Biomedical subjects

K Melén

Publications and source records attributed to K Melén.

At least 19 recordsLinked to original sources

PubMed2002

Effect of profound acid suppression in functional dyspepsia: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Functional dyspepsia (FD) is defined as persistent or recurrent pain/discomfort centred in the upper abdomen, where no structural explanation for the symptoms is found. The role of drug treatment remains controversial. The aim in this study was to evaluate the effect of omeprazole 20 mg twice daily (b.i.d) and to test methods for symptom assessment. METHODS: 197 patients fulfilling the criteria for FD were randomly allocated to double-blind treatment with omeprazole 20 mg b.i.d (n = 100) or placebo (n = 97) for 14 days. Patients with a known gastrointestinal disorder or with main symptoms indicating gastro-oesophageal reflux disease or irritable bowel syndrome were excluded. Helicobacter pylori testing and 24-h intra-oesophageal 24-h pH-metry were performed before randomization. The patients recorded dyspeptic symptoms on diary cards. RESULTS: A stringent endpoint, 'complete symptom relief on the last day of treatment', was the primary efficacy variable. For the APT cohort, this was achieved in 29.0% and 17.7% on omeprazole and placebo, respectively (95% CI of difference (11.3%): -0.4%-23.0%, P = 0.057). Similar figures in the PP cohort were 31.0% and 15.5%, respectively (95% Cl of difference (15.5%): 3.2%-27.7%, P = 0.018). The benefit of omeprazole in the PP cohort was confirmed by secondary endpoints such as, no dyspeptic symptoms on the last 2 days of treatment and overall treatment response. H. pylori status and the level of oesophageal acid exposure did not significantly influence the response to therapy. CONCLUSION: A subset of patients with FD will respond to therapy with omeprazole.

Adult↗

PubMed2000

Inflammatory responses in influenza A virus infection.

Influenza A virus causes respiratory tract infections, which are occasionally complicated by secondary bacterial infections. Influenza A virus replicates in epithelial cells and leukocytes resulting in the production of chemokines and cytokines, which favor the extravasation of blood mononuclear cells and the development of antiviral and Th1-type immune response. Influenza A virus-infected respiratory epithelial cells produce limited amounts of chemokines (RANTES, MCP-1, IL-8) and IFN-alpha/beta, whereas monocytes/macrophages readily produce chemokines such as RANTES, MIP-1alpha, MCP-1, MCP-3, IP-10 and cytokines TNF-alpha, IL-1beta, IL-6, IL-18 and IFN-alpha/beta. The role of influenza A virus-induced inflammatory response in relation to otitis media is being discussed.

Apoptosis↗

PubMed2000

Interferon-induced gene expression and signaling in human hepatoma cell lines.

BACKGROUND/AIM: Interferon(IFN)-alpha alone or combined with other antiviral substances has been extensively used for the treatment of viral infections of the liver. Since the molecular mechanisms of IFN action in liver cells are relatively poorly characterized, we studied IFN-induced gene expression and signaling in human hepatoma, HepG2 and HuH7 cell lines. METHODS/RESULTS: IFN binding to its specific cell surface receptor leads to activation of the Janus family tyrosine kinase (JAK) - signal transducer and activator of transcription (STAT) pathway. We observed that in HepG2 and HuH7 cells IFN-inducible genes were upregulated by IFNs, but relatively high concentrations of IFN-alpha were needed to turn on MxA (an antiviral gene) and MxB gene expression. The basal expression of IFN-alpha receptor (IFNAR1 and IF-NAR2) JAK1 and TYK2 mRNAs was readily detectable, and their expression was not significantly altered by treatment with either IFN-alpha or IFN-gamma. Hepatoma cells possessed relatively low basal expression levels of IFN signaling molecules STAT1, STAT2 and p48, but their expression was strongly upregulated by both types of IFNs. Pretreatment of HepG2 or HuH7 with low IFN-gamma doses, followed by stimulation with IFN-alpha, resulted in a marked enhancement of the formation of IFN-alpha-specific signaling complex ISGF3. CONCLUSION: The results indicate positive feedback mechanisms in the IFN signaling system in hepatoma cells.

