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K Brasch

Publications and source records attributed to K Brasch.

At least 19 recordsLinked to original sources
PubMed1995

Nuclear remodeling in response to steroid hormone action.

Steroid and similar hormones comprise the broadest class of gene regulatory agents known, spanning vertebrates through the lower animals, and even fungi. Not unexpectedly, therefore, steroid receptors belong to an evolutionarily highly conserved family of proteins. After complexing with their cognate ligands, receptors interact with hormone response elements on target genes and modulate transcription. These actions are multifaceted and only partly understood, and include large-scale changes in the structure and molecular composition of the affected cell nuclei. This chapter examines steroid hormone action and the resultant nuclear remodeling from the following perspectives: (1) Where are the receptors located? (2) Which nuclear domains are most affected? (3) Are there extended or permanent nuclear changes? (4) What is the role of coiled bodies and similar structures in this regard? To address these and related questions, information is drawn from several sources, including vertebrates, insects, and malignant tissues. Entirely new data are presented as well as a review of the literature.

Animals↗
PubMed1995

Formation of nuclear bodies in hepatocytes of estrogen-treated roosters.

As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.

Animals↗
PubMed1992

Nuclear bodies (NBs): a newly "rediscovered" organelle.

Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.

Animals↗
PubMed1990

Drug and metabolite-induced perturbations in nuclear structure and function: a review.

Clarification of large-scale organization in cell nuclei is central to a full understanding of the mechanisms which underlie replication, transcription, and interaction with the cytoplasmic compartment. Many drugs and natural biological agents affect such processes directly, through inhibitory or stimulatory action, and often reveal structural and functional aspects in nuclei not otherwise apparent. There is increasing evidence that the pore complex - lamina of the nuclear matrix is a primary structural element during interphase, which is pivotal to most nuclear processes. This communication reviews evidence based on studies with drugs, inhibitors, and natural biological processes, that the nuclear matrix concept is indeed valid. It is further shown that despite diversity in mode of action and affected target cells, many compounds induce related or complementary changes in nuclei and that such uniformity in pattern is attributable to the nuclear matrix. A general model of the functional organization of the nucleus is presented, to account for the perturbations observed after mass interference with either transcription, replication, or protein synthesis.

Animals↗
PubMed1990

Nuclear protein modifications in vitellogenic rooster liver.

The estrogenic regulation of vitellogenesis in chicken liver provides an unique perspective on cellular reprogramming because males can be induced experimentally. Transient exposure to estradiol (ES) completely alters established patterns of gene expression in rooster hepatocytes within 6 hrs., and triggers major structural and compositional changes in cell nuclei by 24 hrs. Concurrently, the total protein content of nuclei increases nearly 50% and the relative proportion of protein within the nucleoplasmic, chromatin and residual compartments, shifts markedly. These bulk quantitative changes in nuclear composition are accompanied by marked alterations in 2-D electrophoretic patterns of cytoplasmic, nuclear and nuclear matrix polypeptides. Although most individual proteins remain unidentified, several components clearly overlap both the cytoplasmic and nuclear compartments. Reciprocal changes in the 2-D patterns are also evident after ES stimulation, with progressive decline in some and relative increase in other proteins. Among known species, the lamins (La and Lb) decrease in prominence after hormone, while RNP-associated polypeptides become increasingly pronounced in the residual matrix fraction. The results are discussed in relation to other systems where large-scale nuclear reprogramming is known to occur.

Animals↗
PubMed1989

Isolation and analysis of nuclear bodies from estrogen-stimulated chick liver.

