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Biomedical subjects

T E Martin

Publications and source records attributed to T E Martin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Nuclear bodies are usual constituents in tissues of hibernating dormice.

In previous studies we demonstrated in several tissues of the hazel dormouse Muscardinus avellanarius that during hibernation cell nuclei contain particular structural constituents absent in euthermia. In the present study we examine the same tissues in euthermic and hibernating individuals of the edible dormouse Glis glis in order to investigate possible modifications of nuclear structural constituents occurring during hibernation in this species. Edible dormice were captured in the wild and maintained in an external animal house. Samples of liver, pancreas, brown adipose tissue and adrenal cortex were taken from three hibernating and three euthermic animals and processed for resin embedding. Ultrastructural and immunocytochemical studies were carried out on cell nuclei of these tissues. The most evident feature of cell nuclei of hibernating dormice was the presence of several nuclear bodies, namely fibro-granular material, amorphous bodies, coiled bodies, perichromatin granule-like granules and nucleoplasmic fibrils, the distribution of which was peculiar to each tissue. No one of these constituents was detectable during euthermia. Immunocytochemical analyses revealed that they contain some splicing factors. Apart from some differences, maybe due to the different characteristics of lethargy, the nuclear bodies found in edible dormice were morphologically and immunocytochemically similar to those previously described in the same tissues of hazel dormice. They therefore seem to be strictly correlated to the hibernating state. If they represent storage and/or assembly sites of splicing factors to be rapidly used upon arousal, they could represent a usual structural feature in cells of hibernating species.

Adipose Tissue, Brown↗
PubMed1999

Terminal differentiation of erythroblasts leads to RNP segregation and formation of heterogeneous ectopic RNP-derived structures.

We show an as yet unnoticed feature of mammalian erythrocyte maturation, i.e., the formation of heterogeneous ectopic RNP-derived structures in the nucleus of erythroblasts, occurring in parallel with chromatin condensation. Inside these structures, RNPs are always recognized by specific antibodies, which demonstrates that the protein moieties of RNPs still preserve (at least partially) their native organization. This phenomenon shares extensive similarity with the segregation and clustering of nuclear RNPs occurring during spontaneous apoptosis of rat thymocytes and in several other cell models in which transcription is physiologically arrested.

Animals↗
PubMed1999

Nuclear phospholipids in human lymphocytes activated by phytohemagglutinin.

Using a specific ultracytochemical technique, the labelling with phospholipase A2-gold complex, we have followed nuclear phospholipids (PL) along the G1 phase in human lymphocytes activated by PHA. Our data point out two main results relating nuclear PL to the transcriptional activity, characteristic of the G1 phase, during which many different molecules necessary both for progression through G1 and for the start of S phase are synthesized. PL quantitative changes parallel those of hnRNPs and snRNPs, which are markers of the levels of transcriptional activity and processing. We found that nuclei of G0 lymphocytes, with a very low transcription level, are poor of PL as well as of RNPs. The amount of PL increases in activated lymphocytes, along all G1, until the beginning of S phase. At the same time, hnRNPs and snRNPs strongly increase and maintain higher levels than in control cells, till the beginning of S phase. PL are localized on nuclear structures where also RNPs involved in transcription and splicing, are located, i. e. perichromatin fibrils, interchromatin granules and the dense fibrillar component of the nucleolus. Since it is known that during S phase nuclear PL decrease, while both the enzyme activities related to their breakdown and their hydrolysis products increase, PL seem to be involved in the generation of signal molecules triggering DNA replication. We suggest that PL in the nucleus can be involved in multiple functions, depending on the phase of the cell cycle.

Cell Cycle Proteins↗
PubMed1999

Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Cell Line↗
PubMed1998

Ultrastructural distribution of the death-domain-containing MyD88 protein in HeLa cells.

