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H Apfel

Publications and source records attributed to H Apfel.

At least 19 recordsLinked to original sources
PubMed1999

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Amino Acid Sequence↗
PubMed1999

Up-regulation of extracellular copper/zinc superoxide dismutase mRNA after transmission of the filarial parasite Acanthocheilonema viteae in the vertebrate host Meriones unguiculatus.

The gene encoding the cytoplasmic copper/zinc superoxide dismutase (AVSOD1) from the filarial parasite Acanthocheilonema viteae was isolated from a genomic DNA library using a degenerate oligonucleotide probe. Additionally, cDNAs of the AVSOD1 and the secreted extracellular SOD (AVSOD2) were both cloned by RT-PCR, and the AVSOD2 was expressed at high levels in E. coli. The amino acid sequence of the AVSOD1 is 89.5 and 87.5% identical to that of the corresponding enzymes of Brugia pahangi and Onchocerca volvulus, respectively. In contrast, the AVSOD2 shows a lower degree of identity to the other filarial SODs and is extensively glycosylated. RT-PCR studies demonstrate the expression of both SOD subtypes in all developmental stages of A. viteae and indicate up-regulation of the AVSOD2 expression after transmission from the vector to the definitive host. This suggests an enhanced requirement for SOD activity in post-infective larval stages and adults of A. viteae. ELISAs performed with purified recombinant AVSOD2 show that the AVSOD2 is not a major target for the immune system in naturally infected jirds.

Amino Acid Sequence↗
PubMed1999

Immunogenicity of the extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae delivered by a two-phase vaccine strain of Salmonella typhimurium.

The recombinant extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae (AVSOD2) was cloned in an expression vector under control of the bacteriophage T7 promoter and the resulting plasmid pLAT7 was introduced in tha aroA attenuated Salmonella typhimurium vaccine strain SL3261:pYZ84. This vaccine strain carries a chromosomally integrated two phase expression system containing inducible T7 RNA polymerase. The recombinant AVSOD2 was efficiently expressed, constituting up to 5% of the total bacterial protein. Furthermore, the plasmid vector containing the AVSOD2 cDNA was shown to be stable over a long period of time in the vaccine strain without antibiotic selection in vitro and in vivo. Jirds which were immunised orally with the recombinant vaccine strain expressing the A. viteae EC-SOD produced a strong humoral immune response.

Administration, Oral↗
PubMed1998

Regulation of ion channels in rat hepatocytes.

Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K+ channel with a mean inward conductance of 55 +/- 6.5 pS (n = 20) and a mean outward conductance of 25 +/- 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl- channel with a mean outward conductance of 30 +/- 4.5 pS (n = 8) and a mean inward conductance of 10 +/- 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca2+ activity on the intracellular side. Accordingly, the Ca2+ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K+ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K+ channel is inhibited by progesterone, while the outwardly rectifying Cl- channel is insensitive to progesterone.

Animals↗
PubMed1997

PCR-based amplification of total cDNA with high fidelity and high yield from minute amounts of parasite RNA.

cDNA, synthesised from total RNA from Acanthocheilonema viteae, was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+)strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

Animals↗
PubMed1996

Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T lymphocytes upon Fas stimulation.

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56(lck) tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56(lck) kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis.

Apoptosis↗
PubMed1995

Electrophysiological effects of progesterone on hepatocytes.

The addition of progesterone (1-100 mumol/l) to the extracellular fluid bathing rat hepatocytes led to a rapid and fully reversible depolarization of the cell membrane. The progesterone-induced depolarization was paralleled by a decrease of potassium selectivity and an increase of cell membrane resistance and was abolished in the presence of the potassium channel blocker barium. Accordingly, in whole cell recordings, progesterone led to a decrease of the cell membrane conductance. 17 alpha-Hydroxyprogesterone and beta-estradiol were less effective by a factor of 10, whereas cholesterol, corticosterone and hydrocortisone did not significantly alter the potential difference across the cell membrane. In conclusion, acute administration of progesterone depolarized rat hepatocytes by decreasing the potassium conductance of the cell membrane.

Animals↗
PubMed1995

Ca2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells.

