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M Ackermann

Publications and source records attributed to M Ackermann.

At least 73 records · Page 4Linked to original sources
PubMed1997

[Importance of AFPplus-screening in daily practice. Evaluation of 2641 analyses].

Since 1991 the measurement of 3 biochemical parameters for the risk assessment of trisomy 21 pregnancy (AFPplus, Alpha-check) is also offered by private laboratories in Switzerland. This work aimed at the proof of reliability of this risk analysis in the hands of practitioners and private laboratories. The investigated cohort included 2,641 pregnant women. An anonymous questionnaire on the outcome of pregnancy furnished informations in 97% of cases (2,561 cases). At a trisomy 21 risk cutoff of one to 380, 9% of the pregnant women had conspicuous AFPplus results (trisomy 21-risk > 1 to 380; age distribution at term: 17 to 45 years; median 29.4 years). Among the conspicuous cases were 8 with trisomy 21, 1 with trisomy 18, 5 spontaneous abortions and 3 special cases. Seven of a total of 9 trisomies were detected by the screening. From these data the following diagnostic parameters for AFPplus have been calculated: specificity 91.0%, sensitivity 77.8%, positive predictive value 99.9%, false positive rate 9.0%, false negative rate 22.2%. The use of AFPplus by a practitioner cooperating with a private laboratory thus reveals a detection rate comparable to that known from larger centers. AFPplus by a practitioner cooperating with a private laboratory thus reveals a detection rate comparable to that known from larger centers. AFPplus allows to individualize the statistical age-related risk of a pregnant women before age 35. Above age 35 AFPplus may support the indication for cases suitable for cytogenetic investigation. Thorough personal information of the patient before the 16th pregnancy week by the practitioner or by genetic counseling has without any doubt first priority.

Adolescent↗
PubMed1997

Small, anionic, and charge-neutralizing propeptide fragments of zymogens are antimicrobial.

Some inactive precursor proteins, or zymogens, contain small, amino terminus, homopolymeric regions of Asp that neutralize the cationic charge of the active protein during synthesis. After posttranslational cleavage, the anionic propeptide fragment may exhibit antimicrobial activity. To demonstrate this, ovine trypsinogen activation peptide, and frog (Xenopus laevis) PYL activation peptide, both containing homopolymeric regions of Asp, were synthesized and tested against previously described surfactant-associated anionic peptide. Peptides inhibited the growth of both gram-negative (MIC, 0.08 to 3.00 mM) and gram-positive (MIC, 0.94 to 2.67 mM) bacteria. Small, anionic, and charge-neutralizing propeptide fragments of zymogens form a new class of host-derived antimicrobial peptides important in innate defense.

Anions↗
PubMed1996

Pathogenesis of ruminant herpesvirus infections.

Ruminants are hosts for members of both Alpha- and Gamma-herpesvirinae. A wide range of disease syndromes is associated with infections by these agents. The associated diseases reflect the biological nature of the causative viruses. Clinically, the symptoms may be mild and localized or include severe generalized disease, leading eventually to death. Much knowledge has been gained concerning the pathogenesis of some alpha-herpesviruses. Initially, these viruses replicate in epithelial cells at the portal of entry. The symptoms of the acute diseases are often associated with the destruction of those epithelial cells. However, as in the case of bovine herpesvirus 1 (BHV-1), the virus may spread in the infected host by viremia, gaining access to a broader range of tissues and organs, and causing a broader variety of diseases. Furthermore, many herpesviruses are capable of entering neuronal cells. There, they may replicate, which may lead to neuronal diseases, for example, encephalitis. In addition, the herpesviruses may establish latency in neuronal or lymphoid cells. During latency, apparently no viral antigens are synthesized but the genomes of the latent viruses are present in the nuclei of long living cells, such as, e.g., neurones of the ganglia corresponding to the sites of peripheral replication. Upon reactivation, the viruses re-establish the lytic cycle of replication. Shielded from the effectors of the immune system, they migrate back to the peripheral tissues where they are excreted and may be transmitted. Although a strong immune response is provoked during primary viral replication, these mechanisms help the herpesviruses to escape from immune surveillance during latency and to a lesser degree during reactivation. It has been observed that certain herpesviruses may behave differently upon infection of different hosts. Relatively little progress has been made concerning the understanding of the pathogenesis of ruminant herpesviruses but much has been learned about viral molecular biology. Many viral proteins have been identified and characterized and the technology to create recombinant viruses has been established. With these tools in our hands, it is now possible to address the really interesting questions concerning pathogenesis. We postulate that herpesviruses contain at least two sets of genes, a first set involved in gene expression and viral replication, and a second set responsible for functions, which may affect pathogenesis, latency, and virus/host interactions. Using recombinant virus technology, it will be possible in the future to design targeted deletions and gene transfers in ruminant herpesviruses in order to study the viral and host factors involved in pathogenesis on the molecular level.