Animals↗

PubMed1999

Impaired antiviral response in human hepatoma cells.

Hepatitis B, C, and D viruses can infect liver cells and in some individuals establish a chronic phase of infection. Presently, relatively little information is available on the antiviral mechanisms in liver cells. Because no good in vitro model infection systems for hepatitis viruses are available, we have used influenza A, Sendai, and vesicular stomatitis (VSV) viruses to characterize interferon (IFN) responses and IFN-induced antiviral mechanisms in human hepatoma cell lines. HepG2 or HuH7 cells did not show any detectable IFN-alpha/beta production in response to influenza A or Sendai virus infections. Treatment of cells with IFN-alpha resulted in upregulation of IFN-alpha-inducible Mx, 2',5'-oligoadenylate synthetase (OAS) and HLA class I gene expression but only with exceptionally high levels of IFN-alpha (>/=100 IU/ml). Accordingly, high pretreatment levels of IFN-alpha, 1000 IU/ml for influenza A and VSV and 100 IU/ml for Sendai virus, were required before any detectable antiviral activity against these viruses was seen. IFN-gamma had some antiviral effect against influenza A virus but appeared to be ineffective against VSV and Sendai virus. IFN-gamma upregulated HLA class I protein expression, whereas Mx or OAS expression levels were not increased. There was a modest upregulation of HLA class I expression during Sendai virus infection, whereas influenza A virus infection resulted, after an initial weak upregulation, in a clear decrease in HLA class I expression at late times of infection. The results suggest that hepatoma cells may have intrinsically poor ability to produce and respond to type I IFNs, which may contribute to their inability to efficiently resist viral infections.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

Efficacy of PCR and other diagnostic methods for the detection of respiratory adenoviral infections.

Five methods were evaluated for the detection of adenovirus directly from nasopharyngeal aspirates (NPA), including conventional and rapid virus culture, two antigen detection tests, and the polymerase chain reaction (PCR). NPA specimens were obtained from 269 military conscripts suffering from an acute respiratory infection during an adenovirus outbreak. In 133 cases, paired blood specimens were also available. Virus culture followed by a hexon-specific immunofluorescence revealed 159 (59%) adenovirus-positive specimens and it was used as a reference method. In comparison to conventional culture, a rapid 2-day culture method had a sensitivity of 71%. The sensitivities of an enzyme immunoassay and time-resolved fluoroimmunoassay were 53% and 46%, respectively. The PCR method employing Ad7 hexon-specific primers showed a high sensitivity of 94%, and revealed an additional 15 (6%) specimens that could not be confirmed by virus culture. Serology based on significant adenovirus antibody rises had a diagnostic efficacy nearly equal to the virus culture and PCR methods, but a relatively high number of discordant results was found. The present study demonstrates that PCR is a very sensitive rapid diagnostic method for detecting adenovirus specific DNA in NPA specimens of adults.

Adenovirus Infections, Human↗

PubMed1998

Kinetics of cytokine and NFAT gene expression in human interleukin-2-dependent T lymphoblasts stimulated via T-cell receptor.

T cells respond to mitogenic or antigenic stimulation by proliferation and by turning on cytokine gene expression. Here we have analysed the kinetics and nature of cytokine production in human peripheral blood-derived T lymphoblasts stimulated with anti-CD3 antibodies or Lens culinaris lectin (LCL). T cells were purified from peripheral blood mononuclear cells (PBMC) and primarily activated with anti-CD3 antibodies and cultured in the presence of interleukin-2 (IL-2). Anti-CD3-restimulated T cells (mainly CD8+) produced IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and low levels of IL-4 and IL-10 transcripts and proteins. No IL-6 gene expression was observed. In LCL-stimulated cells the cytokine production pattern was very similar. Steady-state mRNA levels of IL-2, IL-10 and IFN-gamma peaked at 3 hr after anti-CD3 stimulation and declined rapidly thereafter. The kinetics of TNF-alpha mRNA expression was faster, being at its peak level 1 hr after stimulation. Anti-CD3-stimulated IL-2 gene expression was down-regulated by protein synthesis inhibitor, whereas IL-10, IFN-gamma and TNF-alpha genes were readily induced independent of ongoing protein synthesis. T-cell receptor stimulation also induced a very rapid expression of c-jun, c-fos and NFATc1 (NFATc) genes, the gene products of which are involved in cytokine gene expression. In conclusion, the cytokines synthesized by IL-2-dependent T cells were predominantly IL-2, IFN-gamma and TNF-alpha.

