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PubMed2025

Transcriptomic analysis at 48 h postmortem: a proof of concept for the identification of biomarkers to estimate time since death.

BACKGROUND: The postmortem interval (PMI) refers to the time elapsed between an individual's death and the examination of the body. Tissues undergo a sequence of anatomical changes following death, which are routinely used to estimate the PMI. METHODS: To determine if these anatomical changes are associated with identifiable genomic adaptations that could characterize the PMI more accurately, we analyzed the rat skeletal muscle transcriptome at 0 and 48 h postmortem using Clariom™ S arrays. This study investigates whether specific transcriptomic changes correlate with PMI progression, offering a potential molecular tool to complement established anatomical methods. RESULTS: A total of 3,873 differentially expressed mRNAs were identified, of which 2,787 downregulated and 1,086 upregulated transcripts. The most significantly downregulated mRNA was Tnni1 (FC = -30.95, p = 1 × 10-3), while the most upregulated were mt-ATP6, mt-ATP8, and mt-CO3 (FC > 7.78, p < 1.36 × 10-12). Gene ontology (GO) enrichment analyses revealed that mRNAs upregulated at 48 h in the PMI were primarily associated with vascular and endothelial processes, including nitric oxide transport and angiogenesis. Conversely, downregulated mRNAs were linked to mitochondrial activity and cellular metabolism, reflecting both a transient vascular response and metabolic pathway shutdown in the rat skeletal muscle. CONCLUSION: Our results demonstrate significant transcriptomic changes at 48 h postmortem, highlighting specific genes and biological pathways that may serve as candidate biomarkers for PMI estimation.

Animals↗

PubMed1988

Distribution of acetylated alpha-tubulin in retina and in vitro-assembled microtubules.

We have used the mouse monoclonal antibody 6-11 B-1, specific for acetylated alpha-tubulin, to determine the distribution of acetylated alpha-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated alpha-tubulin. Immunofluorescence, using 6-11 B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for alpha-tubulin, demonstrated the colocalization of both alpha-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated alpha-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.

Acetylation↗

PubMed1982

[Analysis of silicone testicular implants after explantation].

Two juvenile size silicone testicular prostheses were analyzed following replacement by adult size devices after 72 and 99 months in situ. Scanning electron microscopy and electron microprobe studies were carried out on the explanted silicone prostheses and their attached tissue capsules. The material at the surface of the prostheses gave no evidence of biodeterioration. However, localized sites of calcification were observed. The adjacent tissue capsule consisted of two distinct concentric layers with different morphologies; the one adjacent to the prosthesis consisted of dense arrays of collagen fibres oriented parallel to the silicone surface and extending outward to a thickness of 100-120 microns. The outer layer was less dense, more disordered and was uneven in thickness. It contained numerous discrete particles of silicon-rich material. Technical and clinical implications of these results are discussed.

Adolescent↗

PubMed1989

Electromagnetic pulse propagation in dispersive planar dielectrics.

The responses of a plane-wave pulse train irradiating a lossy dispersive dielectric half-space are investigated. The incident pulse train is expressed as a Fourier series with summing done by the inverse fast Fourier transform. The Fourier series technique is adopted to avoid the many difficulties often encountered in finding the inverse Fourier transform when transform analyses are used. Calculations are made for propagation in pure water, and typical waveforms inside the dielectric half-space are presented. Higher harmonics are strongly attenuated, resulting in a single continuous sinusoidal waveform at the frequency of the fundamental depth in the material. The time-averaged specific absorption rate (SAR) for pulse-train propagation is shown to be the sum of the time-averaged SARs of the individual harmonic components of the pulse train. For the same average power, calculated SARs reveal that pulse trains generally penetrate deeper than carrier-frequency continuous waves but not deeper than continuous waves at frequencies approaching the fundamental of the pulse train. The effects of rise time on the propagating pulse train in the dielectrics are shown and explained. Since most practical pulsed systems are very limited in bandwidth, no pronounced differences between their response and continuous wave (CW) response would be expected. Typical results for pulse-train propagation in arrays of dispersive planar dielectric slabs are presented. Expressing the pulse train as a Fourier series provides a practical way of interpreting the dispersion characteristics from the spectral point of view.

Adipose Tissue↗

PubMed1991

Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events.

Hypervariability at minisatellite loci is maintained by spontaneous mutation to new-length alleles. At the most variable loci, mutation rate is directly measurable by pedigree analysis. The mouse minisatellite locus Ms6-hm has a germline mutation rate of 2.5% per gamete and is therefore one of the most unstable loci yet identified in the mouse genome. Mutation events at this locus also occur during early mouse development, resulting in mice mosaic for cells carrying a common non-parental allele in different somatic tissues and the germline. The DNA sequence flanking Ms6-hm is rich in dispersed repetitive elements; the minisatellite array has expanded from within a member of the Mouse Transcript family which is flanked by two additional Mouse Transcript elements, and a B2 element lies further 3' to the minisatellite. To define the characteristics of mutation events at Ms6-hm we have analysed 19 germline and 13 somatic length-change events. Germline mutation events at Ms6-hm are not accompanied by the exchange of flanking markers in three informative mutant alleles analysed.

