Biomedical subjects

P Henklein

Publications and source records attributed to P Henklein.

At least 55 records · Page 3Linked to original sources

PubMed1996

Interaction of cholecystokinin and glutamate agonists within the dLGN, the dentate gyrus, and the hippocampus.

The interaction of sulfated cholecystokinin (CCK-8S) with excitatory amino acids (EAA) was studied on single units of the dorsal lateral geniculate nucleus (dLGN), the dentate gyrus, and the hippocampal CA3 region in rats anaesthetized with urethane. lontophoretic co-administration of small, individually ineffective currents of CCK-8S and kainic acid or N-methyl-D-aspartate repeatedly elicited an increase of the discharge rate in nearly all geniculate and half of the dentate neurons but not in those of the CA3 region. The effect could be reduced by the CCKB receptor antagonist PD 135,158 more often than by the CCKA antagonist KL 1001. The increased firing due to co-administration of CCK and kainate could also be suppressed by the non-NMDA antagonist CNQX but not by the NMDA antagonists CPP or AP-5, which were otherwise able to prevent the neuron from responding to co-administration of CCK and NMDA. It is suggested that in distinct brain regions the effectivity of the "low level" EAA transmission may be enhanced by small amounts of CCK-8S. This is thought to be mediated by a coactivation of CCK and EEA receptors.

Animals↗

PubMed1996

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu).

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.

Amino Acid Sequence↗

PubMed1996

Plasma and urine salsolinol in humans: effect of acute ethanol intake on the enantiomeric composition of salsolinol.

The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R-enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S-enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.

Adult↗

PubMed1995

Resolution of catecholic tetrahydroisoquinoline enantiomers and the determination of R- and S-salsolinol in biological samples by gas chromatography-mass spectrometry.

Tetrahydroisoquinolines (TIQs) might be formed endogenously and can act centrally to promote a mechanism governing alcohol drinking behaviour. The possibility that biosynthesis occurs through a stereospecific enzymatic reaction is considered. Several TIQs were transformed into diastereomers by a two-step derivation with N-methyl-N-trimethylsilyltrifluoracetamide and R-(-)-2-phenylbutyrylic acid and were analyzed by gas chromatography-mass spectrometry (GC-MS). High resolution of the TIQ enantiomers was achieved. This method was applied to the quantification of the enantiomers of salsolinol (SAL) in urine and plasma of healthy humans. Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenyl-boronic phase cartridges and transformed into diastereomers. The sensitivity and specificity of the assay permit the determination of the enantiomeric composition of SAL in plasma and urine. The limit of quantification was found to be 100 pg/ml for each enantiomer. The described method has the advantage that optimal resolution of the SAL enantiomers without peak overlapping between analyte and other compounds can be achieved. Contrary to other findings, our GC-MS studies have demonstrated that endogenously formed SAL is racemic in plasma as well as in urine of healthy subjects.

Deuterium↗

PubMed1995

Novel disulfide-constrained pentapeptides as models for beta-VIa turns in proteins.

The conformational behavior of cyclic peptides of the amino acid sequence Cys-Phe/Ala-Pro-Ala-Cys has been investigated through the combined use of molecular simulation methods and NMR experiments to find models for beta-VIa turns of proteins. Both oxidized (cyclic) peptides and reduced (linear) forms were investigated. At least 95% of the cyclic peptides show a cis conformation of the Xaa-Pro bond in solution in DMSO or water, whereas all other peptide bonds are trans. Furthermore, we observed a hydrogen bond between the NH group of residue Ala4 and the C = O group of residue Cys1. Both properties are indicative of beta-VIa turns. After reduction of the disulfide bridge, the all-trans form of the peptide bonds predominates.

Base Sequence↗

PubMed1995

Inhibitory effects of cholecystokinin on rat's geniculate activity can be blocked by GABA-antagonists.

Cholecystokinin applied iontophoretically as the sulfated octapeptide (CCK-8S) or as highly selective CCKA- and CCKB-agonists induced excitatory as well as inhibitory effects on dorsal lateral geniculate unit activity. Inhibitory effects of CCK could be abolished by administration of GABAergic antagonists, especially bicuculline. Therefore, interneurons seem to be largely excited by CCK, and in this way, they appear to mediate the inhibition of the discharge rate in geniculate relay cells induced by CCK.

Animals↗

PubMed1995

Cyclic cholecystokinin-analog pentapeptide cyclo (Asp-Trp-Met-Asp-Phe): an unexpected solution conformation.