MyD88, a protein implicated in interleukin-1 signaling, was localized in HeLa cells transiently transfected with an epitope-tagged (flag) version of MyD88. Overexpression of MyD88 can induce apoptosis. We have analyzed the fine structural intracellular distribution of MyD88 using immunoelectron microscopy. MyD88 is localized to the nucleus and to the cytoplasm as revealed by immunofluorescence visualization. Ultrastructural immunocytochemistry shows that, in the cytoplasm, this protein is associated with fibrillar aggregates containing beta-actin. In the nucleus, MyD88 was found in fibrillar domains present only in cells not yet displaying morphological signs of apoptosis. These domains are not derived from nucleoli and do not constitute an accumulation site of splicing factors. We suggest that such structures could be involved in the formation of the apoptotic bodies and/or in the modification of the nuclear structure and of nucleocytoplasmic trafficking during apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript.

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

Animals↗
PubMed1998

Cis-acting sequences that affect somatic hypermutation of Ig genes.

We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cis-acting elements are present within the 10-16 kb of a transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may be the BCL6 gene, which was mutated in human but not in mouse B cells. Transcription of a region is required for its mutability. When the transcriptional promoter located upstream of the variable region is duplicated upstream of the constant region, this region also becomes mutable. This suggests a model in which a mutator factor associates with the RNA polymerase at the promoter, travels with the polymerase during elongation, and causes mutations during polymerase pausing. The DNA repair systems, nucleotide excision repair and DNA mismatch repair, are not required. Our recent data with an artificial substrate of somatic mutation suggest that pausing may be due to secondary structure of the DNA or nascent RNA, and the specific mutations to preferences of the mutator factor.

Animals↗
PubMed1998

Immunoglobulin transgenes as targets for somatic hypermutation.

This review describes studies on somatic hypermutation of immunoglobulin genes that were started in the mid-80s in collaboration with Ralph Brinster. Almost all of the experiments were carried out using Ig transgenes as targets for the somatic mutation mechanism. Ig transgenes can be very good targets of somatic mutation, despite many different transgene integration sites. Thus, the required cis-acting elements must be present within the approximately 10 kb of the transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region in unmanipulated sequences. Several Ig gene enhancers are permissive for somatic mutation and they do not have to be associated with the Ig promoter they normally interact with. However, the mutation process does seem to be specific for Ig genes. No mutations were found in several housekeeping genes isolated from cells that had very high levels of somatic hypermutation of their Ig genes. This suggests that the Ig enhancers provide the lg gene specificity. An exception is the Bcl-6 gene, encoding a transcription factor, which was found to be mutated in normal human memory B cells. When the transcriptional promoter that is located upstream of the variable region is duplicated upstream of the constant region, this region is mutated as well. This suggests a transcription coupled model in which a mutator factor associates with the RNA polymerase at the initiation of transcription, travels with the polymerase during elongation, and causes mutations during polymerase pausing. Our recent data with an artificial substrate for somatic mutation suggest that the mutations are increased by increased stability of the secondary structures in the nascent RNA, and the specific nucleotides that are mutated are due to preferences of a mutator factor.

Animals↗
PubMed1998

Using functional magnetic resonance imaging to understand the mechanisms of consciousness.

BACKGROUND: In order to elucidate mental functions that subserve human consciousness, brain activation was investigated in 12 normal, right-handed volunteers who performed tasks of selective attention, working memory, and sensorimotor coordination during the collection of multislice echoplanar functional magnetic resonance images. HYPOTHESIS: These functions are located in (and controlled by) distinct anatomical regions that can be identified by functional magnetic resonance imaging techniques. METHODS: In each subject, 100 10-slice data sets were acquired using a 1.5-T scanner and the blood oxygenation level dependent contrast technique. Time-series regression modeling estimated power in the magnetic resonance signal during the on/off phases of task performance. Comparison between subjects was made possible by the transformation of each data set into standard Talairach space. RESULTS: Activation maps were based on the median value of the fundamental power quotient at each voxel. Results showed the activation of prefrontal and parasagittal cortices in both the selective attention and working memory tasks, but they also revealed activation in both insular cortices and the posterior cingulate gyri. CONCLUSIONS: The results provide evidence for structures in the anterior right hemisphere and left medial frontal lobe for attentional tasks, although there appears to be an engagement of a widespread network of anterior brain structures, possibly with the inhibition of some posterior regions, during task performance. The sensorimotor coordination task showed activation regions similar to those seen in selective attention. Once learned, this task probably demands attention rather than overt conscious motor control. Clearly, the functions of attention, working memory, and sensorimotor coordination are not located in single, discrete brain areas. However, interactions and interplay between related areas were demonstrated, giving supporting evidence that complex mental operations rely on the coordinated activity of widely distributed brain regions that contribute to neural networks.

