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R Waldmann

Publications and source records attributed to R Waldmann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Expression cloning of a protective Leishmania antigen.

Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice. An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II. A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Amino Acid Sequence↗
PubMed1994

Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone.

Long term regulation of the amiloride-sensitive Na+ channel activity by steroid hormones occurs via de novo protein synthesis. The messenger level of RCNaCh1, previously shown by expression cloning to be a component of this channel, was measured in colons from rats fed with a low sodium diet. After 1 week of this diet, the channel activity was increased in an all-or-none fashion, whereas the level of RCNaCh1 messenger remained constant. A cDNA coding for another subunit of the Na+ channel was obtained by polymerase chain reaction. The 650-amino acid protein, entitled RCNaCh2, is 58% homologous to RCNaCh1 and displays a similar structure. It had no intrinsic activity when expressed alone in Xenopus oocytes, but its co-expression with RCNaCh1 increased the channel activity 18 +/- 5-fold. The increase in messenger level for RCNaCh2 during the time course of the diet is likely to explain the positive regulation of the rat colon Na+ channel by steroids. Immunocytochemical localization of the RCNaCh1 subunit revealed an apical labeling in colon from sodium-depleted rats. No labeling was observed in colon from control animals. These results suggest that oligomerization is needed for the proper expression of RCNaCh1 at the cell surface.

Aldosterone↗
PubMed1994

Molecular cloning of a murine N-type calcium channel alpha 1 subunit. Evidence for isoforms, brain distribution, and chromosomal localization.

A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

Amino Acid Sequence↗
PubMed1994

The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning.

Water balance in the lung is controlled via active Na+ and Cl- transport. Electrophysiological measurements on lung epithelial cells demonstrated the presence of a Na+ channel that is inhibited by amiloride (K0.5 = 90 nM) and some of its derivatives such as phenamil (K0.5 = 19 nM) and benzamil (K0.5 = 14 nM) but not by ethylisopropylamiloride. An amiloride-sensitive Na+ channel of 4 pS was recorded from outside-out patches excised from the apical membrane. This channel is highly selective for Na+ (PNa+/PK+ > or = to 10). Isolation of a human lung cDNA led to the primary structure of the lung Na+ channel. The corresponding protein is 669 residues long and has two large hydrophobic domains. An amiloride-sensitive Na(+)-selective current apparently identical to the one observed in lung epithelial cells was recorded after expression of the cloned channel in oocytes. The level of the mRNA for the Na+ channel was highly increased from fetal to newborn and adult stages. This observation indicates that the increased Na+ reabsorption that occurs at birth as a necessary event to pass to an air-breathing environment is probably associated with control of transcription of this Na+ channel. The human gene for the lung Na+ channel was mapped on chromosome 12p13.

Amiloride↗
PubMed1994

[Recent developments in the diagnosis of parapoxviruses].

Neutralizing and non-neutralizing monoclonal antibodies against parapoxviruses (PPV) were generated by immunizing BALB/c-mice with gradient-purified PPV Orf D-1701 or purified envelopes. Epitope specificity studies identified three distinct epitopes localized in the virus envelope. These antigenic sites allowed a differentiation between orf and stomatitis papulosa viruses. For a rapid diagnosis of parapoxviruses transmission-electron microscopy, immunofluorescence- or immunoperoxidase-staining, antigen capture ELISA, and polymerase-chain-reaction were used.

Animals↗
PubMed1993

Multiple mRNA isoforms encoding the mouse cardiac Kv1-5 delayed rectifier K+ channel.