Alphaherpesvirinae↗
PubMed1996

Molecular virology of ruminant herpesviruses.

Molecular virology has served to establish bovine herpesvirus 1 (BHV-1) as the prototype member of ruminant herpesviruses. Based on the genomic sequence of the virus, we aim to identify and characterize virus-specified components, to explain their concerted action, and to predict how the chain of events during the lytic and latent phases of the viral life cycle may be interrupted. The nucleotide sequence of the BHV-1 genome (136 kb) has just been completed by international cooperation (July 1995; except for a small gap in UL36). It comprises 67 unique genes and 2 genes, both duplicated, in the inverted repeats. In general, these genes exhibit strong homology at the amino acid sequence level to those of other alphaherpesviruses (HSV-1, VZV, EHV-1) and are arranged in similar order. A few genes are peculiar to only one or two herpesviruses, e.g. in BHV-1 the circ, UL0.5, UL3.5 and US1.5 genes. Not long ago, the repertoire of BHV-1 proteins under study was restricted to the three major glycoproteins (gB, gC, and gD) and thymidine kinase. The repertoire is now growing rapidly and includes 7 additional glycoproteins (gE, gI, gH, gL, gG, gK and gM), a number of enzymes (e.g. ribonucleotide reductase, DNA Polymerase, dUTPase), and a group of regulatory proteins (BICPO, 4, 22, and 27, alpha TIF). Investigations into the functions of these proteins and comparison with their counterparts in other herpesviruses should reveal which are useful targets for diagnosis, prevention or antiviral treatment. Recombinant viruses containing deletions or replacements of individual genes are being created, aiming at vaccine development and insights into pathogenesis, notably latency, neurotropism, and interference with host functions. Molecular analysis of other ruminant herpesviruses is much less advanced. Over a dozen virus species have been described; most share basic properties with BHV-1 and may be classified as alphaherpesviruses. The gammaherpesviruses are represented by the proposed agent of malignant catarrhal fever, alcelaphine herpesvirus 1, and by bovine herpesvirus 4, whose partial sequences exhibit similarity to herpesvirus saimiri.

Animals↗
PubMed1996

[Use of polymerase chain reaction to reduce the use of animal studies: a project for the expression of the structural proteins of tick-borne meningoencephalitis virus].

Tick-borne encephalitis virus is difficult to propagate because with consecutive passages in cell culture the virus titer will decrease. Stockvirus has to be propagated in young mice. Therefore, every production of virus for research, diagnostic assays or vaccination demands the use of laboratory animals. We decided to clone the part of the viral genome which codes for the structural proteins, and to produce the structural proteins in a suitable expression system. Using reverse transcription, followed by the polymerase chain reaction, we amplified exactly this part of the viral genome, added restriction sites for cloning and a stop-codon. Cloning of this DNA-fragment and expression of the structural proteins of tick-borne encephalitis virus in the baculovirus expression system has thus been possible. Replacement of traditional viral antigen by these recombinant proteins may reduce the need for laboratory animals.

Animals↗
PubMed1996

[Identification and characterization of new and unknown coronaviruses using RT-PCR and degenerate primers].