Exposure of aortic strips from guinea-pigs to hypotonic extracellular fluid is followed by marked vasoconstriction, which is inhibited by D-600 (3 microM), a blocker of voltage-sensitive Ca2+ channels. Conventional electrophysiology, patch-clamp studies, pH determination with 2',7' bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and Ca2+ measurements with Fura-2 have been performed on smooth muscle cells cultured either from rat or human aorta to further elucidate the underlying mechanisms. Exposure of the cells to a 25% hypotonic extracellular fluid leads to a rapid and fully reversible depolarization, paralleled by an increase of the selectivity and conductance of the cell membrane to Cl-, an acidification of the cytoplasm and an increase of intracellular Ca2+ concentration ([Ca2+]i). The latter is inhibited by the Ca2+ channel blocker D-600 (1-3 microM). It is concluded that osmotic cell swelling leads to the activation of an anion channel. The subsequent depolarization of the cell membrane activates voltage-sensitive Ca2+ channels which increases [Ca2+]i, thus stimulating the contraction of vascular smooth muscle cells.

Animals↗
PubMed1994

Protective immunity linked with a distinct developmental stage of a filarial parasite.

Repeated low dose infections of the jird Meriones unguiculatus, with the filarial parasite Acanthocheilonema viteae cause a substantial reduction of the total worm burden, suggesting a parasite-driven immune mechanism that controls super-infections. Quantitative recovery of parasites from tissues of triple infected jirds reveals that the larvae derived from a subsequent challenge infection are inactivated or severely impaired several days after transmission, precisely during their molt from the L3 to the L4 stage. Moreover, only larvae undergoing the molt from L3 to L4 stages are capable of stimulating an immune response directed against the challenge infection, indicating that protective Ag are produced during the molting period. Consistent with this, inactivated L3 or live L4 do not produce the same effect. In contrast to susceptible animals, immune jirds elicit high serum antibody titers against molting Ag. Indirect fluorescence antibody-binding tests with sera from protected jirds reveal specific labeling of the surface of molting L3 and not other larval stages, implying a stage-specific elimination process. The identification of molting L3 as a natural target for host immune mechanisms, emphasizes the central importance of this larval stage for future efforts aimed toward the development of a filarial vaccine.

Animals↗
PubMed1992

Changes in the surface composition after transmission of Acanthocheilonema viteae third stage larvae into the jird.

This study describes the dynamics and the biochemical nature of changes in the surface of the filarial nematode Acanthocheilonema viteae after its transmission into the vertebrate host. Vector-derived third-stage larvae (mL3) were inoculated into naive Meriones unguiculatus and recovered from the tissues at different times post-infection until their moult to fourth-stage larvae (L4). Surface-specific labelling with fluoresceinated lectins revealed that the larvae are covered by a carbohydrate envelope. Although the mL3 envelope was strongly reduced one day after transmission, new surface carbohydrates appeared until the onset of moulting, some of which could also be identified on the surface of L4. In general, surface carbohydrates were partially shed by moving larvae, suggesting a loose association of these components in the epicuticle. The fate of cuticular lipids and proteins of L3 and L4 was monitored by external 125I-labelling and differential extraction of the components. Thin-layer chromatography of surface-labelled lipids revealed only minor changes 1 day after parasite transmission. Afterwards the number of lipids accessible to label decreased further until moulting was complete. Two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of surface-labelled proteins showed a consistent surface exposure of mL3 specific proteins until 1 day post-infection. Thereafter, the composition of surface-labelled proteins changed rapidly, resembling that of the L4 as early as several days before moulting. During this period individual differences in the composition of surface proteins were evident.

Animals↗
PubMed1991

Recovery, distribution, and development of Acanthocheilonema viteae third- and early fourth-stage larvae in adult jirds.

Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.

Abdomen↗
PubMed1990

Active release of surface proteins: a mechanism associated with the immune escape of Acanthocheilonema viteae microfilariae.