The mouse Kv1-5 K+ channel cDNA has been cloned from heart. This channel was highly expressed in heart and, to a lesser extent, in other tissues, including brain and thymus. Two alternatively spliced isoforms were found. The longer form encoded a 602-amino acid protein, while in the short form (Kv1-5 delta 5'), the first 200 amino acids lying upstream the transmembrane segment S1 were deleted. RNase protection experiments showed that both Kv1-5 mRNA isoforms are present in the mouse tissues examined, the longer form being predominant. The short mRNA (Kv1-5 delta 5') arose by an unusual splicing event within the exonic sequence. An additional short cDNA clone (Kv1-5 delta 3') that codes for a carboxyl-terminal truncated protein has been isolated. The gene coding sequence contained a single exon and has been mapped on human chromosome 12 (p13) and on mouse chromosome 6 (band F). Expression in Xenopus oocytes revealed that the long (Kv1-5) and the amino-terminal deleted (Kv1-5 delta 5') isoforms elicited similar K+ currents with a drastically decreased efficacy for Kv1-5 delta 5'. The carboxyl-terminal truncated Kv1-5 delta 3' clone was not functional but inhibited the expression of the long isoform.

Alternative Splicing↗
PubMed1993

Molecular cloning and functional expression of different molecular forms of rat amiloride-binding proteins.

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3' terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 = 10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 microM) approximately ethylisopropylamiloride (K0.5 = 1.6 microM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 microM) approximately ethylisopropylamiloride (K0.5 = 4 microM) approximately benzamil (K0.5 = 6.3 microM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the first members of a new family of epithelia-specific proteins.

Amine Oxidase (Copper-Containing)↗
PubMed1993

Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins.

A complementary DNA encoding an amiloride-sensitive Na+ channel has been cloned and characterized from rat colon. The protein encoded by the cDNA has a sequence of 699 amino acids (79 kDa) containing several putative membrane spanning domains and potential phosphorylation sites. It forms a channel that has the electrophysiological and pharmacological properties characteristic of the epithelial Na+ channel. Homologies (including in transmembrane domains) have been found between a part of the channel sequence and the Mec4 gene product of Caenorhabditis elegans, a protein associated with mutation-induced neuronal degeneration.

Amiloride↗
PubMed1990

Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies.

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.

Amino Acid Sequence↗
PubMed1990

The UCLA-University of Utah epidemiologic survey of autism: genealogical analysis of familial aggregation.

To assess familial aggregation of autism, 86 autistic subjects were linked to the Utah Genealogical Database. Kinship coefficients were estimated for all possible pairs of autistic subjects and then averaged. Fifty replicate sets of matched control subjects (86 members in each set) were drawn randomly from the database, and the average kinship coefficient was computed for all possible pairs of individuals in each set. The average kinship coefficient for the autistic subjects was approximately 1/1,000, while the average kinship coefficients for the 50 control groups ranged from 4/100,000 to 1.6./10,000. These results indicate a strong tendency for autism to cluster in families. When kinship was analyzed by specific degrees of relationship, it was shown that the familial aggregation of autism is confined exclusively to sib pairs and does not extend to more remote degrees of relationship. This finding indicates that a single-gene model is unlikely to account for most cases of autism.

Autistic Disorder↗
PubMed1990

Randomized clinical trial comparing mitoxantrone with epirubicin and with doxorubicin, each combined with cyclophosphamide in the first-line treatment of patients with metastatic breast cancer.

Two hundred and twenty-four patients with advanced breast cancer were enrolled in a multicenter prospective randomized clinical study and received either doxorubicin (40 mg/m2), or epirubicin (40 mg/m2) or mitoxantrone (12 mg/m2) each combined with cyclophosphamide (600 mg/m2) i.v. In the patient collective the following response rates were observed: complete response 12.1%; partial response 30.6%; stable disease 40.5%; progressive disease 16.8%. A complete response was observed significantly less often in patients where more than one organ site was involved as compared to those patients with only one metastatic site. The mean time period required to reach a best response was 3.7 months. There was no significant difference between the response rates in the three arms. In comparing the observed toxicities in 1,434 treatment cycles, there was a significant difference with regard to leukocytopenia (mitoxantrone arm exhibiting more than either epirubicin and doxorubicin) although infections did not occur more frequently in the mitoxantrone arm; with regard to alopecia, mitoxantrone and epirubicin arms both exhibited less than doxorubicin. It is noteworthy that no patient who had previously received adjuvant chemotherapy achieved a complete response (p = 0.006). The overall significance of these findings can only be clearly evaluated when survival times can be measured.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1990

[Recent information about the orf virus of small ruminants].

