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Biomedical subjects

V P Lehto

Publications and source records attributed to V P Lehto.

At least 19 recordsLinked to original sources
PubMed2004

Progression of malignancy in clear cell renal cell carcinomas.

Much progress has recently been obtained in the classification and characterization of RCC by using cytogenetic, gene microarray and proteomic techniques. Pivotal for the understanding of the progression of malignancy of clear cell renal cell carcinomas are findings connecting its biology to inactivation of the von Hippel-Lindau tumour suppressor gene product (VHLp), found in most CC-RCCs. Disruption of VHLp function appears to be involved in altered cell cycle control, resistance to hypoxia, hyperangiogenesis and changes in the organization of cytoskeletal and extracellular matrix proteins in RCC. These changes are reflected in the overexpression of the vascular endothelial growth factor (VEGF) and the subunits of hypoxia-inducible factor (HIF), and other angiogenetic and metastasis-promoting factors. Other changes related to progression of malignancy in RCC are the upregulation of proinflammatory cytokines and changes in cell adhesion proteins.

Carcinoma, Renal Cell↗
PubMed2003

Involvement of the subacromial bursa in impingement syndrome of the shoulder as judged by expression of tenascin-C and histopathology.

Our aim was to evaluate bursal involvement at different stages of the impingement syndrome as judged by conventional histopathological examination and expression of tenascin-C, which is known to reflect active reparative processes in different tissues and disorders. Samples of subacromial bursa were taken from 33 patients with tendinitis, 11 with a partial tear and 18 with a complete tear of the rotator cuff, and from 24 control shoulders. We assessed the expression of tenascin-C, the thickness of the bursa, and the occurrence and degree of fibrosis, vascularity, haemorrhage and inflammatory cells. The expression of tenascin-C was significantly more pronounced in the complete tear group (p < 0.001) than in the partial tear, tendinitis or control groups. It was more pronounced in the tendinitis group than in the control group (p = 0.06), and there was more fibrosis in all the study groups than in the control group. The changes in the other parameters were not equally distinctive. Expression of tenascin-C did not correlate with the conventional histopathological parameters, suggesting that these markers reflect different phases of the bursal reaction. Tenascin-C seems to be a general indicator of bursal reaction, being especially pronounced at the more advanced stages of impingement and this reaction seems to be an essential part of the pathology of impingement at all its stages.

Adult↗
PubMed2001

STAM/EAST/Hbp adapter proteins--integrators of signalling pathways.

STAM/EAST/Hbp family of proteins consists of eight members well conserved from yeast to mammals. The basic domain architecture is comprised of an N-terminal Vps27, Hrs and STAM homology domain, a ubiquitin-interacting motif and a central Src homology-3 domain. Vertebrate members also carry an immunoreceptor tyrosine-based activation motif. STAM/EAST/Hbp proteins become tyrosine-phosphorylated by a variety of cytokines and growth factors. STAM 1 and STAM 2A are involved in cytokine-mediated signalling for DNA synthesis and c-myc induction. EAST and STAM 2A/Hbp play a role in receptor-mediated endo- and exocytosis and probably also in the regulation of actin cytoskeleton. Knockout experiments implicate a role for STAM 1 in neural cell survival. A picture is emerging of STAM/EAST/Hbp proteins acting as integrators of thus far mechanistically disparate cellular signalling events.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Binding of GGA2 to the lysosomal enzyme sorting motif of the mannose 6-phosphate receptor.

The GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Here, the VHS domain of GGA2 was shown to bind to the acidic cluster-dileucine motif in the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CI-MPR). Receptors with mutations in this motif were defective in lysosomal enzyme sorting. The hinge domain of GGA2 bound clathrin, suggesting that GGA2 could be a link between cargo molecules and clathrin-coated vesicle assembly. Thus, GGA2 binding to the CI-MPR is important for lysosomal enzyme targeting.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Dynamic solid-state and tableting properties of four theophylline forms.

