in neurons, cultured primary neurons were depolarized by potassium chloride for 5-24 h, or tetrodotoxin inactivated for 2-6 days. In vivo studies were done on rats monocularly enucleated for 4 days to 2 weeks. Expressions of cytochrome c oxidase subunit mRNAs were measured by real-time quantitative polymerase chain reaction. Results showed that in vitro, all 13 transcripts were significantly up-regulated after 5 h of depolarizing stimulation. With tetrodotoxin blockade, however, the three mitochondrial-encoded transcripts were down-regulated earlier than the 10 nuclear ones (2 days versus 4 days). In vivo, all three mitochondrial-encoded subunit mRNAs were also down-regulated earlier than the nuclear ones in deprived visual cortex (4 days versus 1 week after monocular enucleation). Cytochrome c oxidase activity and protein levels were significantly decreased in parallel after 4 days of deprivation in vitro and 1 week in vivo. Our results are consistent with a coordinated mechanism of up-regulation of all 13 transcripts in response to functional stimulation, but an earlier and more severe down-regulation of the mitochondrial transcripts than the nuclear ones in response to functional deprivation. Thus, the mitochondrial subunits may play a more important role in regulating cytochrome c oxidase protein amount and activity in neurons. Our results also point to the need of all 13 subunits to form a functional holoenzyme.

Analysis of Variance↗
PubMed1993

Rapid genomic typing of BK virus directly from clinical specimens.

A simple and rapid method, PCR-restriction enzyme analysis (PCR-RE) for BK virus (BKV) typing was developed, based on the presence of type-specific restriction enzyme sites in a 327 bp PCR-generated fragment which partially encodes the VP1 protein. The enzymes, Alu I, Xmn I and Ava II, were used to digest the PCR products in two stages. Ethidium bromide banding patterns characteristic of each of four subtypes of BKV were visualized through gel electrophoresis. A total of 37 samples from clinical specimens and culture fluids were successfully subtyped using the PCR-RE. A second method, PCR-sequencing, was applied to samples that generated less than 100 ng of DNA. These were subjected to a second round of PCR and then sequenced from single stranded templates immobilised via a biotinylated primer. The subtypes were assigned on the basis of the type-specific sequences previously characterized.

BK Virus↗
PubMed1999

Application of the RLGS image analysis tool (RAT) to the construction of a genetic linkage map of recombinant inbred strain SMXA.

The construction of a genetic linkage map is the first, fundamental step to analyze the genetic properties of any organism. For this purpose, the restriction landmark genome scanning method (RLGS) can be used and has been shown to have high productivity in various genetic analyses. However, construction of a genetic linkage map by the RLGS method is laborious, because hundreds of spots must be scored, usually by visual observation. In order to reduce human involvement in the data processing, we developed an image analysis software, RAT (RLGS Analysis Tool). We evaluated its accuracy and feasibility by comparing the parental distribution patterns of RLGS spots obtained by RAT and by human observation, using Syrian hamster strain backcross progeny. We then used RAT to construct a genetic linkage map of the recombinant inbred strain SMXA. We were able to obtain 121 progenitor strain-specific spots that were assigned to a specific chromosome.

Animals↗
PubMed2004

Peptidomics.

Peptides occur in the whole animal kingdom, from the least evolved phyla with a very simple nervous system (coelenterates) to the highest vertebrates and are involved in most, if not all, physiological processes in animals. Knowing the amino acid sequence of peptide hormones or neurotransmitters is important since this allows for synthesis of large quantities of peptides to perform further functional analysis. Immunocytochemistry, radioimmunoassays (RIA), enzyme-linked immunosorbant assays (ELISA) and mass spectrometry can then provide information on the temporal and spatial distribution and quantification of the (neuro)peptide. Ever since the 1970s, a wealth of peptides has been discovered and investigated and this flow seems to be far from over. This is partially due to the use of new approaches mainly based on chromatographical purifications as well as molecular biological techniques. Surprisingly, peptides have so far been neglected in most proteomic studies. The finalization of the genome projects has opened new opportunities for rapid identification and functional analysis of (neuro)peptides as well. In analogy with the proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications (PTMs). This technology provides us with a fast and efficient tool to analyze the peptides from any tissue. This paper reviews the approaches that have been used so far to achieve this.

Amino Acid Sequence↗
PubMed2006

Engineering stem cells into organs: topobiological transformations demonstrated by beak, feather, and other ectodermal organ morphogenesis.

