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Biomedical subjects

I Virtanen

Publications and source records attributed to I Virtanen.

At least 19 recordsLinked to original sources
PubMed1995

Tenascin and fibronectin isoforms in human renal cell carcinomas, renal cell carcinoma cell lines and xenografts in nude mice.

We studied the expression of tenascin (Tn) and isoforms of fibronectin (Fn) in human renal cell carcinomas (RCC) and oncocytomas, in RCC cell lines and in their s.c. implanted xenografts in nude mice. In well-differentiated RCCs and oncocytomas extra-domain A (EDA)-Fn and Tn immunoreactivities were confined to the basement membranes and blood vessels, while in less-differentiated RCCs they were also widely seen in the stroma, correlating with the morphological differentiation of the tumor. Expression of EDB-Fn and oncofetal (onc)-Fn was very scarce in most of the RCCs and oncocytomas. Western blotting results demonstrated the predominance of the M(r) 190,000 Tn subunit in most RCCs. Among the 4 RCC cell lines, 3 showed Tn in the extracellular matrix. As xenografts, they formed moderately or poorly differentiated tumors, with abundant Tn. Three of the RCC cell lines also showed secretion of EDA-Fn and 2 of them secretion of onc-Fn and EDB-Fn into the culture medium, while in xenografts there was a strong expression of all Fn isoforms. In xenografts, the expression of Tn closely recapitulated that seen in clinical tumors and in the cell lines in vitro, while the expression of Fn isoforms in cultured cells and their xenografts was highly discordant.

Animals↗
PubMed1995

Mutation of the cytoplasmic domain of the integrin beta 3 subunit. Differential effects on cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis.

The cytoplasmic domain of the beta subunit of the alpha IIb beta 3 integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the beta 3 (I757TYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ile757 partially preserved these functions. We further found that substitution of Tyr747 with Ala also abolished alpha IIb beta a-mediated cell spreading. The effects of these and other mutations on additional functions of alpha IIb beta 3 were also studied. Progressive truncations of beta 3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of alpha IIb beta 3 to preestablished adhesion plaques and a gradual decrease in the ability of alpha IIb beta 3 to mediate internalization of fibrinogen-coated particles. The Tyr747-->Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr759, and at a conserved area close to the transmembrane domain of beta 3, decreased integrin recruitment to preestablished adhesion plaques but allowed alpha IIb beta 3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of beta 3 did not affect the constitutive endocytosis of alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1995

Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.

The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.

Animals↗
PubMed1995

Integrins in human anterior chamber angle.

BACKGROUND: Integrins, which are composed of an alpha and beta subunit, are capable of binding to a number of extracellular matrix proteins and, hence, affect cell adhesion and proliferation. METHODS: The distribution of the integrin beta (beta 1, beta 3-beta 5) and alpha (alpha 1-6 and alpha v) subunits in human anterior chamber angle was studied in eyes from subjects aged 9 months to 81 years using the indirect immunofluorescence technique. RESULTS: Immunoreaction for the beta 1 subunit was found throughout the trabecular meshwork (TM), in the cribriform layer, and in the endothelial lining of Schlemm's canal (SC). Labelling for the alpha 3 subunit was found in the TM and the cribriform layer only. In infant eyes the alpha 5 subunit was present in all three areas with the highest concentration in the cribriform layer, whereas no reaction was observed in adult eyes. The alpha 6 subunit was localized to the endothelium of SC only. Immunoreaction for the alpha v subunit was present in the TM and the cribriform layer of infants and young adults. CONCLUSION: The present results suggest the presence of several integrin heterodimers, acting as potential receptors for laminin, collagen, fibronectin, and vitronectin, in the anterior chamber angle.

Adolescent↗
PubMed1995

Distribution of neurofilament proteins and peripherin in the rat pituitary gland.

The distribution of neurofilament proteins and peripherin in the pituitary gland of the rat was studied with a panel of monoclonal and polyclonal antibodies recognizing different neurofilament subunits. In the posterior lobe, a dense plexus of neurofilament- and peripherin-immunoreactive fibers was seen. In the intermediate lobe neurofilament- and peripherin-immunoreactivity was seen only in nerve fibers in the connective tissue septa, while no immunoreactivity was seen in parenchymal nerve fibers. Bilateral sympathetic ganglionectomy did not affect peripherin-immunoreactivity, indicating that the peripherin-immunoreactive fibers are of central origin. In the anterior lobe, a few solitary neurofilament- and peripherin-immunoreactive fibers were observed. Western blotting confirmed the presence of 150 kD and 200 kD neurofilament proteins in the posterior lobe. No neurofilament protein was detected in the intermediate and anterior lobes. Abundant intermediate filaments were seen with electron microscopy in the nerve fibers of the connective tissue septa in the intermediate lobe. In the parenchymal nerve fibers only microtubules were seen, indicating that the lack of neurofilament immunoreactivity is due to absence of neurofilaments.