A modified method of the reverse transcription followed by polymerase chain reaction (RT-PCR) was developed in order to examine the genome of a recently discovered virus, the porcine epidemic diarrhoea virus (PEDV), which resembled morphologically the coronaviruses. The published sequences of the genomes of various coronaviruses were compared. On the level of the amino acid sequence, conserved regions, common to all coronaviruses, were found in the gene encoding the nonstructural protein 1b as well as in the genes coding for the major structural proteins (S, M, and N). Due to the degeneration of the genetic code, some amino acids may be encoded by different nucleotide triplets. In order to compensate for this degeneration, mixtures of primers were synthesized, containing a variety of nucleotide sequences which together represented all possible codons for the conserved amino acid sequences. This method allowed to amplify and clone approximately 4000 base pairs of the genome of PEDV. An analysis of the genomic sequences revealed that PEDV holds an interesting intermediate position between human coronavirus 229E and Transmissible Gastroenteritis virus. We postulate that the method presented in this contribution may be useful to study and characterize other unknown viruses, especially viruses for which no cell cultures for propagation are available.

Amino Acid Sequence↗
PubMed1995

Identification of proteins specified by porcine epidemic diarrhoea virus.

Up to now, little was known about the proteins of porcine epidemic diarrhoea virus (PEDV). Using (i) metabolic labelling, (ii) antisera directed against synthetic peptides and monoclonal antibodies, and (iii) eukaryotic and prokaryotic expression systems, we have started to identify and characterize these proteins. The nucleocapsid protein (N) of PEDV was identified as a phosphoprotein with a relative mobility (M(r)) of 57 k. No additional phosphoproteins were detected. At least three glycoproteins were virion associated. The 180/200 k band most probably represented the surface glycoprotein (S), whereas the 27 k protein was the membrane protein (M). The 21 k species could not yet be identified. A rabbit anti-M protein antiserum was generated by using synthetic peptides corresponding to the C-terminus of M. The specificity of this serum was reconfirmed by transient expression of the M-gene in Vero cells followed by immunofluorescence. In order to generate antisera against the putative gene products of ORF3 and of the internal N reading frames (I-1, I-2, I-3), additional anti-peptide sera were raised. The putative ORF3 product which had been tagged by a 6 Histidine tail, was expressed in E. coli and purified by nickel chelate affinity chromatography before 2-dimensional polyacrylamide gel electrophoresis and immunostaining with a rabbit anti-peptide serum directed against the N-terminus of the ORF3 product. Transient expression of the I-1 and I-3 reading frames was used to confirm the specificity of the corresponding anti-peptide sera. The immunological tools presented in this paper are now being used to identify the putative corresponding gene products specified by PEDV.

Animals↗
PubMed1995

Identification of the membrane protein of porcine epidemic diarrhea virus.

Sequence information on the genome of porcine epidemic diarrhea virus(PEDV) has only recently been determined. In contrast, very little is known about the viral proteins. In the present report we have identified the membrane glycoprotein (M) of PEDV by use of rabbit anti-peptide sera and transient expression of the cloned M gene in Vero cells and by expression in the baculovirus system. The native M protein of PEDV is incorporated into virions, is N-glycosylated, and migrates with a relative mobility (Mr) of 27 k in polyacrylamide gels. In contrast, the M protein synthesized by recombinant baculoviruses migrates with a Mr of 23 k, that is, with identical mobility as the deglycosylated product of PEDV. Thus, it appears that M protein specified by the recombinant baculovirus is poorly, if at all, glycosylated. Using monoclonal antibodies and rabbit and rabbit antipeptide sera specific for the N and C termini of the M protein, we were able to show that a 19 k band detected in PEDV-infected cells but not in virions represented a fragment of M from which the C terminus had been cleaved off. Finally, by electron microscopy and immunogold labelling, the relative orientation of M within the virion envelope was determined as NexoCcyt. In conclusion, all of these data strongly support the hypothesis that PEDV should be classified with the group I coronaviruses.

Amino Acid Sequence↗
PubMed1995

Effects of endothelins on renin secretion from isolated mouse renal juxtaglomerular cells.