Alleles↗

PubMed1989

Morphometric analysis of gap junctions in rat myocardium after hyperkalemia.

The effect of membrane depolarization was investigated on gap junctions from isolated rat hearts perfused with a modified Krebs-Henseleit solution containing 16 mM K+. After freeze-fracturing, the configuration of the junctional particles in the ventricular myocardium was analysed by measurements of connexon densities and centre-to-centre distances between neighbouring particles. Both in control and hyperkalemic tissue, the gap junctions occur on the intercalated discs as round or oval aggregates of connexons which are closely and regularly packed in small, criss-cross-oriented arrays separated by particle-free aisles. Within the arrays, the mean (+/- SD) centre-to-centre distances between particles from control and hyperkalemic tissue, i.e. 9.17 +/- 1.52 and 9.15 +/- 1.51 mm, respectively, are not significantly different. Similarly, comparison of particle densities after control and high-K+ perfusion, i.e. 8,490 +/- 600 and 8,420 +/- 620 particles/microns 2, respectively, reveals no difference in the proportion of the particle arrays to the empty aisles. The apparently unaltered gap junctional morphology after depolarization of the sarcolemma by high-K+ perfusion provides support for the electrophysiological finding that the conductance of cardiac gap junctions is insensitive to membrane potential.

Animals↗

PubMedDate not supplied

Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells.

A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.

Base Sequence↗

PubMed1985

Proteoglycan-type I collagen fibril interactions in bone and non-calcifying connective tissues.

The association of proteoglycans with type I collagen fibrils in skin, tendon, cornea and bone has been determined by electron microscopy using an electron-dense dye, Cupromeronic blue, in the critical electrolyte concentration mode, backed up by biochemical analysis and digestion by hyaluronidase or keratanase. A major proteoglycan of the soft tissues, containing dermatan sulphate, is shown to be regularly and orthogonally arranged at the surface of the fibrils. Uranyl acetate counterstaining revealed that the main specific binding site is the 'd' band, which previous work indicated is very close to the initial site of calcification of type I collagen fibrils. Bone, demineralized by a 'non-aqueous' technique which preserves the proteoglycan in the tissue, does not contain orthogonal arrays; the interfibrillar proteoglycan filaments are oriented parallel to the fibril axis. The main proteoglycan in bone is chondroitin sulphate-rich. It is suggested that dermatan sulphate proteoglycan plays a role in preventing soft connective tissues from calcifying.

Animals↗

PubMed1984

Demonstration by X-ray microprobe analysis of relationship between chondrocytes and tertiary surface structure of hyaline articular cartilage.

Blocks of hyaline cartilage from the femoral condyles of five young adult beagles dogs were quench-frozen in nitrogen slush at 63 K. The free cartilage surfaces of blocks from three animals were examined in the secondary electron mode; the remaining specimens were cut by cryoultramicrotone (approximately 188 K) tangential to the surface to expose midzone cartilage which was examined in the backscattered electron mode. A random array of gently convex prominences was apparent at the free cartilage surfaces. When X-ray emissions were recorded from tissue immediately below these elevations, the spectra proved to be similar to those derived from midzone chondrocytes. These spectra revealed high count rates for the X-rays characteristic of P and K. By contrast, in areas of free surface remote from these prominences, and in midzone intercellular matrix, larger count rates for the X-rays characteristic of Na, S. Cl and Ca were detected. The evidence supports the hypothesis that the elevations seen on the non-loaded articular cartilage of disarticulated mammalian synovial joints are the surface representations of superficial chondrocytes.

Animals↗

PubMed1988

Molecular evolution of the mouse proline-rich protein multigene family. Insertion of a long interspersed repeated DNA element.