To accomplish regenerative medicine, several critical issues in stem cell biology have to be solved, including the identification of sources, the expanding population, building them into organs, and assimilating them to the host. Although many stem cells can now differentiate along certain lineages, knowledge on how to use them to build organs lags behind. Here we focus on topobiological events that bridge this gap, for example, the regulation of number, size, axes, shape, arrangement, and architecture during organogenesis. Rather than reviewing detail molecular pathways known to disrupt organogenesis when perturbed, we highlight conceptual questions at the topobiological level and ask how cellular and molecular mechanisms can work to explain these phenomena. The avian integument is used as the Rosetta stone because the molecular activities are linked to organ forms that are visually apparent and have functional consequences during evolution with fossil records and extant diversity. For example, we show that feather pattern formation is the equilibrium of stochastic interactions among multiple activators and inhibitors. Although morphogens and receptors are coded by the genome, the result is based on the summed physical-chemical properties on the whole cell's surface and is self-organizing. For another example, we show that developing chicken and duck beaks contain differently configured localized growth zones (LoGZs) and can modulate chicken beaks to phenocopy diverse avian beaks in nature by altering the position, number, size, and duration of LoGZs. Different organs have their unique topology and we also discuss shaping mechanisms of liver and different ways of branching morphogenesis. Multi-primordium organs (e.g., feathers, hairs, and teeth) have additional topographic specificities across the body surface, an appendage field, or within an appendage. Promises and problems in reconstitute feather/hair follicles and other organs are discussed. Finally, simple modification at the topobiological level may lead to novel morphology for natural selection at the evolution level.

Animals↗
PubMed1997

Molecular evolution of the rhodopsin gene of marine lamprey, Petromyzon marinus.

Visual pigment genes have been isolated from a marine lamprey, Petromyzon marinus. We report here the rhodopsin gene, spanning 21.2 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Southern analysis suggests that the lamprey genome contains a single rhodopsin gene. The amino acid (aa) sequence deduced from this gene has 92% sequence similarity with that of the river lamprey rhodopsin. The data reveal that aa substitutions occurred more often in the transmembrane region than in the non-transmembrane region, possibly reflecting functional adaptation of the rhodopsin during the last 500 million years of the jawless fish evolution.

Amino Acid Sequence↗
PubMed1996

Application of molecular biological techniques for detection of epizootic hemorrhagic disease virus (EHDV-318) recovered from a sentinel calf in central Sudan.

Epizootic hemorrhagic disease virus (EHDV), isolate 318 (EHDV-318), an untyped virus recovered from a sentinel calf herd at the Khartoum University farm in central Sudan, was characterized using molecular biological techniques. With dot blot hybridization technique, a cDNA probe derived from genome segment 6 of EHDV-2 (Alberta strain) hybridized with RNA from EHDV-318. Application of serogroup-specific EHDV polymerase chain reaction (PCR) to EHDV-318 RNA resulted in specific amplification of a 387 bp PCR product. Amplification product was visualized on ethidium bromide-stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with a non-radiolabelled internal probe. No amplification product or hybridization signal was detected when the serotype-specific EHDV-1 or EHDV-2 PCR-based assays were applied to RNA from EHDV-318. The scientific data presented in this study indicated that cDNA probes and serogroup-specific PCR-based assay can be used to classify the virus as a member of EHDV serogroup, and as serotypically distinct from EHDV-1 and EHDV-2.

Animals↗
PubMed1990

DNA restriction digest and ribosomal RNA gene patterns of Campylobacter jejuni: a comparison with bio-, sero-, and bacteriophage-types of United Kingdom outbreak strains.

DNA restriction endonuclease (Hae III and Hind III) total digest and 16S and 23S ribosomal (r)RNA gene patterns (ribopatterns) were determined for 18 isolates of Campylobacter jejuni from three separate outbreaks of diarrhoea in the north of England. Strains were also characterized by biotyping, serotyping and phage typing. Comparisons of the DNA patterns by visual and numerical methods revealed five distinct strain groupings with clear differences between isolates from different outbreaks as well as some heterogeneity between strains within the community outbreak and one of the school outbreaks. An excellent correlation was observed between the genomic DNA fingerprints data and the Preston bacteriophage group, both of which gave better discrimination than biotyping and serotyping alone or in combination. Only one phage group (PG 37) was not confirmed by the DNA data. DNA fingerprints therefore provide additional information of value in studying the epidemiology of outbreaks of C. jejuni.

Animals↗
PubMed1993

Polypeptide profiles of human oocytes and preimplantation embryos.