The conformational analysis of the CCK-B binding peptide cyclo (Asp-Trp-Met-Asp-Phe) has been carried out in DMSO-d6 and in a mixture of H2O/DMSO-d6 by NMR spectroscopy and by restrained molecular dynamics methods. In the NMR spectra, only one set of resonance signals was found. The NOE analyses proved the existence of an all-trans conformation for this peptide. Distance constraints of 1H pairs derived from NOE data were used for restrained molecular dynamics simulations, resulting in one conformational family with a very regular orientation of the amino acids and similar dihedral angles for each residue. The dihedrals and the absence of an intramolecular hydrogen bond indicate that there is no common turn formation in the peptide backbone. A submicromolar binding constant for CCK-B receptors point to a similarity with the bioactive conformation.

Amino Acid Sequence↗

PubMed1995

CCKB receptor activation results in increased [3H]thymidine incorporation in rat glioma C6 cells.

Cholecystokinin (CCK) is known to stimulate cell proliferation but involvement of CCKB type receptors has not been exactly demonstrated so far. We examined the effect of CCK-8S and two receptor agonists on rat glioma C6 cells when using different CCKB receptor agonists and antagonists. Both CCK-8S and CCKB receptor agonists BC 264 and Suc-Trp-N(Me)Nle-Asp-Phe-NH2 stimulate [3H]thymidine incorporation. These effects were inhibited by CCKB receptor antagonist L-365,260 over 100-fold more effectively than it was seen by using CCKA receptor antagonist L-364,718. The data indicate that CCKB receptor agonists are potent stimulants of rat glioma C6 cell DNA synthesis suggesting that CCKB receptor activation is involved in cell proliferation within the central nervous system.

Amino Acid Sequence↗

PubMed1995

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

Animals↗

PubMed1995

Effects of two newly synthetized cholecystokinin tetrapeptides on the activity of single hippocampal and thalamic neurons.

The influence of two iontophoretically administered newly developed cholecystokinin (CCK) tetrapeptides with high selectivity and affinity to CCK-B receptors on the impulse activity of single hippocampal and thalamic neurons were tested in in-vivo experiments, in comparison to the effect of the sulfated octapeptide (CCK8S). A very similar responsiveness to the compared drugs was found. Most neurons responded with an increase of their discharge frequency. Only a few suppressive effects were elicited by each drug and in each of the structures. There was a good correspondence between the compared drugs concerning the direction and relative response amplitude, resulting in a highly significant correlation of the effects of both CCK4s with the CCK8S effects. On a subsample of neurons, the blocking effect of the selective CCK-B receptor blocker PD135 was tested and found to be effective in 16 out of 20 CCK4 responses, including also one inhibition. The results show that the new compounds act as effective CCK agonist binding to the B-type CCK receptor. The few inhibitory effects obtained could be explained by possible indirect effects mediated via inhibitory interneurons which are known to exist in both investigated structures.

Animals↗

PubMed1995

Expression of the Huntington disease gene in rodents: cloning the rat homologue and evidence for downregulation in non-neuronal tissues during development.

Huntington's disease (HD) is associated with an expanded and unstable (CAG) > 35 repeat within a gene of unknown function. We isolated the complete coding region of the rat HD gene (rhd) from cDNA libraries and investigated its expression in different developmental stages of rodent tissues. The rat gene exhibits 90% peptide sequence identity to the human and 96% to the murine sequence. The (CAG)n repeat is markedly reduced in the rat compared to the average human (CAG)n block. Northern blot analysis and in situ hybridizations reveal that in rodents the hd gene is already expressed during embryonal development. As in humans, the rhd gene is expressed in two transcriptional isoforms which result from different polyadenylation signals. In mice, however, a third transcript of intermediate size was found predominantly expressed in brain. This transcript is downregulated in later development. At day 14.5 p.c. the level of rhd expression is similar in the brain and in non-neuronal tissues. In contrast, the expression in non-neuronal tissues is markedly reduced in adult animals and corresponds to the restricted distribution of neuropathologic changes observed in HD patients.

Amino Acid Sequence↗

PubMed1995

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling.

The molecular interaction of the Fab fragment of the human monoclonal antibody 3D6, directed against the transmembrane protein gp41 of human immunodeficiency virus (HIV) 1, with its peptide epitope is characterized by a panel of overlapping peptides, a peptide epitope library and molecular modeling techniques. The sequence CSGKLICTTAVPW, corresponding to amino acids 605-617 of gp41, was identified as the best binding peptide (KD = 1 x 10(-8) mol/l). This peptide served as a starting point to prepare a cellulose-bound peptide epitope library in which each residue of the epitope is substituted by all L- and D-amino acids, resulting in 494 epitope peptide variants which were subsequently analyzed for binding 3D6. The library was synthesized to identify residues critical for binding and to obtain information about the molecular environment of the epitope peptide bound to 3D6. Both cysteine residues, as well as isoleucine 6, threonine 8 and proline 12, of the epitope were highly sensitive to substitution. Using the data obtained from the epitope characterization, as well as a low-resolution electron density map of a 3D6 Fab-peptide complex, a 3-D model of the Fab-peptide complex was generated by molecular modeling. The modeling experiments predict binding of the peptide, which is cyclized via the two cysteine residues, to a pocket formed dominantly by the hypervariable loops complementarity determining regions CDR3L, CDR2H and CDR3H.