Adult↗
PubMed1997

Still immunodetectable nuclear RNPs are extruded from the cytoplasm of spontaneously apoptotic thymocytes.

The fate of different nuclear ribonucleoprotein (RNP) components was investigated during spontaneous apoptosis of thymocytes using specific monoclonal antibodies against snRNPs, hnRNPs, and ribosomal proteins at light and electron microscopy levels and by flow cytometry. It was found that, during apoptosis, nuclear RNP-containing structures (perichromatin granules, interchromatin granules, and perichromatin fibrils) segregate in the interchromatin space and cluster into heterogeneous aggregates of granules in which some of the structures may still be recognized morphologically. Along with the progress of apoptosis, the clusters are extruded from the nucleus into the cytoplasm, from which they are finally released via cytoplasmic extrusions. At all these stages, RNPs inside the clusters are always recognized by specific antibodies, even when they bleb out of the cell surface, thus suggesting that degradation of RNPs might be only partial during apoptosis. This could be potentially harmful in genetically susceptible subjects, as the appropriate MHC class II molecules may capture and present normally cryptic self-peptides. It is tempting to speculate that this event might have implications in the etiology of autoimmune diseases.

Animals↗
PubMed1997

Use of body measurements and serum metabolites to estimate the nutritional status of mallards wintering in the Mississippi Alluvial Valley, USA.

We collected mallards (Anas platyrhynchos) from bottomland hardwood habitats on the Bayou Meto Wildlife Management Area and the White River National Wildlife Refuge, Arkansas County, Arkansas during the winter of 1990 to 1991 to determine if measures of physiological condition could be predicted from structural size, serum metabolite levels, or from direct measures of carcass composition. Serum triglyceride levels were correlated (r = 0.57, P = 0.007) with total body fat in males and slightly increased the value (from R2 = 0.64 to 0.76) of intact body mass alone for predicting total body fat in males. Overall, however, serum metabolites appeared to be poor indicators of the magnitude of nutrient masses in mallards. Three potential indices of nutritional status were developed from carcass composition data: protein/total ash, fat/ total ash, and fat/fat-free body mass. Protein masses of male mallards changed over winter (P = 0.02). Consequently, fat-free masses are not constant and represent poor indicators of structural size for mallards wintering in the Mississippi Alluvial Valley.

Adipose Tissue↗
PubMed1996

Localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study.

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42 degrees C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

Animals↗
PubMed1996

Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos.

The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in "compact nucleoli" [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m3G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-3H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-3H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m3G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m3G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.

Animals↗
PubMed1996

Dense granular bodies: a novel nucleoplasmic structure in hibernating dormice.

Dense granular bodies (DGB) are particular structural constituents observed in cell nuclei of different tissues-liver, pancreas, brown adipose tissue, adrenal cortex-of hibernating dormice. They appear as strongly electron-dense clusters of closely packed granules, with thin fibrils spreading out at their periphery. DGB always occur in the nucleoplasm, sometimes making contact with other nuclear structural constituents typical of the hibernating state, such as coiled bodies, amorphous bodies and nucleoplasmic fibrils. DGB are present only during deep hibernation and rapidly disappear upon arousal from hibernation. Cytochemical and immunocytochemical analyses showed that DGB contain ribonucleoproteins and several nucleoplasmic RNA processing factors, suggesting that DGB can represent accumulation sites of splicing factors which are provided to splicing sites when normal metabolic activity is rapidly restored during arousal.