New scientific findings in the field of immunobiology and diagnosis of parapoxvirus ovis (Orf-virus) as the causal agent of a zoonosis are presented. The adaptation of Orf-virus to cell lines and its in vitro multiplication without difficulties offer the possibility for extensive studies into the biology of parapoxviruses. The development of monoclonal antibodies (MAB) against an attenuated Orf-virus strain (D-1701) led to the elaboration of a simplified, cheap and highly sensitive "antigen detection ELISA" as a diagnostic tool. Moreover the MAB's can be used to identify parapoxvirus in cells of infected tissues (indirect immuno-peroxidase technique) which is an important precondition for more detailed surveillance of the pathogenesis of parapoxvirus infections. The immunobiological behavior of the Orf-virus in its natural hosts (small ruminants) as well as in non-susceptible species (e.g. laboratory animals) is explained by the course of cellular and humoral immune reactions following infection or vaccination. The problems of combat measures against contagious ecthyma (scabby mouth) of sheep and the human Orf-virus infections are discussed.

Animals↗
PubMed1989

Cyclic nucleotide elevating vasodilators inhibit platelet aggregation at an early step of the activation cascade.

The mechanism of vasodilator-induced inhibition of platelet aggregation was investigated in human platelets. Cyclic nucleotide-elevating vasodilators stimulated cAMP- or cGMP-dependent protein phosphorylation, inhibited the activation of both protein kinase C and myosin light chain kinase, and inhibited the thrombin-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate without affecting its resynthesis. The results suggest that cAMP- and cGMP-elevating vasodilators both inhibit platelet aggregation at an early step of the activation cascade, presumably at the level of phospholipase C.

Blood Proteins↗
PubMed1988

Intracellular mechanism of action of vasodilators.

Vasodilators that act directly at the level of the vasculature may be classified as either endothelium-dependent or endothelium-independent agents. Endothelium-dependent agents stimulate the endothelium to produce endothelium-derived relaxing factor (recently identified as NO) which relaxes vascular smooth muscle and increases cGMP. Endothelium-independent vasodilators may be divided into calcium antagonists, alpha 1-adrenergic antagonists, cAMP-elevating agents and cGMP-elevating agents. It is generally accepted that calcium-dependent phosphorylation of myosin light chains initiates smooth muscle contraction. A variety of evidence suggests that certain vasodilators inhibit calcium-dependent phosphorylation of myosin light chains and smooth muscle contraction via activation of cAMP- and cGMP-dependent protein phosphorylation. Since vascular smooth muscle cells and platelets have many properties in common, and since cAMP- and cGMP-elevating vasodilators inhibit both smooth muscle contraction and platelet aggregation, human platelets were used to study the effects of vasodilators on protein phosphorylation. It could be demonstrated that cAMP- and cGMP-regulated protein kinases are the intracellular mediators for prostaglandin-E1- and nitroprusside-induced protein phosphorylation, respectively. Furthermore, prostaglandin-E1- and nitroprusside-stimulated protein phosphorylation was found to be associated with an inhibition of the phosphatidylinositol cycle. Cyclic nucleotide-elevating vasodilators may inhibit smooth muscle contraction and platelet aggregation by regulating the flux through the phosphatidylinositol cycle, and the activity of myosin light-chain kinase and of calcium ATPases.

Animals↗
PubMed1987

Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases.

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 microM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masses, Mr, of 240,000, 68,000, 50,000, and 22,000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1-2 microM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 microM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50,000 which was also phosphorylated in response to low concentrations (1-2 microM) of cGMP in platelet membranes. An additional protein (Mr 24,000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50,000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.

Alprostadil↗
PubMed1986

Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets.

Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.

Animals↗