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D Hartz

Publications and source records attributed to D Hartz.

15 recordsLinked to original sources
PubMed2000

Down-regulation of Bgp1(a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus 3 infection.

Together with the evidence that the reduced virus growth and the antiviral state induced by interferon (IFN)-gamma, occurring only in macrophages from resistant animals, correlated with the decrease of MHV3 binding to macrophage membrane proteins, we show here the expression of cellular and viral genes in resistant (A/J) and susceptible (BALB/c) mouse macrophages after IFN-gamma activation/infection. The expression of interferon response gene 47 and interferon regulatory factor 1 genes takes place after IFN-gamma activation in both macrophages, indicating their activation. The expression of the biliary glycoprotein 1(a) (Bgp1(a), the main virus receptor) decreased only in IFN-gamma-activated A/J mouse macrophages, in contrast to the expression of the Bgp2 (alternative receptor), which was not influenced by IFN-gamma activation. The synthesis of both viral mRNA and virus particles was delayed only in IFN-gamma-activated A/J mouse macrophages compared with susceptible BALB/c macrophages. Besides the evidence that IFN-gamma may modulate the expression of the Bgp1(a) isoform of carcinoembryonic antigen family, these data show that IFN-gamma, which induces resistance against MHV3 infection, may be involved in the down-regulation of the main viral receptor expression, a key step forward in our understanding of the molecular basis of resistance against virus infection.

Animals↗
PubMed1995

The molecular interaction of sulfonylureas with beta-cell ATP-sensitive K(+)-channels.

The molecular interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor was investigated by kinetic and steady state binding as well as photoaffinity labeling. The novel sulfonylurea, glimepiride, exhibits a significantly higher exchange rate with the sulfonylurea receptor but a 2.5-3 fold lower binding affinity compared to glibenclamide. [3H]Glimepiride was specifically incorporated into a 65-kDa polypeptide under conditions which led to predominant labeling of a 140-kDa protein by [3H]glibenclamide. Labeling of the 140-kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65-kDa protein by [3H]glimepiride. The 65-kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33-kDa protein. Solubilization of beta-cell tumor membranes led to a shift of specific [3H]glibenclamide-binding from the 140-kDa to the 65-kDa protein, exclusively and to an increased labeling of the 65-kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd-values for binding to the sulfonylurea receptor measured for both sulfonylureas upon solubilization of beta-cell membranes. Photoaffinity labeling of intact cultured beta-cells led also to labeling of a 140-kDa protein by [3H]glibenclamide and of a 65-kDa protein by [3H]glimepiride. These studies suggest that the beta-cell sulfonylurea receptor consists of at least two protein subunits of M(r) 140,000 and 65,000 which bind sulfonylureas of different structure with different binding affinities and kinetic parameters. Furthermore, the exchange rate of a sulfonylurea determines the insulin releasing activity in vitro more closely than the binding affinity.

ATP-Binding Cassette Transporters↗
PubMed1994

Differential interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor. I. Binding characteristics.

Glimepiride is a novel sulfonylurea drug for treatment of non-insulin-dependent diabetes mellitus with higher blood sugar lowering efficacy in diabetic patients than glibenclamide raising the question whether this characteristics is in line with different binding of glimepiride and glibenclamide to the beta-cell sulfonylurea receptor. Scatchard plot analysis of [3H]sulfonylurea binding to membranes isolated from rat beta-cell tumors and (RINm5F) insulinoma cells and to RINm5F cells demonstrated that glimepiride has a 2.5-3-fold lower affinity than glibenclamide. This corresponded well to the 8-9-fold higher koff and 2.5-3-fold higher kon rates of glimepiride compared to glibenclamide as revealed by the dissociation and association kinetics of [3H]sulfonylurea binding and the Kd values calculated thereof. In agreement, the concentrations required for half-maximal displacement of [3H]sulfonylurea bound to beta-cell membranes were significantly higher for glimepiride compared to glibenclamide. However, the binding affinity of glimepiride measured by both equilibrium binding and kinetic binding studies upon solubilization of beta-cell tumor membranes and RINm5F cell membranes increased up to the value for glibenclamide. This was primarily based on a drastic decrease of the dissociation rate constant of glimepiride whereas the kinetics of glibenclamide binding remained largely unaffected upon solubilization. These data suggest that the Kd value alone is not sufficient for characterization of a sulfonylurea drug, since the kinetic binding parameters may also determine its acute blood sugar lowering efficacy.

