Spatial relationship between transcription sites and chromosome territories.

We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

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.

Localization and quantification of the insulin-like growth factor-1 receptor in mouse articular cartilage by confocal laser scanning microscopy.

Insulin-like growth factor-1 (IGF-1) is an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-1 Type 1 receptor on the chondrocyte membrane. We have localized and quantified in situ IGF-1 receptor expression in intact articular cartilage of normal mice. The IGF-1 receptor was detected immunohistochemically with antibodies to the IGF-1 receptor and visualized with conventional light microscopy and confocal laser scanning microscopy (CLSM). CLSM analysis enabled us to distinguish IGF-1 receptor immunoreactivity on the chondrocyte cell membrane from intracellular staining. We have established two approaches to quantify in confocal images low levels of fluorescence intensity of the immunolocalized IGF-1 receptor at the chondrocyte membrane, i.e., mean pixel measurement and area measurement. The majority of IGF-1 receptor fluorescence intensity was localized on chondrocytes in the middle and deeper zones of cartilage, whereas chondrocytes in the surface zone exhibited negligible fluorescence. The variable distribution of IGF-1 receptor in chondrocytes of articular cartilage suggests that effects of IGF-1 on chondrocytes may be distinctly heterogeneous in the different mouse articular cartilage zones.

The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.

The effects of interleukin-1 on articular cartilage destruction as observed in arthritic diseases, and its therapeutic control.

At present there is substantial evidence to suggest that interleukin 1 (IL-1) may act as a key mediator in the normal physiologic regulation of cartilage as well as in the pathogenesis of cartilage destruction in arthritic disorders. IL-1 induces stimulation of chondrocyte catabolism and alters chondrocyte biosynthesis in articular cartilage. These actions of IL-1 may lead to destruction and inappropriate repair following degradation of the cartilage matrix. Moreover, IL-1 induced biological activities in chondrocytes may be influenced by growth factors (e.g. fibroblast growth factor, insulin-like growth factor, transforming growth factor-beta), guanine nucleotide proteins, or other cytokines. With respect to the widely suggested potential significance of IL-1 in arthritis, pharmacological control of IL-1 action is of important clinical relevance. Today the therapeutic control of IL-1 induced effects in articular cartilage destruction as observed in arthritic diseases can be divided into drugs which affect IL-1 production, drugs which modify or block the IL-1 effect before stimulation of the target cell, or drugs that interfere with the IL-1 induced effects, e.g. steroidal drugs, non-steroidal anti-inflammatory drugs, immunoregulatory drugs or class-specific proteinase inhibitors. However, these drugs do not specifically block IL-1 activity. For the development of therapeutic agents capable of specifically blocking IL-1 effects, a better understanding of IL-1 induced activities is needed. In conclusion, knowledge about chondrocyte metabolic and regulatory alterations would be beneficial in unraveling the events that take place in arthritic diseases and would favor therapeutic research for agents that might arrest the progressive destruction of articular cartilage in pathological conditions.

Learning sensory maps with real-world stimuli in real time using a biophysically realistic learning rule.

We present a real-time model of learning in the auditory cortex that is trained using real-world stimuli. The system consists of a peripheral and a central cortical network of spiking neurons. The synapses formed by peripheral neurons on the central ones are subject to synaptic plasticity. We implemented a biophysically realistic learning rule that depends on the precise temporal relation of pre- and postsynaptic action potentials. We demonstrate that this biologically realistic real-time neuronal system forms stable receptive fields that accurately reflect the spectral content of the input signals and that the size of these representations can be biased by global signals acting on the local learning mechanism. In addition, we show that this learning mechanism shows fast acquisition and is robust in the presence of large imbalances in the probability of occurrence of individual stimuli and noise.

Chondrocyte IGF-1 receptor expression and responsiveness to IGF-1 stimulation in mouse articular cartilage during various phases of experimentally induced arthritis.

