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Biomedical subjects

W Brandt

Publications and source records attributed to W Brandt.

At least 19 recordsLinked to original sources
PubMed2000

Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor.

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp(2)-Tat(1-9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1-9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1-9) induces the production of the immunosuppressive cytokine transforming growth factor-beta1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.

Dipeptidyl Peptidase 4↗
PubMed2000

The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress.

The LEA-like protein HSP 12 was identified as having a plasma membrane location in yeast. Gold particles, indicative of the presence of HSP 12, were observed on the external side of the plasma membrane when yeast grown to stationary phase were subjected to immunocytochemical analysis. Growth of yeast in the osmolyte mannitol resulted in an increased number of gold particles that were now observed to be present on both sides of the plasma membrane. No gold particles were observed using a mutant strain of the same yeast that did not express HSP 12. A model liposome system encapsulating the fluorescent dye calcein was used to investigate the protection by HSP 12 of membranes during desiccation. HSP 12 was found to act in an analogous manner to trehalose and protect liposomal membrane integrity against desiccation. The interaction between HSP 12 and the liposomal membrane was judged to be electrostatic as membrane protection was only observed with positively charged liposomes and not with either neutral or negatively charged liposomes. The ability of the wild-type and mutant yeast to grow in media containing ethanol was compared. It was found that yeast not expressing the HSP 12 protein were less able to grow in media containing ethanol. HSP 12 was shown to confer increased integrity on the liposomal membrane in the presence of ethanol. Ethanol, like mannitol, was found to induce HSP 12 protein synthesis. However, yeast grown in both ethanol and mannitol showed a decreased HSP 12 response compared with yeast grown in the presence of either osmolyte alone.

Cell Membrane↗
PubMed2000

N-terminal HIV-1 Tat nonapeptides as inhibitors of dipeptidyl peptidase IV. Conformational characterization.

Compared to the N-terminal nonapeptide of the HIV-1 Tat protein as inhibitor of activity of DP IV which is supposed to mediate the immunosuppressive effects of HIV-1 Tat, the Ile5 and Leu6 analogues showed strongly reduced inhibitory activity. Interestingly, replacement of Asp2 with Gly or Lys led to compounds with considerably enhanced inhibition. Therefore, we have applied 1H NMR spectroscopy and restrained molecular dynamics calculations to elucidate the molecular conformation of a series of Tat nonapeptides. Conformational backbone differences of these peptides as well as the nature and the arrangement of the side chains per se at significant positions preventing effective binding to DP IV might explain their different inhibitory activity on DP IV.

Amino Acid Substitution↗
PubMed2000

Effects of nonapeptides derived from the N-terminal structure of human immunodeficiency virus-1 (HIV-1) Tat on suppression of CD26-dependent T cell growth.

The human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and exerts immunosuppressive effects. Interestingly, Tat inhibits dipeptidyl peptidase IV (DP IV) activity of the T cell activation marker CD26. The short N-terminal nonapeptide Tat(1-9), MDPVDPNIE, also inhibits DP IV activity and suppresses DNA synthesis of tetanus toxoid-stimulated peripheral blood mononuclear cells (PBMC). Here, we present the influence of amino acid exchanges in the first three positions of Tat(1-9). For instance, the replacement of D2 of Tat(1-9) by G or K generated peptides, which inhibit DP IV-catalyzed IL-2(1-12) cleavage nearly threefold stronger. Similar effects were observed on the suppression of DNA synthesis of Tetanus toxoid-stimulated PBMC. This correlation suggests that Tat(1-9)-deduced peptides mediate antiproliferative effects at least in part via specific DP IV interactions and supports the hypothesis that CD26 plays a key role in the regulation of lymphocyte growth.

Acetylation↗
PubMed2000

Development and validation of homology models of human cathepsins K, S, H, and F.

Models of the tertiary structures of cathepsins K, S, H, and F were constructed by using homology protein modelling methods and refinements by interactive graphics and energy minimisation. The predicted structures yield information regarding their substrate binding sites and indicate the residues surrounding these sites. The ligand binding sites were characterised and compared with each other by means of calculated molecular electrostatic surface potentials. This will allow designing and development of new ligands specific for these cathepsins in future investigations.

Cathepsin F↗
PubMed2000

Development of a tertiary-structure model of the C-terminal domain of DPP IV.

Based on the recently published structure of prolyl oligopeptidase (POP) a model of the C-terminal part of dipeptidyl peptidase IV (DPP IV) which contains the active site has been developed. The structure of the model of DPP IV shows considerable similarity to the structure of POP particularly in the active site. A hydrophobic pocket (Tyr666, Tyr670, Tyr 631, Val556) forms the S1-binding site for recognition of proline. Tyr547 may stabilise the oxyanion formed in the tetrahedral intermediates by a strong hydrogen bond. The positively charged N-terminus of ligands of DPP IV is recognised by forming a salt bridge with the acidic side chain Glu668. A second hydrophobic pocket (S2' to S5') may represent an important binding site for HIV-1 Tat-protein derivatives, chemokines and others.