This study was performed to examine the effects of endothelin (ET)-1, ET-2, and ET-3 on renin secretion from cultured mouse renal juxtaglomerular (JG) cells. Although different ETs had no consistent effect on basal renin secretion, they equipotently inhibited adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated renin release with a concentration of approximately 3 nM inhibiting 50% of maximal response. ETs did not significantly affect renin release stimulated by the nitric oxide donor sodium nitroprusside (100 microM) or that stimulated by low [2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] or high (3 mM CaCl2) extracellular calcium. The inhibitory effect of ETs on cAMP-dependent renin secretion was abolished by lowering extracellular calcium concentration to the nanomolar range. However, the action of ETs was not changed by the ETA receptor antagonist BQ-123 (100 nM) and was mimicked by ETB receptor agonists IRL-1620 (1 microM), sarafotoxin S6b (1 microM), and [Ala1,3,11,15]ET-1 (1 microM). All ETs induced calcium oscillations in JG cells that were dependent on extracellular calcium and were associated with prominent calcium-activated chloride currents. These findings suggest that ETs inhibit rather selectively the cAMP-activated pathway of renin secretion through a calcium-sensitive process. The action of ETs on renal JG cells appears to be mediated via ETB receptors and is presumably related to activation of phospholipase C and subsequent events.

Animals↗
PubMed1995

Endothelin inhibits cAMP-induced renin release from isolated renal juxtaglomerular cells.

We examined the effects of endothelin (ET)-1, -2, and -3 on renin secretion from cultured mouse renal juxtaglomerular cells. Although different ETs had no consistent effect on basal renin secretion, they equipotently inhibited cAMP-stimulated renin release with an IC50 value of approximately 3 nM. ETs did not significantly affect renin release stimulated by the nitric oxide (NO)-donor nitroprusside (100 microM) or that stimulated by low (2 mM EGTA) or high (3 mM CaCl2) extracellular calcium. The inhibitory effect of ETs on cAMP-dependent renin secretion was abolished by lowering extracellular calcium concentration to the nanomolar range. However, the action of ETs was not changed by the ETA receptor antagonist BQ 123 (100 nM) and was mimicked by the ETB receptor agonists IRL 1620 (1 microM), sarafotoxin S6c (1 microM), and [Ala1,3,11,15]endothelin-1 (4 Ala-ET-1, 1 microM). These findings suggest that ETs selectively inhibit the cAMP-activated pathway of renin secretion through a calcium-sensitive process. The action of ETs on renal juxtaglomerular cells appears to be mediated via ETB receptors.

Animals↗
PubMed1994

Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF.

In order to investigate the genome organization of the porcine epidemic diarrhea virus (PEDV) further, cDNA clones covering the region between the nucleocapsid and the spike (S) protein genes were independently constructed and sequenced for the two virulent isolates Br1/87 and CV777. Of the three major ORFs identified, two were found to encode the major and minor coronavirus membrane proteins M and sM. A potentially single ORF, designated ORF3 according to the pattern of the viral subgenomic mRNAs, could be identified between the S and sM genes. A striking variability, essentially generated by short deletions clustered in a few loci, was observed in the ORF3 of both isolates. The largest predicted polypeptide of 223 amino acids showed homology with polypeptides potentially encoded by other members of the same genetic subset, including two shorter polypeptides of human respiratory virus HCV 229E and one of transmissible gastroenteritis virus TGEV. This homology suggests that the two HCV ORFs may have originated from a single precursor. The function of these polypeptides is not known, but the predicted products of the PEDV ORF3 and related ORFs share features suggestive of a membrane-associated protein.

Amino Acid Sequence↗
PubMed1994

Endothelium-mediated regulation of renin secretion.