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

Blastocyst↗
PubMedDate not supplied

Tandem repeat sequence variation and length heteroplasmy in the mitochondrial DNA D-loop of the threatened Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi.

Genetic variability within the Suwannee River, Florida, population of Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi, was assessed by examining sequence and length variation within the control region, or D-loop, of the mitochondrial genome. Although once abundant throughout the Gulf of Mexico, Gulf sturgeon are now listed as a threatened species by the U.S. Fish and Wildlife Service. Mitochondrial DNA was analyzed for length variation from 168 individual Gulf sturgeon by PCR amplification and visualization of PCR products using ethidium bromide-stained agarose gels. Of the 168 individual Gulf sturgeon, 31 (18.5%) were heteroplasmic for one to four copies of an 81-base pair, tandemly repeated sequence in the D-loop region. However, no individuals homoplasmic for multiple copies of the repeat sequence were observed. The existence and nature of these tandem repeats in heteroplasmic individuals was confirmed by direct sequencing of the PCR products for a subset of 22 individuals. The results are consistent with the apparent nature and mechanism of heteroplasmy observed in a congeneric species, A. transmontanus. In addition, sequences for 187 base pairs outside of the tandem repeats were identical among all 16 individuals assayed for this region. Lack of variable sequences is concordant with earlier studies involving mtDNA restriction fragment length profiles of Gulf sturgeon found in the Suwannee River. The absence of sequence variation exclusive of the tandem repeats is consistent with the hypothesis that the subspecies has undergone a population or evolutionary bottleneck.

Animals↗
PubMed2025

Polyamines buffer labile iron to suppress ferroptosis.

Polyamines are essential and evolutionarily conserved metabolites present at millimolar concentrations in mammalian cells. Cells tightly regulate polyamine homeostasis through complex feedback mechanisms, yet the precise role necessitating this regulation remains unclear. Here, we show that polyamines function as endogenous buffers of redox-active iron, providing a molecular link between polyamine metabolism and ferroptosis. Using genome-wide CRISPR screens, we identified a synthetic lethal dependency between polyamine depletion and the key ferroptosis suppressor, GPX4. Mechanistically, we show that polyamine deficiency triggers a redistribution of cellular iron, increasing the labile iron pool and upregulating ferritin. To directly visualize this iron buffering in living cells, we developed a genetically encoded fluorescent reporter for redox-active iron. Live-cell analysis revealed a striking inverse correlation between intracellular polyamine levels and redox-active iron at single-cell resolution. These findings reposition polyamines as key regulators of iron homeostasis, with implications for ferroptosis-linked disease states and cellular redox balance.

Journal Article↗
PubMed2000

A high association with cone dystrophy in Fundus albipunctatus caused by mutations of the RDH5 gene.

PURPOSE: To analyze the RDH5 gene in patients with fundus albipunctatus with and without cone dystrophy and to determine whether the disease is stationary or progressive and whether the cone dystrophy is a part of fundus albipunctatus or a separate disease. METHODS: Fourteen patients from 12 separate Japanese families with fundus albipunctatus were examined. Six of the patients from 6 families also had a cone dystrophy. Genomic DNA was extracted from leukocytes of the peripheral blood, and exons 2, 3, 4, and 5 of the RDH5 gene were amplified by polymerase chain reaction and were directly sequenced. A complete ophthalmic examination was performed including best-corrected visual acuity, slit-lamp examination, indirect ophthalmoscopy, fundus photography, and electroretinography. RESULTS: In all the patients, either a homozygous mutation or compound heterozygous mutations in the RDH5 gene were identified. The identified mutations were nucleotide position (nt) 103 G to A (Gly35Ser), nt 319 G to C (Gly107Arg), nt 394 G to A (Val132Met), nt 719 G insertion (frame shift), nt 839 G to A (Arg280His), nt 841 T to C (Tyr281His), and nt 928 C to GAAG (Leu310 to GluVal). All these mutations except the Arg280His were new. The nt 928 C to GAAG mutation was detected in patients with and without cone dystrophy. Cone dystrophy was most frequently seen in patients over 40 years old. CONCLUSIONS: Fundus albipunctatus either with or without cone dystrophy is caused by mutations of the RDH5 gene. Cone dystrophy is frequently observed in elderly patients with fundus albipunctatus. The conclusion was reached that the mutations of the RDH5 gene caused a progressive cone dystrophy as well as night blindness.

Adolescent↗
PubMed2004

Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles.

OBJECTIVE: To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). METHODS: The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization. RESULTS: The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg. CONCLUSION: The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes↗