Proline-rich proteins (PRPs) in the salivary glands of mice, rats, and hamsters are encoded by tissue-specific inducible multigene families. Mouse PRP genes are located on chromosome 8, and transcription is dramatically induced (about 70-fold) by isoproterenol treatment. Clones containing two nonallelic PRP genes (MP2 and M14) were isolated from cosmid and phage libraries of CD-1 mouse genomic DNA. The cloned regions comprise a contiguous block of 77 kilobase pairs of the mouse genome. Restriction mapping established the physical lineage of PRP genes MP2 and M14, and they are tandemly arrayed. The DNA sequence analysis presented in this report suggests that genes M14 and MP2 (Ann, D. K., and Carlson, D. M. (1985) J. Biol. Chem. 260, 15863-15872) arose via a gene duplication of a common ancestor. Two major differences between M14 and MP2 were observed. PRP gene MP2 has 13 tandemly arrayed 42-nucleotide repeats in exon II, whereas M14 has 17 repeats, and PRP gene M14 has an insertion by transposition of a 2-kilobase pair member of the long interspersed repeated DNA (LINE) family (LIMd) into intron I. The evolution of this PRP multigene family has been dominated by intra-exonic amplification of repeating nucleotide units coding for these and other proline-rich repeated peptides and by gene duplication. The LIMd element gives rise to heterogenous EcoRI, BamHI, and HindIII restriction enzyme patterns, and this insertion is also present in BALB/c, C57BL/6J, and DBA/2J mice.

Amino Acid Sequence↗

PubMed1991

How do geometric factors influence epidural spinal cord stimulation? A quantitative analysis by computer modeling.

Effects of both anatomic and electrode geometry on the recruitment of rostrocaudal fibers in the spinal cord were investigated by computer simulation of epidural spinal cord stimulation. A three-dimensional model was used, representing the geometry and electrical conductivity of the spinal cord and surrounding tissues, in combination with a model representing the electrical properties of a myelinated nerve fiber. Recruitment contours in the dorsal columns were calculated at various spinal geometries as a function of electrode position, combination and area. Cathodal position appeared to be most significant. Recruitment areas resulting from different contact combinations of a mediodorsal array were almost identical. It was shown that perception threshold largely depends on both dorsal cerebrospinal fluid width and fiber size. The usual bipolar contact separation appeared to approximate the theoretically optimal value, resulting in maximum fiber recruitment at minimum stimulus.

Analgesia, Epidural↗

PubMed1990

In situ hybridization analysis of TGF beta 3 RNA expression during mouse development: comparative studies with TGF beta 1 and beta 2.

To date, three closely-related TGF beta genes have been found in the mouse; TGF beta 1, TGF beta 2 and TGF beta 3. Previous experiments have indicated that TGF beta 1 and TGF beta 2 may play important roles during mouse embryogenesis. The present study now reports the distribution of transcripts of TGF beta 3 in comparison to the other two genes and reveals overlapping but distinct patterns of RNA expression. TGF beta 3 RNA is expressed in a diverse array of tissues including perichondrium, bone, intervertebral discs, mesenteries, pleura, heart, lung, palate, and amnion, as well as in central nervous system (CNS) structures such as the meninges, choroid plexus and the olfactory bulbs. Furthermore, in several organ systems, TGF beta 3 transcripts are expressed during periods of active morphogenesis suggesting that the protein may be an important factor for the growth and differentiation of many embryonic tissues.

Animals↗

PubMed1986

Antigenic interrelationship between the 40-kilodalton cytokeratin polypeptide and desmoplakins.

We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.

Animals↗

PubMed1991

Cytochrome c oxidase subunit VIIa isoforms. Characterization and expression of bovine cDNAs.

Subunit VIIa of bovine cytochrome c oxidase occurs in two forms, the so-called heart and liver isoforms, which have been shown by protein analysis to differ in 35% of their amino acids. We have isolated and characterized cDNAs for each isoform. The derived heart-type processed protein is 59 amino acids long, with a 21-residue presequence; the immediate C terminus differs from the established protein sequence. The liver-type processed protein is 60 residues long, with a 23-amino acid presequence. Both presequences are traditional in that they are positively charged and appear amphiphilic when helically arrayed. The presequences are only 22% identical, but they both contain conserved residues indicative of two-step processing of the precursor proteins. Southern blot analysis reveals that the bovine genome contains five to six copies of the liver-type gene as opposed to the presence of a single copy heart-type gene. Transcriptional analysis shows that heart-type message is detectable only in heart and skeletal muscle; the liver type is also seen in heart and muscle and, additionally, in the other tissues examined (liver, brain, and lung). The amino acid sequence EKQKLFQED is conserved in rat and in both isoforms in cow and human and may represent a domain of core subunit function.

Amino Acid Sequence↗

PubMed1990

Hepatic copper concentrations in purebred and mixed-breed dogs.