Animals↗
PubMed1995

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy.

To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.

Actinin↗
PubMed1995

Lack of effect of hormonal treatment and smoking on cytokeratin expression in buccal mucosa.

Differences in cytokeratin expression of clinically normal buccal mucosa were studied in 50 healthy women by indirect immunofluorescence staining with monoclonal antibodies. The subjects were divided into four groups: control group (N = 18), smokers (N = 8), oral contraceptive users (N = 8) and smokers receiving oral contraceptives (N = 16). Our findings indicate that cytokeratin expression in noncornified stratified epithelium is not influenced by smoking or oestradiol/progesterone treatment. Only cytokeratin No. 19 showed variable patterns of expression but the differences could not be ascribed to smoking or contraceptives. Cytokeratin No. 19 gave a positive reaction in the basal and suprabasal layers in 34 subjects (68%). In 9 (18%) specimens, the staining was positive in the basal cells and showed a positive heterogeneous cytoplasmic reaction in the suprabasal cells. Interestingly, cytokeratin No. 7 was recognized in all epithelial cells except the basal cells. Our results suggest that changes in the serum oestradiol levels do not affect the cytokeratin pattern in buccal mucosa.

Adult↗
PubMed1995

Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading.

The C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.

CSK Tyrosine-Protein Kinase↗
PubMed1995

Modulation of fibronectin and tenascin production in human bronchial epithelial cells by inflammatory cytokines in vitro.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix glycoproteins participating in tissue morphogenesis and repair. The regulation of their synthesis and deposition during airway inflammation and their possible contribution in asthma are poorly understood. In this study, modulation of Fn and Tn production was investigated in transformed human bronchial epithelial cells in culture. The cells were treated with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), a combination of these cytokines, interleukins 3 and 6 (IL-3 and IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), and a combination of IL-3 and IL-6 for 48 h. Immunofluorescence and immunoblotting methods with monoclonal antibodies to Fn and Tn antibodies suggested the production of some Fn and Tn in the untreated cells. Fn was minimally induced in response to IFN-gamma and TNF-alpha, when compared with the untreated cells, whereas TNF-alpha and especially the IFN-gamma plus TNF-alpha combination resulted in a prominent Tn induction. Interleukins and GM-CSF did not induce Fn or Tn in any case. These results show that human bronchial epithelial cells are capable of producing Fn and Tn. The modulation of Fn and Tn may have an important impact on the pathology of epithelial cells during airway inflammation in vivo.

Bronchi↗
PubMed1995

Transforming growth factor-beta regulates the expression of fibronectin and tenascin in BEAS 2B human bronchial epithelial cells.

We used monoclonal antibodies to study expression and extracellular matrix (ECM) incorporation of tenascin (Tn) and isoforms of fibronectin (Fn) in BEAS 2B immortalized human bronchial epithelial cells and the regulation of their synthesis by transforming growth factor (TGF)-beta 1 and -beta 2. In immunofluorescence microscopy, the control cells appeared negative for Tn. Extradomain A (EDA)-Fn was mainly seen in association with ECM fibers and, in a few cells, in an intracellular location. Immunoreactivity for oncofetal (onc)-Fn and extradomain B (EDB)-Fn was only seen in a few cells. In TGF-beta 1- and -beta 2-treated cells, a greatly enhanced immunostaining for Tn and three isoforms of Fn was seen both as to the number of positive cells and to the amount of immunoreactive material around them. In Western blotting of the untreated cells, EDA-Fn and onc-Fn were detected in the cell-free ECM and in the culture medium, whereas EDB-Fn was not detectable. An enhanced secretion and deposition of both EDA-Fn and onc-Fn and also secretion of EDB-Fn was seen upon treatment with TGF-beta s. In TGF-beta-treated cells, Tn was found exclusively in the ECM and not in the culture medium as shown by Western blotting of cell-free ECM and culture medium, respectively. Accentuation of tenascin staining in TGF-beta-treated cells was due to a greatly enhanced production of M(r) 280,000 and M(r) 190,000 isoforms of Tn.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1995

Differential expression of laminin polypeptides in developing and adult human kidney.

