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H Lankinen

Publications and source records attributed to H Lankinen.

34 records · Page 2Linked to original sources
PubMed1995

Effect of interferon-alpha and cell differentiation on Puumala virus infection in human monocyte/macrophages.

Pathogenesis of hantavirus infections is poorly understood. Puumala virus (PUU) is the etiologic agent of nephropathia epidemica, a form of hemorrhagic fever with renal syndrome common in Europe. We have studied PUU infection in primary human monocyte/macrophages and specifically the role of interferon alpha (IFN-alpha) and cell differentiation in it. PUU infection proceeded at a low level in monocyte/macrophages, and nucleocapsid (N) protein accumulation started 2 days postinfection. IFN-induced antiviral MxA protein was detected 3 days postinfection, suggesting IFN-alpha production in culture. IFN-alpha titers remained low, proposing that PUU is a poor IFN inducer. However, the PUU-induced IFN had an inhibitory effect on virus production as was shown by the effect of anti-IFN-alpha. Pretreatment of cells with IFN-alpha caused a dose-dependent inhibition of PUU N accumulation and reduced the yield of infectious virus. Monocytic U-937 cells overexpressing MxA protein were susceptible to PUU, suggesting that, unlike in some other negative strand RNA virus infections, MxA does not mediate resistance to PUU infection. Differentiation of monocyte/macrophages in culture and treatment of THP-1 promonocytic cells with phorbol 12-myristate 13-acetate made the cells more susceptible to PUU. The increased susceptibility of mature macrophages to PUU suggests that after differentiation to tissue macrophages they might function in the spread of the virus during PUU infection.

Animals↗
PubMed1995

Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied.

Animals↗
PubMed1994

Morphological differentiation of human SH-SY5Y neuroblastoma cells inhibits human immunodeficiency virus type 1 infection.

We have studied human immunodeficiency virus type 1 (HIV-1) infection in human SH-SY5Y neuroblastoma cells at various stages of morphological differentiation. Two days' treatment of the cells with retinoic acid (RA) or dibutyryl cAMP (db-cAMP) resulted in the appearance of elongated neurites and enhanced production of 160K to 200K neurofilament proteins as shown by indirect immunofluorescence. DNA synthesis was reduced only in RA-treated cells as detected by 5-bromo-2'-deoxyuridine incorporation. The cells were infected with two T-lymphotropic virus strains (IIIB and NDK) and two fresh isolates (39001 and 46001) from bronchoalveolar lavage samples of AIDS patients. The latter two isolates were unable to form syncytia in infected CD4-positive T-lymphoblastoid C8166 cells which was in contrast to our T-lymphotropic virus strains. Interphase in situ hybridization showed that 14 to 16% of SH-SY5Y cells become positive for HIV-1 DNA. Regardless of the virus strain, morphological differentiation of the cells with RA or db-cAMP inhibited infection by 50% at a single cell in situ resolution. Nested PCR confirmed the presence of proviral DNA in the infected cells. These results show that human neuroblastoma cells, tumour cells of neuroectodermal origin, can be infected by different HIV-1 isolates and that the infection is inhibited by neurotypic cell differentiation.

Base Sequence↗
PubMed1994

Tula virus: a newly detected hantavirus carried by European common voles.

A novel hantavirus has been discovered in European common voles, Microtus arvalis and Microtus rossiaemeridionalis. According to sequencing data for the genomic RNA S segment and nucleocapsid protein and data obtained by immunoblotting with a panel of monoclonal antibodies, the virus, designated Tula virus, is a distinct novel member of the genus Hantavirus. Phylogenetic analyses of Tula virus indicate that it is most closely related to Prospect Hill, Puumala, and Muerto Canyon viruses. The results support the view that the evolution of hantaviruses follows that of their primary carriers. Comparison of strains circulating within a local rodent population revealed a genetic drift via accumulation of base substitutions and deletions or insertions. The Tula virus population from individual animals is represented by quasispecies, indicating the potential for rapid evolution of the agent.

Amino Acid Sequence↗
PubMed1993

Susceptibility of human cells to Puumala virus infection.

Nephropathia epidemica involves several organs including kidney, lung, liver and brain. To investigate the susceptibility of putative target cells to the agent responsible, Puumala virus, we screened established human cell lines of lung (WI-38, A-427, CCD-11Lu), kidney (A-704), liver (Hep G2), pharynx (Detroit 562), submaxillary gland (A-253) and neural (SK-N-MC, SH-SY5Y) origin as well as primary human kidney glomerular cells, endothelial cells and peripheral blood monocytes/macrophages. Propagation of the Sotkamo strain of Puumala virus was also tested in the primary kidney, spleen and lung cells of bank voles (the natural host of the virus). All of the primary cells and most of the established cell lines expressed viral protein, synthesized viral RNA and secreted infectious virus, except the neural SK-N-MC and SH-SY5Y cells. None of the tested cell types except the primary bank vole kidney cells could propagate the virus as efficiently as the Vero E6 cells. The observed host cell range is wide and consistent with a multiorgan involvement of Puumala virus. No cytopathic effects were seen in any of the infected cell cultures.