Antibodies↗

PubMed1997

Nuclear cotransport mechanism of cytoplasmic human MxB protein.

Interferon-alpha/beta-inducible Mx proteins belong to the family of large GTPases and share high sequence homology with dynamins in their N-terminal GTP-binding domains. In addition, Mx proteins have a conserved C-terminal leucine zipper element that is involved in their oligomerization. Cytoplasmic human MxA protein mediates resistance to multiple RNA viruses, whereas no antiviral activity has been found for human MxB protein. We have previously shown that MxB protein exists as a nuclear 78-kDa and as a cytoplasmic 76-kDa form in interferon-alpha-induced human cells. Using various influenza hemagglutinin epitope-tagged MxB gene constructs in transient transfection experiments in COS-1 cells, we show that the cytoplasmic 76-kDa MxB protein forms hetero-oligomers with the nuclear 78-kDa MxB protein via the C-terminal leucine zipper element. This enables the cytoplasmic form of MxB protein to be translocated into the nucleus together with the nuclear form of MxB protein. This finding was confirmed in interferon-alpha-induced HEp-2 and T98G cells transfected with various MxB gene constructs. Cell fractionation studies also suggest that a considerable amount of the cytoplasmic MxB protein is also found in the nucleus. Using confocal laser microscopy, we also demonstrate that the cytoplasmic MxA and the nuclear MxB proteins do not colocalize/oligomerize with each other, and both of these proteins are retained in their specific cellular compartments.

Animals↗

PubMed1997

Regulation of IFN-alpha/beta, MxA, 2',5'-oligoadenylate synthetase, and HLA gene expression in influenza A-infected human lung epithelial cells.

The epithelial cells of the respiratory tract are the primary sites of virus replication in influenza A virus infections. We infected human alveolar epithelium-like A549 cells and fibroblast-like human fetal lung (HFL1) cells with a pathogenic influenza A virus (A/Beijing/353/89), and studied the kinetics of infection and the expression of host IFN-alpha/beta, MxA, OAS (2',5'-oligoadenylate synthetase), and HLA class I and II genes. Viral mRNA and protein synthesis was clearly seen in virus-infected lung cells. A549 and HFL1 cells produced only small amounts of IFN-alpha/beta, whereas virus-infected macrophages produced type I IFN very efficiently. The kinetics of IFN-beta gene expression in A549 cells was rapid, as shown by reverse-transcriptase PCR, and IFN-beta mRNA expression levels correlated well to the kinetics of nuclear factor-kappa B transcription factor activation. In influenza A virus-infected A549 and HFL1 cells, MxA gene induction was mediated by IFN-alpha/beta released into the cell culture supernatant, and was prevented by anti-type I IFN Abs. HLA class I Ag expression, which could be activated by IFN in noninfected A549 and HFL1 cells, was not induced in these cells by virus infection. The results suggest that type I IFN are essential for the activation of the antiviral response in lung epithelial cells.

2',5'-Oligoadenylate Synthetase↗

PubMed1997

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Acute-Phase Proteins↗

PubMed1996

Inhibition of puumala and tula hantaviruses in Vero cells by MxA protein.