Relationships between solid-state, densification and compact properties of theophylline monohydrate (TMO), a mixture of forms (TMIX), and anhydrous polymorphs I (TA-I) and II (TA-II) were evaluated. Solid-state identification of powders and compacts was accomplished by powder X-ray diffraction. A compaction simulator was used to assess deformation behaviour of the powders and to prepare compacts. Porosity and tensile strength of the compacts were determined after 1,24, and 168 h of storage at 22% relative humidity. TA-II was stable, whereas TA-I, TMIX and TMO partially transformed to the TA-II form during storage. All theophylline modifications primarily deformed by plastic flow. Increased water content decreased resistance towards densification and deformation of TMIX and TMO when compared to TA-II or TA-I, demonstrating viscoelasticity. Permanent densification behaviours of TMIX and TMO approached to that of TA-II during storage. Tensile strength of the different theophylline forms were practically equal after 1 h of storage. Tensile strength and porosity of TMIX and TMO compacts increased during the storage. Dynamic solid-state transformations from TMO, TMIX and TA-I to TA-II were associated with parallel changes in their densification and compact properties. The extent of these changes was also dependent on the materials' water content.

Bronchodilator Agents↗
PubMed2001

The small myelin-associated glycoprotein binds to tubulin and microtubules.

The myelin-associated glycoprotein (MAG) exists as two isoforms, differing only by their respective cytoplasmic domains, that have been suggested to function in the formation and maintenance of myelin. In the present study, a 50 kDa protein binding directly to the small MAG (S-MAG) cytoplasmic domain was detected and identified as tubulin, the core component of the microtubular cytoskeleton. In vitro, the S-MAG cytoplasmic domain slowed the polymerization rate of tubulin and co-purified with assembled microtubules. A significant sequence homology was found between the tau family tubulin-binding repeats and the carboxy-terminus of S-MAG. Our results indicate that S-MAG is the first member of the Ig superfamily that can be classified as a microtubule-associated protein, and place S-MAG in a dynamic structural complex that could participate in linking the axonal surface and the myelinating Schwann cell cytoskeleton.

Animals↗
PubMed2001

Vear, a novel Golgi-associated protein, is preferentially expressed in type I cells in skeletal muscle.

Vear is a novel Golgi-associated protein with a domain structure characteristic of many vesicular transport-associated proteins. It has been suggested that Vear is involved in vesicle transport through trans-Golgi. In this study, we have determined the localization of Vear in skeletal muscle. The staining for Vear in normal human muscle revealed a distribution pattern similar to that of type I fibers. We conclude that Vear is preferentially expressed in type I fibers in human muscle, presumably indicative of a specific function that remains to be identified.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Podocyte-specific expression of a novel trans-Golgi protein Vear in human kidney.

BACKGROUND: Vear is a recently identified Golgi apparatus-associated protein. It has been suggested to be involved in vesicular trafficking between the Golgi and the vacuolar/lysosomal system. Proteins similar to Vear have also been shown to interact with activated ARF proteins (ADP ribosylation factor), and they are probably involved in membrane trafficking from the trans-Golgi network (TGN). We have previously shown that Vear is widely distributed in human tissues, with an especially high level of mRNA in the kidney. This study further characterizes the distribution and subcellular localization of Vear in normal adult kidney and shows its association with glomerulogenesis in fetal kidney. METHODS: Immunofluorescence and immunoelectron microscopy were used to study the expression of Vear in fetal and adult kidney. The expression of Vear in isolated glomeruli was shown by immunoblotting. The distribution of its mRNA was analyzed by using in situ and Northern hybridization. RESULTS: In situ hybridization and immunofluorescence microscopy showed that in the kidney, Vear is present in glomerular structures. By fluorescence microscopy, the immunoreactivity for Vear was found only in podocytes, as judged by its distinct colocalization with podocalyxin and vimentin, well-established marker proteins of podocytes. Its specific expression in the glomeruli versus other compartments of the kidney was also verified by Western blotting. By using immunogold electron microscopy, Vear was seen in the Golgi apparatus, tubulovesicular structures, and membranes adjacent to the Golgi complex. In fetal kidney, expression of Vear coincided with the formation of segmental structures of the glomeruli. It was first seen close to the undifferentiated luminal cells at the vesicular stage and increasingly in the differentiating podocytes at the more advanced stages of glomerulogenesis. CONCLUSIONS: In the kidney, Vear shows a distinct, specific, and developmentally regulated expression in glomerular podocytes. This suggests that Vear has a specific function in podocytes. It could be associated with the known high secretory and synthetic activity of the podocytes, especially the production of the basement membrane components, which are critically involved in the glomerulogenesis and the maintenance of the glomerular function.