Animals↗
PubMed1993

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase.

The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources. We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein. A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed. They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305. The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site. Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.

Amino Acid Sequence↗
PubMed1992

The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity.

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

Amino Acid Sequence↗
PubMed1991

The large subunit of herpes simplex virus type 1 ribonucleotide reductase: expression in Escherichia coli and purification.

The open reading frame of the large subunit (R1) of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been positioned downstream of the phage T7 gene 10 promoter in the expression vector, pET. Transformation of this recombinant plasmid into Escherichia coli BL21 DE3 cells containing the T7 RNA polymerase, under the control of the lac UV5 promoter, allows expression of the subunit on induction of the T7 RNA polymerase by isopropyl thiodigalactoside. The expressed protein is soluble and can be purified with yields up to 0.5 mg of R1 per litre of bacterial culture. The subunit can complement R2 produced in BHK cells or E. coli to give specific activities comparable to that produced in BHK cells infected with HSV-1. Enzyme activity reconstituted from E. coli-expressed R1 and R2 is inhibited by the nonapeptide YAGAVVNDL with an IC50 comparable to that obtained with enzyme extracted from BHK cells infected with HSV-1. Results suggest that the E. coli produced enzyme is a good source of protein for further structural and functional studies.

Animals↗
PubMed1991

Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli.

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.

Amino Acids↗
PubMed1990

Inhibition of equine herpesvirus type 1 subtype 1-induced ribonucleotide reductase by the nonapeptide YAGAVVNDL.

The synthetic nonapeptide YAGAVVNDL [identical to the nine carboxy-terminal amino acids of the small subunit of herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR)] was found to inhibit the RR activity induced by equine herpesvirus type 1 subtype 1 (EHV-1). Parallel experiments with HSV type 1 (HSV-1)-encoded RR established that the concentration of peptide required to inhibit 50% of the RR activity was 28 microM for both enzymes. The optimum pH for the EHV-1 enzyme was found to be between 8.0 and 8.1 which is the same as that of HSV-1 RR. By use of antisera made against peptides corresponding to different regions of the large subunit (RR1) of the HSV-1 enzyme and monoclonal antibodies directed against HSV-1 RR1 we have obtained evidence which suggests that the EHV-1 large subunit has an Mr of approximately 90,000 and lacks the N-terminal domain which is so far unique to HSV.

Amino Acid Sequence↗
PubMed1989

The unique N-terminal domain of the large subunit of herpes simplex virus ribonucleotide reductase is preferentially sensitive to proteolysis.

Using antisera made against peptides corresponding to different regions of the large subunit of herpes simplex virus type 1 ribonucleotide reductase we have probed proteolytic fragments of this protein and found that at least a part of its unique N-terminal domain is not necessary for enzyme activity. This non-essential region encompasses the domain previously predicted to be composed of beta sheets with a well buried core of hydrophobic residues. Truncated forms of the large subunit are generated in vivo and are located almost exclusively in the nucleus.

Amino Acid Sequence↗
PubMed1987

The herpes simplex virus type 1 ribonucleotide reductase is a tight complex of the type alpha 2 beta 2 composed of 40K and 140K proteins, of which the latter shows multiple forms due to proteolysis.

We have isolated two monospecific monoclonal mouse antibodies directed against the HSV-1 ribonucleotide reductase. When immobilized to Sepharose, both antibodies remove enzyme activity from solution. However, on immunoblots of crude extracts of HSV-1-infected cells, one antibody only detects a 140K protein and the other antibody only a 40K protein. Neither antibody recognizes the cellular ribonucleotide reductase or the related pseudorabies virus-induced enzyme. Therefore, our data strongly suggest that the HSV-1 ribonucleotide reductase consists of a 140K and a 40K protein. The 140K protein is sequentially degraded to 110K, 93K, and 81K proteins by a Vero cell-specific, N alpha-p-tosyl-L-lysine chloromethyl ketone-sensitive protease. Of the different proteolytic products, at least the 93K species seems to be enzymatically active, suggesting that part of the 140K protein may have functions not related to ribonucleotide reduction. There is a very high affinity between the 140K and 40K proteins as evident from affinity chromatography on antibody-Sepharose and sedimentation velocity centrifugation in a glycerol gradient. The 140K and 40K proteins cosediment with the HSV-1 ribonucleotide reductase activity at 17 S. This indicates that the active form of the HSV-1 reductase consists of the 140K and 40K proteins forming a tight complex of the alpha 2 beta 2 type.

Antibodies, Monoclonal↗
PubMed1982

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.

Allosteric Regulation↗
PubMed1981

Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells.

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form. By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity. The amount of M1 protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit. We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein. It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.

Animals↗
PubMedDate not supplied

Effect of temporin A modifications on its cytotoxicity and antimicrobial activity.

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.

Amino Acid Sequence↗