Human MxA protein is a type I Interferon-inducible intracytoplasmic protein, which mediates antiviral actions against a variety of negative-strand RNA viruses including influenza A, measles, and vesicular stomatitis viruses. Recently, it has also been shown that several members of the Bunyaviridae family are inhibited by MxA protein. The hantavirus genus in the Bunyaviridae family includes important human pathogenic viruses, e.g., Puumala (PUUV), Hantaan, and Sin Nombre viruses. Tula virus (TULV) is a new member of the genus, but its pathogenicity in man remains to be determined. As assumed by the similarities in replication strategy. MxA would be a good candidate molecule for antiviral action against these viruses, also. To gain more insight into the MxA action on PUUV, we studied PUUV and TULV replication in stably MxA genetransfected Vero cells. We show that MxA protein has the capacity to inhibit both viral protein and RNA accumulation in virus-infected cells. We also studied PUUV and TULV infection in MxA-transfected U-937 cell clones. In these cell lines both hantaviruses grew poorly, independent of whether the cells were expressing MxA or not Whether cell line-specific differences in the antiviral activity of MxA protein against hantaviruses exist cannot be conclusively determined due to the lack of productive infection of PUUV and TULV in U-937 cells.

Animals↗

PubMed1996

Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope.

Interferon-inducible Mx proteins belong to the family of large GTPases and are highly homologous with dynamins within their GTP-binding domain. Cytoplasmically localized human MxA protein mediates resistance to influenza and several other viruses, whereas human MxB protein has not been found to have any antiviral activity. Here we show that MxB protein is found both in the cytoplasm and in the nucleus, where it is localized in a granular pattern in the heterochromatin region beneath the nuclear envelope. Transfection experiments in COS cells of N-terminally deleted MxB constructs revealed a functional nuclear localization signal within the first 24 N-terminal amino acids. Nuclear 78-kDa and cytoplasmic 76-kDa forms of MxB protein were found in all of the cell lines studied and in human peripheral blood mononuclear cells. MxB protein proved to be a functional GTPase with activity comparable to that of MxA protein. N-terminally truncated (delta1-82) MxB protein lacking both the nuclear localization signal and a proline-rich domain had almost completely lost its GTPase activity. Analysis of peripheral blood mononuclear cells suggested that MxB protein expression is strictly regulated by interferon-alpha. This is the first documentation that human Mx protein resides in the nucleus. It also emphasizes that there are considerable differences in the localization and structure of functional domains within Mx proteins.

Amino Acid Sequence↗

PubMed1996

Early infant grasping involves radial fingers.

Halverson's hypothesis states that prehension gradually develops from ulnar to forefinger holding. This hypothesis was tested by measuring the grip force of individual fingers (digits II to V) in 36 healthy children from 2.5 to 7 months of age. The results dismissed Halverson's hypothesis, since it was shown that from an early age the index finger played a leading role in grasping, usually contacting and initiating the grasp prior to the other fingers and producing a stronger grip force.

Child Development↗

PubMed1995

Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme.

Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coli and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms. The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms. Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4-dihydroxybenzoic acid was studied in detail. Here, the catalytic number (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The Km values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher Km values for catecholamines than MB-COMT. The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L-methionine (AdoMet). The average dissociation constants Ks were 3.4 and 20.2 microM for MB-COMT and S-COMT, respectively. Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed. Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 [Lundström et al. (1991) DNA Cell. Biol. 10, 181-189] or Met-108 [Bertocci et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1416-1420].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Mutational analysis of murine Mx1 protein: GTP binding core domain is essential for anti-influenza A activity.