Amino Acid Sequence↗
PubMed2000

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii.

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.

Amino Acid Sequence↗
PubMed2000

Effects of inhibition of calcium and potassium currents in guinea-pig cardiac contraction: comparison of beta-caryophyllene oxide, eugenol, and nifedipine.

1. To investigate the effects of the clove oil constituents beta-caryophyllene oxide and eugenol on the heart muscle, experiments were performed on isolated papillary muscles and on ventricular myocytes of the guinea-pig. The results were compared with those obtained with the dihydropyridine, nifedipine. 2. All three substances exerted negative inotropic effects in heart muscle although with different potencies and different influences on the time course of the contraction curve. 3. They all reduced rested-state contractions (RSCs) in the presence of isoprenaline which are thought to be due to the Ca(2+) current (I(Ca)). 4. beta-Caryophyllene oxide, eugenol and nifedipine inhibited the I(Ca) in single cells from the guinea-pig ventricle in a concentration-dependent, reversible way, but with different potencies. 5. In addition to the I(Ca)-inhibiting effect, beta-caryophyllene oxide strongly inhibited and eugenol slightly inhibited the potassium current. 6. The action potential of papillary muscles at a 1 Hz contraction frequency was greatly shortened by nifedipine, slightly shortened by eugenol, but not changed by beta-caryophyllene oxide. 7. The inhibition of the potassium current by beta-caryophyllene oxide obviously prevents a shortening of the action potential due to the diminution of the I(Ca). Accordingly, the negative inotropic effect of beta-caryophyllene oxide is closely related to the inhibition of I(Ca). In contrast, eugenol and nifedipine, which shorten the action potential, exert stronger negative inotropic effects than expected from their influence on I(Ca). 8. The results show that the negative inotropic effect of a calcium channel blocker can be attenuated by an additional inhibition of potassium channels.

Action Potentials↗
PubMed2000

Development of a pulmonary phenytoin-associated hypersensitivity reaction despite concomitant dexamethasone and prednisolone administration.

OBJECTIVE: This is the report about a very early onset of phenytoin hypersensitivity reaction showing respiratory insufficiency, fever, hepatic and skin reactions in a 68-year-old male patient who was concomitantly treated with high dose dexamethasone/prednisolone because of brain edema. CASE REPORT: The patient developed a hypersensitivity reaction one week after starting a phenytoin treatment, 250 mg daily intravenously, because of a focal epileptic seizure after a transsphenoidal resection of a pituitary tumor. The drug hypersensitivity was diagnosed first clinically and was confirmed by an in vitro rechallenge using the lymphocyte transformation test three months later. Phenytoin itself did not stimulate the lymphocytes' proliferation. The test was performed successfully using human reference sera. CONCLUSIONS: The results suggest that protein-bound reactive drug metabolites which are assumed to occur in the sera may be responsible for the reaction. Corticosteroids did not prevent the reaction and even seem to mask laboratory and clinical findings. Dexamethasone might accelerate the accumulation of potential allergotoxic epoxide metabolites. Both phenytoin and dexamethasone should be withdrawn urgently in the case of suspected hypersensitivity syndrome.

Aged↗
PubMed1999

Sequence-ready contig for the 1.4-cM ductal carcinoma in situ loss of heterozygosity region on chromosome 8p22-p23.

We report the construction of an approximately 1.7-Mb sequence-ready YAC/BAC clone contig of 8p22-p23. This chromosomal region has been associated with frequent loss of heterozygosity (LOH) in breast, ovarian, prostate, head and neck, and liver cancer. We first constructed a meiotic linkage map for 8p to resolve previously reported conflicting map orders from the literature. The target region containing a putative tumor suppressor gene was defined by allelotyping 65 cases of sporadic ductal carcinoma in situ with 18 polymorphic markers from 8p. The minimal region of loss encompassed the interval between D8S520 and D8S261, and one tumor had loss of D8S550 only. We chose to begin physical mapping of this minimal LOH region by concentrating on the distal end, which includes D8S550. A fine-structure radiation hybrid map for the region that extends from D8S520 (distal) to D8S1759 (proximal) was prepared, followed by construction of a single, integrated YAC/BAC contig for the interval. The approximately 1730-kb contig consists of 13 YACs and 27 BACs. Fifty-four sequence-tagged sites (STSs) developed from BAC insert end-sequences and 11 expressed sequence tags were localized within the contig by STS content mapping. In addition, four unique cDNA clones from the region were isolated and fully sequenced. This integrated YAC/BAC resource provides the starting point for transcription mapping, genomic sequencing, and positional cloning of this region.