We studied the expression of laminin chains in embryonic and adult human kidney by indirect immunofluorescence with monoclonal antibodies (MAbs). In embryonic human kidney, immunoreactivity for laminin alpha 1, beta 1, and gamma 1 chains was found in basement membranes (BMs) of primary vesicles, in comma- and S-shaped bodies, and in more mature stages of glomeruli and in tubules. The beta 2 chain of laminin was absent in the early glomerular structures but was prominent in BMs of maturing glomeruli (GBMs) and Bowman's capsule (BCBMs) and was also detectable in some tubules. Both the beta 2 and alpha 2 chains were variably seen in medullary tubule BMs. In adult human kidney, laminin alpha 1 chain was seen in GBMs and all tubule BMs (TBMs) as well as in arterial smooth muscle BMs (SMBMs). Laminin beta 1 chain reactivity was found in all TBMs, but not in GBMs or SMBMs. In the glomerulus, a distinct mesangial type of reaction was revealed with the MAbs to beta 1 and alpha 2 chains. The GBMs and SMBMs reacted with MAbs to the beta 2 chain, but reactivity was lacking in BCBMs. Laminin gamma 1 chain immunoreactivity was weakly present in BCBMs, GBMs, and SMBMs. The alpha 3 and beta 3 chains could not be detected in developing or adult human nephron. The results show that during development the BMs in human nephron undergo distinct changes, laminin beta 1 chain being transiently co-expressed with alpha 1 chain during early glomerular development and then becoming replaced by the beta 2 chain, which, on the other hand, disappears from the BCBMs on maturation. The alpha 2 chain appears to emerge in the mesangium late during development.

Embryonic and Fetal Development↗
PubMed1995

Differential expression of laminin chains and their integrin receptors in human gastric mucosa.

The proliferating cells of the gastric mucosa are found among the pit and mucous neck cells. These cells migrate upward to renew the surface epithelium and downward to restitute the glandular cells. As the epithelial basement membranes (BMs) function as substrate for cell adhesion and migration as well as signals for their differentiation, we studied, by indirect immunofluorescence microscopy, the distribution of different laminin chains and their integrin receptors in adult human stomach. The immunoreactivity for laminin alpha 2 chain localized to the BMs of glands and the lower parts of the gastric pits whereas the laminin alpha 3 chain (laminin-5/kalinin) immunoreactivity was strictly confined to BMs underneath the surface epithelium and the upper parts of the pits. Proliferating mucosal epithelial cells, identified by Ki-67 antibodies, were confined to the areas containing both alpha 2 and alpha 3 laminin chains. The alpha 1, beta 1, and gamma 1 laminin chains were found in all BMs of the mucosa whereas the beta 2 chain was prominent in mucosal blood vessels and also detectable in some glands. Among the laminin integrin receptors, the alpha 3 and beta 4 subunits were seen to be expressed in cells along the BMs with the alpha 3 laminin chain. The alpha 6 integrin, on the other hand, was seen in all gastric epithelia. The present results demonstrate that in the adult human stomach laminin alpha 2 and alpha 3 chains show zonal distribution in BM underlying gastric mucosal epithelium whereas other laminin chains show a more general distribution.

Adult↗
PubMed1994

Isoforms of cellular fibronectin and tenascin in amniotic fluid.

Amniotic fluid (AF) obtained from second trimester pregnancies presented extradomain (ED) A, B and an oncofetal (onc-f) domain containing isoforms of cellular fibronectin (cFn) in Western blotting of gelatin-bound polypeptides and directly of AF. Western blotting after sequential immunoprecipitation suggested at least three Fn molecules: one containing EDA and the onc-f domain and another minor component distinctly containing all the domains, and a third one only containing EDA. The immunoblotting results for EDA-cFn and onc-f-cFn were closely similar to that for total Fn, whereas in plasma samples of normal and pregnant women only traces of EDA-cFn and onc-f-cFn, but no EDB-cFn, were found. Western blotting of AF also indicated the presence of three isoforms of tenascin (Tn), M(r) 190,000 and 280,000 polypeptides earlier found in many cells, and a M(r) 200,000 polypeptide, novel for AF and not present in plasma. The results suggest a novel extracellular matrix polypeptide composition for AF.

Amniocentesis↗
PubMed1994

Entry of coxsackievirus A9 into host cells: specific interactions with alpha v beta 3 integrin, the vitronectin receptor.