The target cells of most steroid hormones contain prominent structures termed nuclear bodies (NB). Estrogen (ES) responsive tissues have very distinctive NB, whose development has been associated with both the level of ES receptors and their retention times in nuclei. It has been suggested that these NB may be large-scale ES binding centers at transcriptionally hyperstimulated sites. We examined the formation of NB in ES-stimulated rooster liver, after primary induction of vitellogenesis and during subsequent decay of this response. Distinct NB appear 6 h postinduction, initially as compact spheroids with ring-like profiles and later on as more complex morphotypes. Nuclei average 15-17 NB each at peak induction (24-48 h), but this declines again gradually as the hormonal response abates. This transient pattern is paralleled closely by the [3H]ES binding capacity of nuclei in vitro and the calculated number of specific hormone receptors present. There is no direct numerical correspondence, however, between the NB and the ES receptors, either on a one-to-one basis or as some simple ratio. Hence any links between ES binding activity in nuclei and the NB must be both complex and indirect. Nuclear bodies were isolated following lysis of nuclei by sonication and separation of subfractions on discontinuous sucrose gradients. The resultant NB-enriched fraction consisted primarily of protein, with traces of DNA and RNA. Analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a complex pattern of polypeptides, some apparently unique and probably NB-derived.

Animals↗
PubMed1988

Changes in structure and protein composition of bovine lymphocyte nuclear matrix during concanavalin-A-induced mitogenesis.

A major component of nuclear change in concanavalin-A-stimulated bovine lymphocytes is a severalfold increase in interchromatinic volume, which coincides with nuclear swelling and extensive structural remodelling. Large-scale ultrastructural changes in isolated nuclei and nuclear matrices (NM) reflect those occurring within nuclei in situ during mitogenesis. While nonchromatinic nuclear material embedded within nuclease- and salt-extracted whole cells closely resembled in situ interchromatinic matrices, large NM isolated in solution shrank after chromatin was extracted. Numerous perinuclear filaments persisted throughout NM isolation and cytoskeletal proteins were identified in two-dimensional (2-D) gels of such preparations. Taken together these data indicated that the lymphocyte cytoskeleton is likely continuous with the nuclear matrix and could play a role in maintaining nuclear organization. A wide range of lymphocyte NM proteins were resolved in 2-D gels. Significant changes in protein composition coincided with nuclear structural remodelling. Lamin B was prominent at each stage of nuclear development, whereas lamins A and C were only found in stimulated lymphocyte matrices. Lymphoblast NM contained more large basic proteins. Progressively increasing polypeptide complexity of these NM arose by de novo protein synthesis and posttranslational modifications throughout concanavalin A stimulation. NM from stimulated lymphocytes also contained more ribonucleoproteins, possibly indicating the presence of significant amounts of transcriptional material.

Animals↗
PubMed1987

Does the interchromatin compartment contain actin?

Transient blockage of transcription in chick liver by alpha-amanitin results in a temporary clearing of nucleoplasmic regions in affected nuclei and concurrent exposure in situ of a discrete interchromatin framework. This is accompanied by sharp decreases in total nuclear RNA (approximately 28%) and nonhistone protein content (approximately 18%). Electrophoretic analysis of matrix fraction proteins from such highly denuded nuclei shows a marked relative enrichment in actin and RNP-derived polypeptides. These observations suggest that actin is a real constituent of the interchromatin region and supports various reports that this protein may have a basic structural role in nuclei.

Actins↗
PubMed1985

Nuclear development in locust fat body: the influence of juvenile hormone on inclusion bodies and the nuclear matrix.

The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components greater than 150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.

Adipose Tissue↗
PubMed1985

Nuclear protein, matrix and structural changes in rooster liver after estrogenic induction of vitellogenesis.