Histomorphologic, histochemical, and atomic absorption analysis studies were performed on liver tissue from 623 clinically normal purebred and mixed-breed dogs to assess the range of hepatic copper concentrations, the histologic distribution of copper, and the histomorphologic changes associated with the various copper concentrations that were found. Atomic absorption analysis revealed a continuous numerical spectrum ranging from less than 100 parts per million on a dry weight basis (ppm dw) to more than 2,000. No decisive numerical criterion was found that could be used to separate normal from abnormal copper concentrations because of this continuous array. The threshold for histochemical demonstration of copper-containing granules using rhodanine or rubeanic acid staining was 400 ppm dw. At this concentration the copper-containing granules were located in the centrilobular hepatocytes (zone 3 of Rappaport). Copper-containing granules were found in the midzonal and periportal hepatocytes (zones 2 and 1 of Rappaport) in livers with 1,000 ppm dw and higher copper concentrations. The majority of the 623 livers were normal histomorphologically. Multifocal hepatitis characterized by mixed inflammatory cell accumulation and centrilobular distribution was found to be associated with copper concentrations in the range of 2,000 ppm dw or higher. An appropriate upper limit for normal hepatic copper concentration in the dog was not determined based on the present study.

Animals↗

PubMed1982

Reflectometric mapping of microregional blood flow and blood volume in the brain cortex.

A reflectometric indicator-dilution method has been developed for mapping the parenchymal minute volume flow and blood content over tiny superficial areas of the brain cortex at 625 or 2500 locations, respectively. About 0.4 ml dextran-saline solution was used as nondiffusible indicator and injected into the feline cerebral circulation for each measurement. The subsequent cerebrocortical transit of the hemodiluted bolus was detected as a temporary change in the tissue optical density [OD(t)] and interpreted as indicator dilution, C(t). This gave the data necessary to calculate the microregional blood volume (mrCBV), mean transit time of the bolus (mrMTT), and the microregional blood flow (mrCBF = mrCBV/mrMTT). A two-dimensional record of the OD(t) function was made on Kodak SO 115 film by 16-mm cinematography, as a reflectometric tool, over an exposed area of the brain cortex during the bolus perfusion. Later, the microregional OD(t) functions were retrieved for analysis in a square array from the developed film by computer-controlled, frame-by-frame scanning densitometry. Maps of mrCBF, mrCBV, and mrMTT were presented as square arrays of gray-scaled pixels. The maximal spatial and temporal resolution of the method was 0.015 mm2 (mrCBF), 0.004 mm2 (mrCBV), 6 maps/min (mrCBF), and 600 maps/min (mrCBV).

Animals↗

PubMed1983

Repeated nucleotide sequence arrays in Balbiani ring 1 of Chironomus tentans contain internally nonrepeating and subrepeating elements.

Balbiani rings in Chironomus are large puffs on salivary gland polytene chromosomes that contain functionally related, but nonidentical genes that code for tissue-specific secretory polypeptides. In situ hybridization was used to select a recombinant plasmid (pCtBR1-1) that contained an insert of Chironomus tentans genomic DNA that originated from Balbiani ring 1. Mapping with restriction endonucleases demonstrated that the insert was 385 bp (base pair) and it contained duplicate clusters of certain cleavage sites about 250 bp apart. This repeat was shown to be part of tandem sequence arrays in the genome by hybridization of radioactive pCtBR1-1 to nitrocellulose blots containing limit and partial restriction endonuclease digests of nuclear DNA. Subsequent sequence analysis of the cloned DNA confirmed the presence of one complete copy of a 246-bp repeat comprised of a 114-bp internally nonrepeating segment and a 132-bp segment containing four 33-bp subrepeats. The subrepeats apparently evolved from a simple 9-bp sequence encoding a consensus tripeptide (Lys-Pro-Ser) in which the first two codons (AAA-CCA) were highly conserved at the nucleotide level. Comparisons between intragenic and interspecific (BRb in Chironomus thummi) copies of corresponding sequences revealed that, during the evolution of these tandemly repeated protein-coding sequences, internally nonrepeated segments were highly conserved and most likely became interspersed by variable segments containing subrepeats that arose from reduplication and divergence of 9-bp repeats.

Amino Acid Sequence↗

PubMed1979

Histones and histone-DNA ratios in diploid and polyploid cottons.

Histones are nuclear proteins which repress gene transcription and modify chromosome structure. They are remarkably conservative in structure throughout a wide evolutionary array of plants and animals; however, quantitative histone differences have been detected by cytological means in species having extra chromosomes. The purpose of this study was to: 1) isolate and characterize the histones of several Gossypium species and 2) relate differences to known differences in vigor, ploidy level, and genome constitution or size. Histones extracted from isolated nuclei of leaf tissues were characterized by electrophoresis on polyacrylamide gels into 14 subfractions of the five major histone classes. The subfractions were identified by various means including co-electrophoresis with known histone standards. Densitometric analysis revealed only slight quantitative differences in subfraction ratios between species. Histone-DNA ratios were significantly higher in the pentaploid species. This observed increase is considered a result of genome imbalance. These data support the premise that histones may function as generalized gene deactivators in plant species having multiple genomes.

Diploidy↗