Living Acanthocheilonema viteae microfilariae obtained from peripheral blood of parasitised Meriones unguiculatus were surface-labelled with 125I. Four major surface exposed proteins of approximately 14.50, 14.55, 17.5, 19 kDa and one less abundant protein of 40 kDa were identified. Under non-reducing conditions the low-molecular-weight (LMW) proteins were isolated as multimers suggesting the presence of intermolecular disulphide linkages. In gels containing Triton X-100 the labelled epicuticular proteins behaved lipophilically. By cultivation of surface-labelled and metabolically labelled microfilaria in vitro, a continuous shedding of two LMW proteins was demonstrated. These proteins were produced in large amounts and released into the culture supernatant as monomeric and pentameric molecules. Concomitant with this release, one of the proteins appeared to lose its lipophilic character, giving rise to a hydrophilic 14.50-kDa entity. Although most of the extracted surface proteins reacted with sera from patent jirds, these sera failed to recognise the surface of living microfilariae. However, microfilariae pretreated with glutaraldehyde or attenuated with Na-azide could be labelled with surface specific antibodies.

Acanthocephala↗
PubMed1988

Acetylcholine induces overdrive excitation in sheep Purkinje fibres.

The hypothesis that in the presence of acetylcholine overdrive may induce spontaneous repetitive activity (overdrive excitation) was tested by recording the transmembrane potentials and contractile force in sheep cardiac Purkinje fibres perfused in vitro. The results were: (a) acetylcholine 10(-8) to 10(-4) mol.litre-1 increased the action potential duration, the diastolic depolarisation slope and amplitude, and the twitch amplitude in a dose dependent manner; (b) at concentrations of 10(-5) to 10(-4) mol.litre-1 the interruption of a 60 beat.min-1 drive often induced repetitive spontaneous activity which was fastest immediately after the drive, slowed gradually, and ceased abruptly when an oscillatory potential failed to attain the threshold; (c) in the presence of acetylcholine, in quiescent preparations, applying drives of different durations and at different frequencies resulted in a steeper and larger diastolic depolarisation, oscillatory potentials, and a prolonged afterdepolarisation; (d) when overdrive excitation was induced it was faster and longer after faster or longer overdrives; (e) the cessation of overdrive was often associated with an aftercontraction; (f) in the presence of acetylcholine and a high extracellular calcium concentration (10.8 mmol.litre-1) overdrive excitation was preceded by a short period of inhibition; (g) strophanthidin (5 x 10(-8) mol.litre-1) facilitated acetylcholine induced overdrive excitation (without an initial inhibition); (h) lowering the extracellular sodium concentration (-50% NaCl) antagonised overdrive excitation; and (i) atropine 10(-5) mol.litre-1 prevented acetylcholine induced overdrive excitation. It is concluded that acetylcholine induces overdrive excitation by causing a larger diastolic depolarisation, oscillatory potentials, and a transient prolonged afterdepolarisation; that overdrive excitation requires sodium as a charge carrier underlying the electrical events leading to the attainment of the threshold; and that acetylcholine induces excitation by acting on a muscarinic receptor.

Acetylcholine↗
PubMed1985

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.

Animals↗
PubMed1985

Molecular cloning and a stable amplification of the DNA molecules heavily methylated at CpG sequences using a new E. coli cell system (GC3).

Fish lymphocystis disease virus (FLDV) DNA is heavily methylated in CpG sequences and therefore the amplification of recombinant DNA of FLDV inserted into a bacterial plasmid vector and/or a bacteriophage system poses severe problems. The problem was solved using a newly constructed and developed bacterial system (E. coli GC-3 system), which allowed the amplification of foreign DNA material heavily methylated at CpG sequences. E. coli GC-3 (Gc-1, rifd) is derived from E. coli GC-1 (K 12, hsd-, MDU) after transformation with phage lambda drifd 18 Hind III-B-fragment. A defined and complete gene library of the FLDV DNA sequences was established by insertion of FLDV DNA fragments (Eco RI, Eco RI/Bam HI, Eco RI/Hind III) into the corresponding restriction sites of bacterial plasmid vector pAT 153. Bacterial colonies harbouring recombinant plasmids were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridizing plasmid DNA to viral DNA. The analyses revealed that sequences representative for the complete viral genome were cloned.

Animals↗