The aim of the present study was to investigate the endothelial influence on renin secretion of isolated juxtaglomerular cells. Specifically the role of nitric oxide (NO) and of endothelin was studied. Coculture of primary cultures of juxtaglomerular cells with aortic and microvascular endothelial cells decreased renin secretion. Inhibition of NO formation by absence of l-arginine or presence of N omega-nitro-l-arginine caused a marked decrease in cGMP accumulation and a reduction in renin secretion in cocultures. Exogenous NO (NO liberators sodium nitroprusside/SIN 1) stimulated the 20-hour renin secretion from juxtaglomerular cells markedly, too. The effect of NO on renin secretion was biphasic: short-time inhibition and long-time stimulation of renin release. NO's stimulatory effect on renin secretion is dependent on extracellular calcium, but independent on cAMP or cGMP accumulation. Endothelin 1, 2, and 3 did not affect basal renin secretion, but inhibited cAMP stimulated renin release to a similar extent. Endothelin's action is not mediated via the subtype A endothelin receptor, but seems to involve calcium mobilization in juxtaglomerular cells that is dependent on extracellular calcium and associated with prominent calcium activated chloride channels. Taken together, coculture of juxtaglomerular cells with endothelial cells inhibits renin secretion despite the stimulatory effect of native NO released from endothelial cells. cAMP stimulated renin secretion is inhibited by all three endothelin isoforms thus contributing to the inhibition of renin secretion in coculture.

Animals↗
PubMed1994

Identification of the bovine herpesvirus 1 circ protein, a myristylated and virion-associated polypeptide which is not essential for virus replication in cell culture.

We have recently reported immediate-early (IE) transcription over covalently joined genome ends of bovine herpesvirus 1 (BHV-1). A spliced 1.5-kb IE RNA (IER1.5) is coterminal with an unspliced 1.1-kb late RNA (LR1.1) which is transcribed from the left end of the genome. Sequence analysis reveals an open reading frame common to IER1.5 and LR1.1 predicted to encode the 247-amino-acid circ polypeptide. This paper reports on the identification of circ as a protein. Using a rabbit antiserum raised against a synthetic oligopeptide representing the carboxy terminus of the predicted circ polypeptide for Western blot (immunoblot) analyses and immunofluorescence assays, we identified a 34-kDa virion-associated protein which accumulated in the cytoplasm of infected cells. To confirm that LR1.1 indeed encoded the 34-kDa polypeptide, we inserted a DNA fragment containing circ coding sequences into the Autographa californica baculovirus genome. A group of recombinant polypeptides with sizes of 32, 34, and 35 kDa were identified by their reactivity with the antipeptide serum. Chicken egg yolk antibodies raised against total proteins of insect cells infected with the recombinant baculovirus identified the 34-kDa circ protein specified by BHV-1. The recombinant circ polypeptides and the circ protein specified by BHV-1 were both myristylated, as determined by radiolabeling with [3H]myristic acid. It was noted that the circ gene could be deleted from the BHV-1 genome without impairing virus replication in cell culture.

Amino Acid Sequence↗
PubMed1994

Identification and zinc dependence of the bovine herpesvirus 1 transactivator protein BICP0.

Bovine herpesvirus 1 (BHV-1) specifies and unspliced early 2.6-kb RNA (ER2.6) which is 3' coterminal with exon 2 of the 2.9-kb immediate-early (IE) RNA. The two transcripts have a common open reading frame (676 codons). The predicted protein, designated BHV-1 infected cell protein 0 (BICP0), contains a zinc finger domain with homology to ICP0 of herpes simplex virus type 1 and protein 61 of varicella-zoster virus, and depending on the promoter, it acts as a strong activator or as a repressor in transient expression assays. In situ immunoadsorbent assays using antisera against synthetic oligopeptides demonstrated that BICP0 accumulates in nuclei of BHV-1-infected cells, as expected for an IE gene product involved in gene regulation. Western blots (immunoblots) revealed a BHV-1-specific 97-kDa protein which was detectable during the IE phase and also at later periods of infection, indicating that the kinetics of BICP0 synthesis is consistent with the switch from IER2.9 to ER2.6. To confirm that ER2.6 encoded the 97-kDa BICP0 protein, a DNA fragment containing BICP0-coding sequences was inserted into the Autographa californica baculovirus genome. A recombinant protein, identified by its reactivity with antipeptide sera, exhibited the same electrophoretic mobility as BICP0 specified by BHV-1. We microinjected Xenopus oocytes with a BICP0 effector plasmid and a promoter-chloramphenicol acetyltransferase plasmid. BICP0-induced stimulation of this promoter was strongly reduced when intracellular zinc was chelated by thionein, indicating that the effect of BICP0 is zinc dependent.

Amino Acid Sequence↗