Adaptor Proteins, Vesicular Transport↗
PubMed2000

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues.

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.

Adult↗
PubMed2000

S100beta inhibits the phosphorylation of the L-MAG cytoplasmic domain by PKA.

The myelin-associated glycoprotein (MAG) is a cell adhesion molecule expressed by myelinating glia, existing as two isoforms that differ only by their cytoplasmic domains. We have studied the in vitro phosphorylation of recombinant rat MAG cytoplasmic domains by three kinases for which consensus sequences exist within this domain, revealing phosphorylation of the L-MAG-specific domain by protein kinase A (PKA). Phosphorylation of the L-MAG cytoplasmic domain by PKA was decreased in the presence of S100beta, providing a functional significance to the interaction between L-MAG and S100beta, and further indicating that L-MAG may play a role in myelinating glial cell signalling processes.

Animals↗
PubMed2000

Vear, a novel Golgi-associated protein with VHS and gamma-adaptin "ear" domains.

The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to gamma-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker gamma-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane- and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.

Adaptor Protein Complex gamma Subunits↗
PubMed2000

Estimation of total ribonucleic acid quantity from dilute samples by nondenaturing electrophoresis and silver staining.

Often, the amount of RNA that can be isolated from a defined tissue is very small. A method consisting of nondenaturing polyacrylamide gel electrophoresis and silver staining is described that can be used to evaluate the concentration of very dilute RNA samples. The method is a good starting point for assays dealing with small amounts of RNA, such as semiquantitative reverse transcription polymerase chain reaction (RT-PCR), making it possible to perform parallel assays from similar amounts of total RNA when quantitation by other methods is too insensitive. The method has been used successfully to monitor the amount of total RNA isolated from rat sciatic nerve and the rat C6 glioma cell line.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Localization of salmon cardiac peptide (sCP) in the heart of salmon (Salmo salar L.).

We have previously cloned and characterized a novel cardiac hormone from the salmon (Salmo salar) which has a uniquely heart-specific distribution and a low structural similarity with any other known natriuretic peptides. Specific antibodies were raised in goat against the salmon cardiac peptide. For localization and quantification, four different methods were applied: immunohistochemistry (avidin-biotin peroxidase), transmission electron microscopy, cryoimmunoelectron microscopy (protein A-gold), and a specific radioimmunoassay. Both atrial and ventricular myocytes stained immunohistochemically. The staining was similar in all myocytes and no specific myoendocrine cells were found. Within a single myocyte, both atrial and ventricular, the staining was stronger near the nucleus. Transmission electron microscopy revealed that both the atrium and the ventricle contained small sarcoplasmic granules of similar type with a diameter of 100 to 200 nm and an electron-dense core with a clear halo. The granules were typical vesicles which can be found in secretory cells utilizing the regulatory pathway. The highest number of granules was found near the nucleus, but granules were located also near the Golgi apparatus, between myofilament bundles, and in subsarcolemmal positions. Gold particles, conjugated to antibodies raised against the salmon cardiac peptide, were deposited on similar sarcoplasmic granules found in transmission electron microscopy. Among the sarcoplasmic granules with gold particles there were granules which did not show any cardiac peptide immunoreactivity. A significantly (Student's t test, P < 0.05) higher concentration of cardiac peptide was found in the heart atrium than in the ventricle, 16.2 +/- 3.5 pmol/mg tissue (n = 8) and 4.5 +/- 1.7 pmol/mg tissue (n = 8), respectively. The findings show that the salmon cardiac peptide is localized in secretory granules in both compartments of the heart. The morphology of the granules suggests that both the atrium and the ventricle utilize the regulatory pathway to release salmon cardiac peptide.