The estrogenic induction of vitellogenesis in avian and amphibian liver has been well characterized at the target gene level. Comparatively little however, is known about cognate nuclear events associated with the response, particularly those involving large-scale structural changes and the non-histone proteins (NHP). We have examined these aspects further in primary stimulated roosters. In the first 24 hr post induction with estradiol, hepatocyte nuclei enlarged by 50% and exhibited sharp rises in total protein and RNA content. In particular, the mass of residual NHP rose about 40%. Extensive internal reorganization was evident, including partial disaggregation of chromatin, proliferation of interchromatin components and de novo appearance of prominent "nuclear bodies". These changes were accompanied by quantitative fluctuations in nucleoplasmic and several matrix fraction proteins. A marked relative decrease was evident in all three lamins, as well as approximately 75 and approximately 175 kD proteins. Hn-RNP-associated polypeptides however, and various unidentified components became much more prominent. By 24 hr, cells were fully differentiated for bulk export of vitellogenin and low density lipoproteins. All changes persisted for several days before gradually regressing to normal over a 2-4 week period. Many key nuclear modifications, however, did not regress fully, including persistent enlargement, elevated NHP content and modified matrix fraction proteins. Collectively, these may reflect part of the "memory" effect, commonly observed in steroid target tissues, whereby a second, more pronounced response can be triggered long after primary induction has subsided.

Animals↗
PubMed1982

Age- and ploidy-related changes in rat liver nuclear proteins as assessed by one- and two-dimensional gel electrophoresis.

Hepatocyte nuclei from young (3--5 week), mature (8--12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (greater than 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.

Aging↗
PubMed1982

Suspension cultures of adult rooster hepatocytes with a sustained vitellogenic response.

Primary suspension cultures of hepatocytes from adult roosters were prepared by collagenase perfusion. The cells remained viable for better than 12h as assessed by vital dye exclusion, electron microscopy and protein synthesis. In addition to the spectrum of proteins observed in control cultures, hepatocytes from animals pretreated with 17 beta-oestradiol synthesized vitellogenin, low density lipoprotein and several other unidentified peptides. Vg production, as determined by direct immunoprecipitation, was maintained at levels comparable to those observed in vivo with oestrogen treated roosters. Attempts to attain secondary induction of vitellogenesis in vitro, however, using cells from animals in which the primary response had subsided, were not successful. Nonetheless, the results show as a first step, that suspension cultures of liver cells from adult birds can be prepared which remain viable and morphologically differentiated for an extended period.

Animals↗
PubMed1980

Endopolyploidy in vertebrate liver: an evolutionary perspective.

Hepatocyte nuclei in several species of vertebrates were examined, to establish the frequency of polyploidy and related parameters along evolutionary lines. Nuclei were compared in terms of volume, DNA content, ploidy ranges and internal organization. Several trends emerged. When present, heterochromatin occupied 20--25% of nuclear profile areas, irrespective of nuclear volume and poloidy; the volume of heterochromatin, however, increased in direct proportion to ploidy level. Regardless of internal organization, ploidy and species, a direct correlation emerged between the volumes of nuclei and their absolute DNA content. Results are discussed in terms of structural and genic DNA.

Animals↗
PubMed1979

The morphology of normal and denatured polytene chromosomes from Drosophila melanogaster.

Normal and denatured polytene chromosomes from Drosophila were compared by light, transmission (TEM) and scanning (SEM) electron microscopy, under different conditions of pretreatment, fixation and specimen preparation. Apart from variations in contrast, acid-alcohol squashes of normal chromosomes were quite comparable in terms of resolution and image quality, regardless of mode of visualization, i.e. stained, unstained, phase contrast, etc. These chromosomes do not appear markedly different from the unfixed or "native" chromosomes isolated by other workers. Thus at the light microscope level at least, any soluble components extracted by acid-alcohol fixation seem to play minor structural roles only. SEM examination of similar squash preparations provided useful topographic views of selected chromosome regions, particularly in favourable spreads where metal coating and tilt angles were optimized. Information on the internal organization of polytene chromosomes was sought by subjecting isolated preparations to alkali-urea denaturation. Under these conditions, chromosomes gradually extend laterally to give rise to lampbrush-like structures, remarkably similar to the meiotic chromosomes of amphibian oocytes. This effect is at least partially reversed by return of alkali-urea treated preparations to physiological Ringer solution, where chromosomes contract again and near-normal banding patterns are re-established. It is suggested that apart from bulk differences due to polyteny, the basic structure of polytene chromosomes and true lampbrush chromosomes may be rather similar.

Acetates↗