Attachment and entry of coxsackievirus A9 (CAV-9) to GMK cells were previously shown to be dependent on an arginine-glycine-aspartic acid (RGD) motif in the capsid protein VP1, suggesting integrins as candidate receptors for the virus. We have pursued the matter further and show that antibodies specific for the alpha v and/or beta 3 integrin subunits protect GMK cells from CAV-9 infection. Affinity purification of radioiodinated cell surface proteins using CAV-9 or virus-specific peptide (RRRGDL) columns confirmed that the alpha v beta 3 heterodimer, known as the vitronectin receptor, is recognized by the virus in GMK cells. Other proteins, of lower molecular weight (less than 40 kDa), were also bound to and specifically eluted from the columns, but their possible role in attachment and entry of CAV-9 remains to be elucidated by further studies. Of several other related viruses studied, only echovirus 22, which also has an RGD motif in the VP1 capsid protein, was found to compete for cell surface binding with CAV-9.

Animals↗
PubMed1994

Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands.

A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28-37) of alpha-human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28-37 of alpha-human CGRP and to a slightly lesser degree whole alpha-human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the beta-human CGRP but not the alpha-rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of alpha-human CGRP lacking the C-terminal part of the molecule. Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Tenascin expression in inflammatory, dysplastic and neoplastic lesions of the human stomach.

We studied the expression of tenascin (Tn) in human stomach. In the normal mucosa of the antrum and body, Tn reaction was only seen in the muscularis mucosae, in the region of the pyloric sphincter and in the duodenum, a Tn-immunoreactive rim was seen underlying surface epithelial cells. Antral gastritis, irrespective of the degree of inflammation, showed a rim-like Tn expression under the surface epithelial cells but no Tn reaction was seen in mild chronic gastritis of the body. In some moderate and severe examples of chronic gastritis a delicate Tn-reactive line was seen to underline the surface epithelium focally and the neck regions of gastric pits. Discontinuous Tn immunoreactivity was sometimes seen beneath hyperplastic epithelium in both parts of the stomach. A Tn-immunoreactive line was seldom seen surrounding glands showing intestinal metaplasia. In both benign and malignant ulcers prominent Tn immunoreaction was seen at the base of ulcers extending deep into the underlying muscularis. Only severely dysplastic lesions displayed Tn in the lamina propria, in close association with capillaries. In early forms of diffuse gastric cancer (DGCA) raggedly increased Tn staining was seen in the lamina propria underlying affected surface epithelial cells. In advanced forms of DGCA consistent Tn expression was seen in the tumour stroma. A distinct Tn reaction was seen surrounding invasive tumour cell nests of intestinal type gastric cancer (IGCA) in the submucosa, whereas in early forms of the tumour enhanced Tn reaction was noted predominantly in the upper part of the lamina propria in the vicinity of dysplastic elements. Notably, while most invading DGCA tumour cell nests showed no Tn in the submucosa and muscle cell layer, invading IGCA islets showed prominent expression of Tn. The most conspicuous Tn enhancement in the stomach is seen in invasive tumours and in ulcers suggesting that Tn is an important stromal component in malignant growth and in lesions undergoing active repair and remodelling.

Biomarkers, Tumor↗
PubMed1994

Cytokeratin inclusions in alcoholic liver disease and their relation to the amount of alcohol intake.

In the present study, the frequency and the distribution pattern of immunoreactive hepatocytic cytokeratin (CK) inclusions was investigated using the monoclonal antibody (MAb) CAM 5.2 detecting CKs 8, 18 and 19. The CK antigenicity of the inclusions was confirmed on frozen sections with MAbs for the CKs 7, 8, 17, 18 and 19. The frequency of hepatocytic CK aggregates was compared to the presence of non-alcoholic and alcoholic liver disease (ALD) as well as to the average all-year daily ethanol intake of 195 consecutive males subjected to medico-legal autopsy. Hepatocytic CK inclusions were infrequent in patients with normal liver histology, portal fibrosis and/or portal inflammation. In ALD, however, inclusions were observed in 35.6% of patients with fatty liver, in 63.2% of patients with alcoholic hepatitis, in 30.8% of patients with bridging fibrosis and in 60.0% of patients with liver cirrhosis. In all specimens studied, the inclusions were reactive only for CKs 8 and 18, CKs 7, 17, and 19 being unreactive. The frequency of inclusion bodies increased, paralleling increasing average all-year daily alcohol consumption. Compared to non-drinkers, a daily intake of between 40 and 80 g of absolute alcohol was associated with a statistically significantly (relative risk, RR = 6.6) increased risk of formation of aggregates. Similarly, daily consumption exceeding 80 g was related to increased (RR = 6.0) frequency of CK aggregates. The frequency of full-blown "classical" Mallory bodies (MBs) was, however, increased (RR = 8.9) only in patients with a daily intake exceeding 160 g.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