Interferon-induced resistance to influenza virus in murine cells is mediated by the Mx1 protein, which inhibits viral mRNA synthesis in the nucleus. Murine Mx1 protein is a GTPase specifically targeted into the cell nucleus and it has a C-terminal leucine zipper domain that mediates its oligomerization. In order to determine functionally important elements of the protein we created several substitution, linker insertion, and deletion mutants of murine Mx1 protein. The antiviral activity of mutant Mx1 proteins was analyzed by a transient transfection/influenza A infection assay in COS cells by indirect immunofluorescence. Mx1 proteins carrying mutations in the vicinity or within the consensus GTP binding elements exhibited markedly reduced, but not completely lost, antiviral activity. Baculovirus produced, GTP binding element substitution mutant Mx1 proteins showed very low (< 10%) GTPase activity as compared to wild-type Mx1 protein. Mutations in other portions of the molecule had less effect on antiviral activity, except one mutant, which was situated six amino acids from the C-terminal end. This mutation evidently interrupted the nuclear localization signal rendering the protein cytoplasmic and clearly reduced the anti-influenza activity. Deletions of various sizes and locations further suggested that the N-terminal half of the molecule is more important in the antiviral activity than other regions of the molecule. These results indicate that the GTP binding domain of Mx1 protein is essential for its anti-influenza activity, correlating to the low GTPase activity of the GTP binding element substitution mutants, but other portions of the molecule such as the leucine zipper and the nuclear localization signal are of importance, too.

Animals↗

PubMed1994

Enzymatic characterization of interferon-induced antiviral GTPases murine Mx1 and human MxA proteins.

Interferons induce a number of different proteins which mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. Interferon-induced Mx proteins, which confer resistance to influenza, vesicular stomatitis, and measles viruses, contain consensus GTPase sequence elements. Insect cell-produced purified murine Mx1 and human MxA proteins were found to hydrolyze GTP with Km = 65 microM (Vmax, 7.1 min-1) and 62 microM (Vmax, 3.1 min-1), respectively. The GTPase activity of Mx1 and MxA proteins was strictly dependent on Mg2+ ions. Murine Mx1 protein was inactivated at 10 degrees C lower temperatures than MxA protein. As analyzed, by filter binding assay, Mx1 protein (at 1 microM) showed a relatively high affinity for GDP (Kd = 1.0 x 10(-7) M) and approximately 340-fold lower affinity for guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) (Kd = 3.4 x 10(-5) M). The Kd values for MxA protein were 2.0 x 10(-7) M for GDP and 5.9 x 10(-6) M for GTP gamma S, showing approximately a 30-fold affinity difference. ATP, UTP, or CTP did not inhibit the Mx protein-dependent GTPase activity, suggesting that Mx1 and MxA proteins are highly specific for guanosine nucleotides. In conclusion recombinant nuclear murine Mx1 and cytoplasmic human MxA proteins show clear differences in their enzymatic activities and nucleotide binding characteristics. How these differences influence their cellular functions and antiviral potential is presently not known.

Animals↗

PubMed1993

Control of IFN-inducible MxA gene expression in human cells.

MxA is an IFN-induced human protein which is located in the cytoplasm of induced cells. MxA makes the cells resistant to infection by influenza and vesicular stomatitis viruses. In the present work we used baculovirus expression system to produce MxA protein. The protein was purified to homogeneity and highly specific polyclonal anti-MxA antibodies were prepared. In human mononuclear cells, and A549 lung carcinoma cells expression of MxA protein is induced by very low (< 1 IU/ml) doses of leukocyte IFN-alpha (nIFN-alpha), whereas IFN-gamma does not seem to induce it or potentiate the induction by IFN-alpha. In mononuclear cells stimulated with high doses of leukocyte IFN-alpha concentrations, the amount of MxA mRNA was induced 10-fold at 4 h after IFN induction and up to 10-fold higher MxA protein levels were observed at 24-48 h postinduction. The gene can be reinduced by IFN-alpha 24 h after the initial induction suggesting for a lack of negative feedback after this time point. The protein is very stable, the half-life being approximately 2.3 days. Flow cytometric analysis revealed that monocytes have higher basal and induced MxA protein levels than lymphocytes but the dose-dependency of MxA expression is very similar in both cell types. Granulocytes are producing very low amounts of MxA protein.

Animals↗

PubMed1992

Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper.

Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.

Animals↗

PubMed1991

Retroperitoneal malignant cyst. Case report.

Forty years after removal of a benign mucinous cyst from the left retroperitoneal space, a malignant cyst with both sarcomatous and carcinomatous components was removed from the same site in a 75-year-old woman. Within 6 months the lesion recurred as a carcinoma penetrating the left colon and the abdominal wall.

Adult↗