OBJECTIVE: To examine the distribution of insulin like growth factor-1 (IGF-1) receptors and the biological response to IGF-1 stimulation in articular cartilage of normal mouse knee joints and arthritic joints taken at various stages of experimentally induced arthritis. METHODS: In situ IGF-1 receptor expression and responsiveness to IGF-1 stimulation were examined in murine articular cartilage at different phases in two models of experimentally induced arthritis. IGF-1 receptor expression was visualised in joint sections with the use of anti-IGF-1 receptor antibodies and quantified by confocal laser scanning microscopy. Chondrocyte proteoglycan (PG) synthesis was measured by incorporation of 35S-sulphate. RESULTS: In control cartilage, the majority of IGF-1 receptors were found on chondrocytes localised in the middle and deeper zones of the cartilage, whereas receptor expression in surface zone chondrocytes was very low. During culture of normal articular cartilage, IGF-1 was able to maintain chondrocyte PG synthesis at the in vivo level. Concurrently with the development of arthritis, cartilage lost its capacity to react to IGF-1, but IGF-1 stimulation recovered when the inflammatory response waned. Shortly after induction of arthritis, IGF-1 receptor expression initially declined, but it had returned to normal levels by day 1 and remained increased thereafter. CONCLUSION: The distribution of IGF-1 receptor expression in the different zones of normal articular cartilage reflects IGF-1 stimulation and metabolic activity of chondrocytes in these layers. This correlation is disturbed in arthritic cartilage, suggesting inadequate or overruled signalling.

Macrophages and dendritic cells during the early stages of antigen-induced arthritis in rats: immunohistochemical analysis of cryostat sections of the whole knee joint.

The appearance of different macrophage subpopulations, Ia-positive antigen-presenting dendritic cells and of T and B lymphocytes was studied in early phases of antigen-induced arthritis in rat knee joints. Cryostat sections of whole knee joints were analysed with immunohistochemical techniques using monoclonal antibodies against rat macrophages, Ia-antigen, and lymphocyte subpopulations. The results showed that in the early phases of the development of arthritis, the synovium was already infiltrated by many monocytes, young macrophages, granulocytes, perivascular Ia-positive non-lymphoid cells, some mature tissue macrophages, and only few T lymphocytes. In later phases not only monocytes, young macrophages and Ia-positive cells became more prominent but also the more mature ED2 positive macrophages and the ED3 positive macrophages that are normally confined to lymphoid organs became increasingly important. The T-cell population increased to some extent in later phases of arthritis induction, possibly induced by clustering with the Ia-positive cells.

Histochemical analysis of insulin-like growth factor-1 binding sites in mouse normal and experimentally induced arthritic articular cartilage.

Insulin-like growth factor-1 (IGF-1) plays a key role in regulation of chondrocyte metabolism. We examined the localization of IGF-1 binding sites on chondrocytes in cartilage from normal and experimentally induced arthritic mouse knee joints. Cryostat sections from patellar cartilage were incubated either with IGF-1 receptor antibody or biotinylated IGF-1. Subsequently confocal laser scanning microscopy was applied to compare the two staining procedures qualitatively and quantitatively. This approach allowed detailed analysis of membrane-associated and intracellular staining. Using IGF-1 receptor antibody, IGF-1 receptors were found on the cell membrane of chondrocytes in the middle and deeper cartilage zones, whereas intracellular staining was highest in chondrocytes of superficial zones. After incubation with biotinylated IGF-1, distinct membrane staining was not present and fluorescence was localized homogeneously in the middle and deeper zones but not in superficial zones. In cartilage from inflamed knee joints staining with the use of IGF-1 receptor antibody did not change significantly, whereas a pronounced increase in staining was noted with biotinylated IGF-1 in chondrocytes of the middle and deeper zones of cartilage. It is concluded that the staining patterns obtained with the use of IGF-1 receptor antibody and biotinylated IGF-1 are remarkably different, suggesting that the latter also detects IGF-binding proteins. The results suggest that joint inflammation has no consistent effect on IGF-1 receptor expression but may induce a significant upregulation of IGF-binding proteins in chondrocytes of the middle and deeper zones of cartilage.

Stimulation of proteoglycan synthesis by triamcinolone acetonide and insulin-like growth factor 1 in normal and arthritic murine articular cartilage.