Breast Neoplasms↗
PubMed1999

A molecular mechanism for the cleavage of a disulfide bond as the primary function of agonist binding to G-protein-coupled receptors based on theoretical calculations supported by experiments.

A model of the binding site of delta-opioids in the extracellular region of the G-protein-coupled opioid receptor based on modelling studies is presented. The distance between Asp288 and the disulfide bridge (Cys121-Cys198) formed between the first and second extracellular loops was found to be short. This model is consistent with site-directed mutagenesis studies. The arrangement of the ligands found in the receptor led to the development of a reaction mechanism for the cleavage of the disulfide bond catalysed by the ligands. Semi-empirical quantum chemical PM3 and AM1 calculations as well as ab initio studies showed that the interaction between the carboxylic acid side chain of aspartic acid and the disulfide bond leads to the polarization of, and withdrawal of a proton from, the protonated nitrogen of the ligand to one of the sulfur atoms. A mixed sulfenic acid and carboxylic acid anhydrate is formed as an intermediate as well as a thiol. The accompanying cleavage of the disulfide bond may produce a conformational change in the extracellular loops such that the pore formed by the seven-helix bundle opens allowing entrance of the ligand, water and ions into the cell. Cleavage of the disulfide bond after opioid administration was demonstrated experimentally by flow-cytometric measurements employing CMTMR and monobromobimane-based analyses of membrane-located thiols. The suggested mechanism may explain, in a consistent way, the action of agonists and antagonists and is assumed to be common for many G-protein coupled receptors.

Animals↗
PubMed1999

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Amino Acid Sequence↗
PubMed1998

Elucidation of the amino acid sequence of a crustacean hyperglycaemic hormone from the spiny lobster, Jasus lalandii.

We have isolated a peptide from extracts of sinus glands of Jasus lalandii, a South African spiny lobster, by high-performance liquid chromatography (HPLC) and identified it as crustacean hyperglycaemic hormone (cHH) by (i) a conspecific bioassay measuring glucose elevation in the haemolymph and (ii) an immunoassay using an antiserum raised against cHH of the American lobster. The J. lalandii peptide has a free N-terminus as evidenced by sequencing the first 30 amino acid residues of the intact peptide. Further primary structural data were obtained from sequencing HPLC-purified tryptic and Asp-N proteolytic digests and by cyanogen bromide cleavage of the native, unreduced peptide. In this way, less than 400 sinus glands were used to provide the full sequence. Mass spectrometric analysis in conjunction with inferences based on interspecies sequence homology of cHH molecules unequivocally assigned the complete primary structure of cHH in a member of the crustacean infraorder Palinura for the first time. Our results show 51-76% homology with cHHs known from other decapod infraorders, the major difference being a free N-terminus and several amino acid substitutions interspersed in the non-conserved regions of the molecule. The J. lalandii cHH sequence presented here differs from a partial cHH sequence previously reported from allegedly the same species.

Amino Acid Sequence↗
PubMed1998

1H NMR conformational study on N-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure-activity relationships.

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1-9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, 1H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1-9), I5-Tat(1-9) and L6-Tat(1-9) and to examine the influence of amino acid exchanges on structural features of these peptides. The defined structures revealed differences in the conformations what might be the reason for different interactions of these Tat(1-9) analogues with certain amino acids of the active site of DP IV.

Amides↗
PubMed1998

HSP 12 is a LEA-like protein in Saccharomyces cerevisiae.

LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae). The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised. However, the antibody failed to recognise the predominant protein present after heating the extract at 80 degrees C for 10 min. This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 degrees C for 10 min. Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 degrees C compared with growth at 30 degrees C. However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol.

Amino Acid Sequence↗
PubMed1998

A uniform molecular model of delta opioid agonist and antagonist pharmacophore conformations.

On the basis of a model of the pharmacophore conformations of agonist of the delta-opioid receptor the corresponding delta-antagonist conformations were determined by means of force field calculations. The results explain the unusual behavior of several cyclic beta-casomorphin analogues on the molecular level. Thus, for instance, the model helps to understand why Tyr-c[D-Orn-2-Nal-D-Pro-Gly] is a mixed mu-agonist and delta-antagonist. Furthermore, the model is consistent with low energy conformations of other delta-antagonists such as Tyr-Tic-Phe, Tyr-Tic-Phe-Phe, naltrindole and BNTX. The occupation of a special spatial area by bulky groups close to the protonated N-terminus of opioid peptides is assumed to be highly critical for the switch from agonist to antagonist behavior.

Analgesics, Opioid↗