ATP-Binding Cassette Transporters↗
PubMed1994

Differential interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor. II. Photoaffinity labeling of a 65 kDa protein by [3H]glimepiride.

Glimepiride is a novel sulfonylurea for the treatment of type II-diabetic patients exhibiting different receptor binding kinetics to beta-cell membranes with 8-9-fold higher koff rate and 2.5-3-fold higher kon rate compared to glibenclamide (see accompanying paper (Müller, G. et al. (1994) Biochim. Biophys. Acta 1191, 267-277)). To elucidate the molecular basis for this differential behaviour of glimepiride and glibenclamide, direct photoaffinity labeling studies using beta-cell tumor membranes were performed. [3H]Glimepiride was specifically incorporated into a membrane polypeptide of M(r) = 65,000 under conditions, which led to predominant labeling of a 140 kDa protein by [3H]glibenclamide (Kramer, W. et al. (1988) FEBS Lett. 229, 355-359). Labeling of the 140 kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65 kDa protein by [3H]glimepiride. The 65 kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33 kDa protein. Competitive Scatchard analysis of [3H]glimepiride-binding and [3H]glibenclamide-binding to RINm5F cell membranes using glibenclamide and glimepiride, respectively, as heterologous displacing compounds yielded non-linear plots. These findings may be explained by cooperative interactions between the 140 and 65 kDa sulfonylurea-binding proteins. The possibility that sulfonylureas of different structure have different access to the 140 and 65 kDa receptor proteins due to the beta-cell membrane barrier was investigated by photoaffinity labeling of solubilized beta-cell membrane proteins. Interestingly, solubilization of beta-cell tumor membranes led to a shift of specific [3H]glibenclamide binding from the 140 kDa to the 65 kDa binding protein, exclusively, and to an increased labeling of the 65 kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd values measured for both sulfonylureas upon solubilization of beta-cell tumor and RINm5F cell membranes (see accompanying paper). Furthermore, competitive Scatchard plots of [3H]glimepiride binding to solubilized RINm5F cell membrane proteins in the presence of glibenclamide and vice versa approximate linearity suggesting loss of cooperativity between the 140 kDa glibenclamide-binding and 65 kDa glimepiride-binding proteins upon solubilization. The physiological significance of the differential interaction of glimepiride and glibenclamide with different binding proteins was also substantiated by photoaffinity labeling of RINm5F cells leading to labeling of a 140 kDa protein by [3H]glibenclamide and of a 65 kDa protein by [3H]glimepiride.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1994

Immunization of mice with Escherichia coli containing an antigen of the malaria parasite Plasmodium chabaudi chabaudi expressed in lambda gt11 induces high antibody titres.

Escherichia coli containing a recombinant malarial protein expressed in lambda gt11 have been evaluated as an antigen delivery system in vivo. They were generated by infecting non-suppressing E. coli cells with a clone from a cDNA library of Plasmodium chabaudi chabaudi in lambda gt11. This clone (termed clone 6) expresses part of a 93 kDa blood-stage antigen of P. c. chabaudi as beta-galactosidase fusion protein. Immunization of C57B1/6 mice with these infected E. coli cells resulted in an antibody response to the malarial part of the fusion protein comparable to that obtained with purified fusion protein preparations. This method, therefore represents a rapid secondary screening of clones from lambda gt11 expression libraries for immunogenic and potentially protective components. In addition, the administration of whole infected E. coli obviates the need for an adjuvant.

Adjuvants, Immunologic↗
PubMed1993

Cloning and sequencing of a cDNA fragment from Plasmodium chabaudi chabaudi that contains repetitive sequences coding for a potentially lysine-rich aspartic acid-rich protein.