OBJECTIVE: During experimentally induced arthritis, inhibition of proteoglycan synthesis is one of the mechanisms leading to cartilage destruction. Disturbed anabolic signalling might contribute to this impaired chondrocyte proteoglycan synthesis. We investigated the effects of insulin-like growth factor 1 (IGF-1) and the glucocorticoid, triamcinolone acetonide, on in vitro chondrocyte proteoglycan synthesis of articular cartilage obtained from normal and arthritic mouse knee joints. METHODS: Proteoglycan synthesis was measured by 35S sulfate incorporation and the hydrodynamic volume of newly synthesized proteoglycans was analyzed with gel chromatography. RESULTS: Culturing normal cartilage with IGF-1 resulted in significant enhancement of chondrocyte proteoglycan synthesis. Concerning the hydrodynamic volume of newly synthesized proteoglycans after culture with IGF-1, proteoglycan monomers with large hydrodynamic size, similar to those synthesized immediately after dissection were observed. In arthritic cartilage, IGF-1 failed to stimulate proteoglycan synthesis and only proteoglycans with relatively small dimensions were produced. However, in the presence of the steroid triamcinolone acetonide, synthesis of hydrodynamically large proteoglycans were found in arthritic as well as normal cartilage. CONCLUSION: Our observations indicate that steroids may play a critical role in maintaining cartilage integrity in both normal and arthritic cartilage.

Responsiveness of articular cartilage from normal and inflamed mouse knee joints to various growth factors.

OBJECTIVE: Disturbed anabolic signalling might contribute to the decreased chondrocyte proteoglycan (PG) synthesis during joint inflammation. Articular cartilage obtained from mouse knee joints with experimentally-induced arthritis exhibits a state of nonresponsiveness towards stimulation of chondrocyte PG synthesis by insulin-like growth factor-1 (IGF-1). Investigations were carried out on the role of other growth factors apart from IGF-1 on regulation of chondrocyte PG synthesis under pathological conditions, that is, during repair after IL-1 exposure as well as during early and later arthritis. METHODS: Mouse patellae were obtained from normal knee joints and joints injected with IL-1 or zymosan. The patellae were cultured with basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], epidermal growth factor [EGF] or transforming growth factor beta [TGF beta] for 24 hours in the presence or absence of IGF-1. Chondrocyte PG synthesis was measured by 35S-sulphate incorporation. RESULTS: In normal cartilage none of the tested growth factors elicited stimulatory effects on the chondrocyte PG synthesis as caused by IGF-1. EGF and TGF beta even caused significant inhibition of chondrocyte PG synthesis. Combination of bFGF or PDGF with IGF-1 exerted significant additional stimulation of the 35S-sulphate incorporation. IL-1 exposed cartilage displayed reactivity to IGF-1 as well as to the other growth factors similar to control cartilage. Cartilage obtained from joints with experimentally-induced arthritis exhibited a state of nonresponsiveness towards all individually tested growth factors as well as growth factor combinations. CONCLUSION: Arthritis causes nonresponsiveness to stimulation of chondrocyte PG synthesis by the tested growth factors, which might be caused by a general receptor function defect.

IL-1 has no direct role in the IGF-1 non-responsive state during experimentally induced arthritis in mouse knee joints.

OBJECTIVE: To investigate the involvement of interleukin-1 (IL-1) in the induction or maintenance of the insulin-like growth factor 1 (IGF-1) non-responsive state of chondrocytes during experimental arthritis in mouse knee joints. METHODS: To characterise IGF-1 nonresponsiveness during arthritis, we measured chondrocyte proteoglycan (PG) synthesis by assaying incorporation of 35S-sulphate into mouse patellar cartilage, obtained from knee joints with experimentally induced arthritis and normal knee joints, cultured with IGF-1. We investigated whether suppressive mediators produced by the arthritic synovium or chondrocytes abolished the IGF-1 stimulation of normal cartilage, and used IL-1 primed cartilage to mimic the arthritic in vivo state. Specific inflammatory mediators responsible for the maintenance of the suppressed IGF-1 response were sought. We measured IGF-1 responsiveness in normal and arthritic patellae cultured with antibodies against tumour necrosis factor (TNF) or IL-1 alpha/beta, with IL-1 receptor antagonist (IL-1ra), and with several inhibitors of proteolytic enzymes or reactive oxygen species, and analysed the role of IL-1 in the development of IGF-1 non-responsiveness by studying IGF-1 responses in cartilage treated with IL-1 antibodies in vivo, at the onset of arthritis. RESULTS: Mediators from the surrounding tissue of both normal and arthritic cartilage suppressed chondrocyte IGF-1 responses. Priming the cartilage with IL-1 did not directly induce IGF-1 non-responsiveness, but enhanced the ability of suppressive mediators from synovium or chondrocytes to downregulate the IGF-1 responsive state. IL-1ra, IL-1 alpha/beta antibody, TNF antibody, or the inhibitors tested did not markedly improve the disturbed IGF-1 response, but treatment with anti-IL-1 at the onset of arthritis prevented the development of IGF-1 non-responsiveness. CONCLUSION: IL-1 alone does not induce IGF-1 non-responsiveness and is not critical in the maintenance of this phenomenon. However, IL-1 does appear to be an important cofactor in the generation of the IGF-1 non-responsive state.