Animals↗
PubMed2000

Simultaneous determination of the heat and the quantity of vapor sorption using a novel microcalorimetric method.

PURPOSE: In this study, instrumentation for measuring vapor sorption enthalpies and sorption uptakes simultaneously with an isothermal microcalorimeter is introduced. Various pharmaceutical model substances undergoing phase transitions when exposed to humid conditions (25 degrees C), were employed to evaluate the usefulness and sensitivity of the constructed experimental method. METHODS: The sample is placed in the sample vessel of a RH cell and the moisture content of the air flow is controlled. From the RH cell the air flow is conducted into a subsequent perfusion cell in which a saturated salt solution has been loaded. The RH cell and perfusion cells are positioned in the sample sides of two twin calorimetric units. Depending on the moisture content in the outlet flow leaving the preceding RH cell, the heat flow signal from the subsequent perfusion cell will vary. By means of blank measurement with identical settings, the rate of water sorption can be calculated and, by integration, the amount of sorbed water is obtained. RESULTS: Amorphous lactose and cefadroxil undergo recrystallization when the moisture level in the surroundings exceeds the threshold values specific to each compound. During the sorption phase, heat is evolved fairly linearly as a function of consumed moisture, and also after the recrystallization, the heats indicate linear behavior. The heat values for the desorption phase of amorphous lactose and the adsorption of crystalline lactose coincide. With the different anhydrous forms of theophylline, the hydration takes place more rapidly in the metastable form 1, and generally, the process is more energetic in form 1. In all cases, the gravimetric results agree with the water sorption uptakes calculated from the calorimetric data. CONCLUSIONS: The technique introduced offers a rapid and sensitive method to gain new insights into the transitions in which vapors are involved. In addition, different kinds of surfaces with various energetics can now be studied more closely.

Calorimetry↗
PubMed1999

Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion.

The expression pattern of laminin (Ln) alpha1 chain has been a controversial topic due to discrepancies between mRNA and protein studies. Recently it was reported that the monoclonal antibody 4C7, previously thought to recognize Ln alpha1 chain, actually detects Ln alpha5 chain. This finding makes it necessary to reestimate the role of Ln alpha1 chain and to compare the expression and functions of Ln alpha1 and alpha5 chains. We studied the expression of Ln alpha1 and alpha5 chains and production of Ln-1 and Ln-10 in cultured human carcinoma cells. Ln alpha1 chain mRNA was detected in JAR choriocarcinoma cells and in all four renal cell carcinoma cell lines studied. In contrast, pancreatic, colon, and lung alveolar carcinoma cell lines did not express or produce Ln alpha1 chain, suggesting that Ln-1 (alpha1 beta1 gamma1) is produced only by certain carcinoma cells. Ln alpha5 chain mRNA was expressed in all carcinoma cells, but was not incorporated into extracellular matrix in vitro, as shown with JAR cells. Immunoprecipitation of metabolically labeled cells showed that cells expressing Ln alpha1 mRNA also produced 400-kDa Ln alpha1 chain, whereas all cells produced 380-kDa Ln alpha5 chain. Adhesion to Ln-1 was inhibited by a functionally blocking antibody against alpha6-integrin subunit, whereas adhesion to Ln-10 was inhibited by an antibody against alpha6-integrin in JAR cells and by an antibody against alpha3-integrin in PANC-1 cells. The results suggest that Ln-10 is a ubiquitously expressed Ln isoform in carcinoma cells, and the mechanism of adhesion to Ln-10 is cell-type specific.

Blotting, Northern↗
PubMed1999

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state of kidney epithelial cell lines.

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton.

Animals↗