Amino Acid Sequence↗

PubMed1995

Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and 1H NMR spectroscopy.

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu41-58, Vpu52-74 and Vpu63-81, by a combination of 2D 1H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu41-58 has an alpha-helix from residues 42 to 50 while a second helix is found for Vpu52-74 from residues 57 to 69. Vpu63-81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two alpha-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus.

Amino Acid Sequence↗

PubMed1994

Type B cholecystokinin receptors on rat glioma C6 cells. Binding studies and measurement of intracellular calcium mobilization.

To our knowledge, no brain derived cell line has been shown as yet to bear cholecystokinin(CCK)B receptors. In this paper, CCK binding sites were identified on rat C6 glioma cells. Pharmacological characterization demonstrated a single class of high affinity binding sites (Kd = 1.7 +/- 0.3 x 10(-10) M) and a binding capacity of 6.1 +/- 1.8 fmol/mg protein. These CCK binding sites displayed a typical CCKB pharmacological profile as shown in competition studies by using several CCK-related compounds and nonpeptide CCK antagonists discriminating between CCKA and CCKB sites. In order to demonstrate that CCK binding sites constitute a functional receptor CCK-8S induced mobilization of free intracellular calcium was investigated in single C6 cells by using a laser scanning confocal imaging system. Since rise in [Ca2+]i noted by stimulation of C6 cells with CCK-8S could be blocked by the CCKB receptor antagonist L-365,260 (100 nM) but not by the CCKA receptor antagonist L-364,718 (100 nM), CCK induced calcium signal is triggered by activation of CCKB receptors in C6 cells. The rat C6 glioma cell line may serve as a useful model for studying CCKB receptor in brain.

Animals↗

PubMed1994

The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Amino Acid Sequence↗

PubMed1994

CCKB receptor signaling in rat pituitary GH3 cells. CCK-8S-induced intracellular calcium mobilization by Ca2+ release and Ca2+ influx.

We describe the effect of sulphated cholecystokinin octapeptide (CCK-8S) on [Ca2+]i in rat pituitary GH3 cells. Investigations were performed on fluo-3 loaded cells by using a confocal imaging system MRC-600 (Bio-Rad). Because CCK-8S mobilized intracellular calcium in cells bathed in Ca(2+)-free buffer it must be able to release calcium from internal stores. Furthermore, influx of Ca2+ from outside the cells seems to contribute to CCK-8S induced increases in [Ca2+]i as demonstrated by calcium mobilization in GH3 cells preincubated with thapsigargin in Ca2+ containing buffer.

Animals↗

PubMed1994

Comparative 3H-CCK-8S binding studies on CCKB-type receptors in guinea-pig cortex and continuous cell lines.

The present study was undertaken to compare binding characteristics of CCKB-type receptors in guinea-pig cortex, Jurkat T-cells, GH3 cells and C6 cells. The rank order of potency of a variety of CCK agonists and antagonists in inhibiting specific [3H]CCK-8S binding was highly correlated for the 4 CCKB receptor models as demonstrated by a computer-assisted statistical analysis. Taking the ligand binding profiles as the criterion it is concluded that CCKB receptors in guinea-pig cortex, Jurkat T-cells, pituitary GH3 cells and rat glioma C6 cells share identical pharmacological properties.

Animals↗

PubMed1994

HLA-B27 binding peptides derived from the 57 kD heat shock protein of Chlamydia trachomatis: novel insights into the peptide binding rules.

In this study we investigate the 57 kD heat shock protein of Chlamydia trachomatis for potential HLA-B27 restricted T cell epitopes. This protein is known to elicit T cell immunity, as judged by delayed type hypersensitivity. We synthesized 24 peptides containing the B27 anchor amino acid arginine at position 2, according to the rules previously described for peptide binding to MHC class I molecules. The nonamer peptides were tested in an in vitro assembly assay; six out of the 24 peptides bind to HLA-B27 although their sequences only partially match the HLA-B27 binding motif. Two of these six peptides carry negatively charged amino acids which apparently fit into the P1 pocket and in three out of the six a positively charged amino acid fits into the P3 pocket. In addition, two octamer peptides stabilized the HLA-B27 molecule without containing an appropriate amino or carboxy terminus. Therefore our data suggest that current binding rules will need to be refined before they can be used to accurately predict potential T cell epitopes. Furthermore our HLA-B27-binding peptides should prove useful probes for the study of the processing and presentation of this bacterial antigen, and of changes in the T cell repertoire induced by this form of infection.

Amino Acid Sequence↗