Localization of insulin-like growth factor-1 receptor in human normal and osteoarthritic cartilage in relation to proteoglycan synthesis and content.

Insulin-like growth factor-1 (IGF-1) plays a key role in the regulation of chondrocyte proteoglycan (PG) metabolism. We investigated whether chondrocyte PG synthetic activity correlates with the presence of chondrocyte IGF-1 receptor in the surface, middle and deeper zones of normal human articular cartilage and in cartilage known to display a shift in chondrocyte metabolism, i.e. cultured cartilage or osteoarthritic (OA) cartilage. Cartilage specimens were obtained post mortem from human knees within 18 h after death from donors without known clinical OA history. The samples were taken from macroscopically normal looking regions as well as from damaged regions with osteoarthritic appearance, yielding a range of OA grades from mild to moderate and severe OA. We examined chondrocyte PG synthesis by in situ autoradiography of incorporated [35S]sulphate and chondrocyte IGF-1 receptor localization by immunohistochemistry, followed by confocal laser scanning microscopical (CLSM) analysis in the same cartilage samples. In normal cartilage, both the amount of chondrocyte PG synthesis and the level of chondrocyte IGF-1 receptor localization are at low levels in the surface zone chondrocytes, but both are high in middle and deeper zone chondrocytes. Furthermore, after culture, the increase in chondrocyte PG synthesis in the surface layer coincides with increase in IGF-1 receptor expression. However, in mild OA particularly high levels of chondrocyte synthetic activity were found in the upper cartilage layer, whereas IGF-1 receptor expression was low in this layer, suggesting that factors other than IGF-1 are involved. High chondrocyte PG synthetic activity and chondrocyte IGF-1 receptor staining were found in the upper and deeper layers of moderate OA cartilage, whereas both low levels of chondrocyte activity as well as IGF-1 receptors were observed in cases of severe OA. Our data indicate that IGF-1 displays cellular heterogeneity in chondrocyte stimulation in the various cartilage zones in normal cartilage. Clear zonal correlation is lost in OA cartilage, and patterns of chondrocyte IGF-1 receptor expression and PG synthesis vary with the stage of OA.

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes.

Rheumatoid arthritis, a disease of unknown aetiology, is characterized by joint inflammation and, in its later stages, cartilage destruction. Inflammatory mediators may exert not only suppression of matrix synthesis but also cartilage degradation, which eventually leads to severe cartilage depletion. Systemically and locally produced growth factors and hormones regulate cartilage metabolism. Alterations in levels of these factors or in their activity can influence the pathogenesis of articular cartilage destruction in arthritic joints. The main topic of the present review is the role of the anabolic factor insulin-like growth factor-1 in the regulation of chondrocyte metabolic functions in normal and in diseased cartilage. This is the most important growth factor that balances chondrocytes proteoglycan synthesis and catabolism to maintain a functional cartilage matrix. A brief overview of how chondrocytes keep the cartilage matrix intact, and how catabolic and anabolic factors are thought to be involved in pathological cartilage destruction precedes the review of the role of this growth factor in proteoglycan metabolism in cartilage.

Local UV-induced DNA damage in cell nuclei results in local transcription inhibition.

UV-induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.