Screening of a cDNA library (prepared in lambda gt11) of the blood stages of Plasmodium chabaudi chabaudi (AS) with immune serum has revealed an antigen the elicits a strong antibody response in infected mice. The clone (clone 6) expressing that antigen contains a 0.7 kb insert and produces a beta-galactosidase fusion protein of about 150 kDa. In Western blot analysis performed on parasite extracts, monoclonal antibodies and polyclonal sera prepared against the fusion protein revealed that the fusion protein contains part of a malarial protein of 93 kDa. Northern hybridization with clone 6 insert as probe detected a plasmodial RNA of about 3.2 kb, which could well code for a protein of this size. The insert hybridized to a single EcoRI fragment and a single HindIII fragment in genomic Southern blotting, suggesting that the gene is present in one copy in the P. chabaudi genome. The DNA sequence of clone 6 insert predicts a hydrophilic, acidic polypeptide consisting of seven repeats of 23-34 amino acids rich in lysine (24%) and aspartic acid (17.5%).

Amino Acid Sequence↗
PubMed1991

Influence of mRNA determinants on translation initiation in Escherichia coli.

We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation.

Anticodon↗
PubMed1991

Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA.

Binary complexes between messenger RNA and E. coli ribosomes were examined. A ribosome-mRNA binary complex on T4 gene 32 mRNA withstood inhibition by antibodies against ribosomal protein S1. Anti-S1 blocks ternary complex formation, as measured by "extension inhibition" or "toeprinting" analysis, only when preincubated with ribosomes prior to mRNA addition and not when anti-S1 was added after preincubation of ribosomes and mRNA. The ribosome was directly localized in a binary complex on two translation initiation sites by toeprinting analysis. In the absence of tRNA the ribosome halted cDNA synthesis by reverse transcriptase close to the Shine and Dalgarno sequence. Binary complex formation was inhibited by an oligodeoxynucleotide competitor of the Shine and Dalgarno sequence.

Base Sequence↗
PubMed1990

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12.

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000 X g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000 X g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of Mr 26,000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.

Amobarbital↗
PubMed1990

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3.

Initiation factors are used by Escherichia coli to select the initiator tRNA over elongator tRNAs during translation initiation. IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon. The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit. Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation. IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU.

Bacterial Proteins↗
PubMed1989

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.

Bacteriolysis↗
PubMed1989

Selection of the initiator tRNA by Escherichia coli initiation factors.

We have developed a new technique, called 'toeprinting,' which has allowed a study of the tRNA-binding properties of Escherichia coli translation initiation complexes. In response to natural mRNAs, the initiator tRNA and a variety of elongator tRNAs bind to the same tRNA-binding site on the 30S ribosomal subunit as long as a cognate codon is present near the Shine and Dalgarno sequence. The selection of the initiator tRNA in 30S initiation complexes is accomplished by initiation factors IF2 and IF3. 70S ribosomes accept both initiator tRNA and elongator tRNAs on natural mRNAs, much like 30S ribosomal subunits; IF3 and IF2 do not, however, select the initiator tRNA on 70S initiation complexes unless the initiation factor IF1 is present.

Base Sequence↗
PubMed1989

Ribosome-binding sites and RNA-processing sites in the transcript of the Escherichia coli unc operon.

The polycistronic mRNA encoding the nine genes of the unc operon of Escherichia coli was studied. We demonstrated the ribosome-binding capabilities of six of the nine unc genes, uncB, uncE, uncF, uncH, uncA, and uncD, by using the technique of primer extension inhibition or "toeprinting." No toeprint was detected for the other genes, uncI, uncG, and uncC. The lack of a toeprint for uncG suggests that this gene is expressed by some form of translational coupling, such that either uncG is read by ribosomes which have translated the preceding gene, uncA, or translation of uncA is required for ribosome binding at the uncG site. RNA sequencing and primer extension in the regions of uncI and uncC, the first and last genes in the operon, respectively, gave less intense signals than those obtained for the other unc genes. This suggested that there are fewer copies of those regions of the transcript and that processing of the unc transcript occurred. Using primer extension and RNA sequencing, we identified sites in the unc transcript at which processing appears to take place, including a site which may remove much of the uncI portion of the transcript. Northern (RNA) blot analysis of unc RNA is consistent with the presence of an RNA-processing site in the uncI region of the transcript and another in the uncH region. These processing events may account for some of the differential levels of expression of the unc genes.

Base Sequence↗