Adrenal Glands↗
PubMed1996

The cell nucleus in early bovine and caprine preimplantation embryos: fine structural cytochemistry and immunoelectron microscopy.

Fine structural cytochemistry and immunocytochemistry were used to study nucleic acids and nuclear proteins in nuclear bodies (NB) of pronuclear and 2-cell bovine and caprine embryos on ultrathin sections of paraformaldehyde fixed and Lowicryl K4M or LR White embedded specimens. The most striking feature detected in some of these nuclear bodies (NBs) was the presence of non-nucleolar proteins known to be involved in pre-mRNA splicing. One category of such intranuclear bodies (showing a rather dense finely fibrillar composition and named here dense body-DB) contained the Sm-antigen (an antigen common to a major group of nucleoplasmic spliceosomal snRNPs). Another, more numerous category of NBs differed morphologically from the former one by a much looser composition of fibrillogranular elements (loose body-LB). Moreover, it showed the presence of the non-snRNP splicing factor SC-35, in addition to the Sm-antigen. Both categories of these nuclear bodies were distinguished clearly from the nucleolar precursor bodies (NPBs) by an absence of immunolabeling of NPB with antibodies against nuclear proteins involved in splicing. Moreover, the former NBs are not stained with silver, while NPBs already in pronuclei exhibit strong affinity to silver. In addition to the immunolabeling in prominent (approx. 0.2-2.0 microns) NBs, regularly occurring high concentration of snRNP was revealed in very small (approx. 0.05 micron), morphologically poorly defined areas (named here small snRNP-enriched areas-SSA), harboring moreover a set of nuclear proteins similar to that of the coiled body. Numerous observations of the presence of these small areas in nuclear bodies and in their close vicinity, in nucleoplasm, in proximity of the nuclear envelope and also in ooplasm suggested that they are possible carriers of certain nuclear proteins moving between nuclear bodies, nucleoplasm and cytoplasm. A functional relationship of all these embryonic subnuclear elements has not been elucidated so far but their mutual relation is suggested, since the NPBs and other nuclear bodies usually occur in a close association. Fine structural and immunoelectron microscopic observations further suggest a similarity of the nuclear bodies in the early ruminant embryo with specific intranuclear bodies ("snurposomes") known from Xenopus laevis oocytes. A new and striking feature emerging from these observations is a possible involvement of a group of nucleoplasmic proteins in a yet unknown way in the differentiation processes concomitant with early embryonic nucleologenesis.

Animals↗
PubMed1996

Immunoelectron microscope analyses of rat germinal vesicle-stage oocyte nucleolus-like bodies.

Germinal vesicle (GV) stage oocytes isolated from rat ovaries were investigated by immunoelectron microscopy for the presence of several nuclear proteins in the prominent 'compact nucleoli' (nucleolus-like bodies named here NLB). Specific spliceosomal components including the Sm antigen of the nucleoplasmic small nuclear ribonucleoproteins (snRNP) and the non-snRNP splicing factor SC-35 were clearly detected in the dense finely fibrillar mass of the NLB. Moreover, the presence of small nuclear RNA (snRNA) in the NLB was demonstrated by means of an antibody which recognized the m3G-capped snRNA. The level of immunolabelling for the nuclear proteins fibrillarin and p80-coilin was relatively lower in the NLB. p80-coilin was distinctly localized, in small, poorly morphologically defined structural constituents in the nucleoplasm. Aggregates of intranuclear granules having similar antigenic composition to the NLB were also detected. Our observations suggested that the 'compact nucleoli' of rat GV oocytes represented nuclear compartments containing significant amounts of non-nucleolar, spliceosomal components. These NLB have a molecular composition closer to the composition of certain nuclear bodies than to the functional nucleoli of somatic cells. The NLB may represent a compartment in the mammalian oocyte, akin to the sphere organelle of the amphibian oocyte (also reported to contain spliceosomal components and a p80-coilin-related protein). Both structures may serve as temporary storage organelles for different maternal macromolecules, which support early embryonic development, inter alia of that involved in the maturation of pre-mRNA to be transcribed from the embryonic genome.

Animals↗