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W Neugebauer

Publications and source records attributed to W Neugebauer.

At least 37 records · Page 2Linked to original sources
PubMed1994

Membrane interaction and conformational properties of the putative fusion peptide of PH-30, a protein active in sperm-egg fusion.

A peptide representing the putative fusion domain of PH-30, a sperm surface protein involved in sperm-egg fusion, was synthesized, and its interaction with model lipid membranes was characterized by biophysical methods. While the peptide binds to the vesicles composed of both neutral and acidic lipids, the apparent affinity is significantly higher for the latter lipid class. The intervesicular lipid mixing assay suggests that the synthetic peptide is able to induce fusion of large unilamellar vesicles. Circular dichroism and Fourier-transform infrared spectroscopy show that while in an aqueous buffer the peptide exists in an essentially unordered conformation, binding to the membranes results in a conformational transition to a beta-structure. These data indicate that the fragment identified on the alpha-subunit of PH-30 as a putative fusion peptide is indeed a good candidate for this role. However, in contrast to what has been proposed for some viral fusion peptides, the PH-30 fusion domain is highly unlikely to act as an insertional "sided" helix.

ADAM Proteins↗
PubMed1994

Further definition of the protein kinase C activation domain of the parathyroid hormone.

The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28-34 region of the molecule. We have now shown that PTH-(29-32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1-84) and the fully bioactive PTH-(1-34) fragment, there were two peaks in the PKC response to PTH-(29-32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28-31) and PTH-(29-31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1-31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1-34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.

Adenylyl Cyclases↗
PubMed1994

Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase.

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1-34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1-31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1-34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1-34). Injections of equimolar amounts of desamino-hPTH(1-34) [N-propionyl(2-3)hPTH-amide], which stimulated PKC as strongly as hPTH(1-34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8-84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.

Adenylyl Cyclases↗
PubMed1994

C-terminal fragments of parathyroid hormone-related protein, PTHrP-(107-111) and (107-139), and the N-terminal PTHrP-(1-40) fragment stimulate membrane-associated protein kinase C activity in rat spleen lymphocytes.

Membrane-associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C-terminal parathyroid hormone-related protein fragments, PTHrP-(107-111) and PTHrP-(107-139), at concentrations from 10(-3) to 10(4) pM. By contrast, the same concentrations of PTHrP-(120-139), without the 107-111 TRSAW (-Thr-Arg-Ser-Ala-Trp-) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N-terminal PTHrP-(1-40) fragment also stimulated membrane-associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response.

Animals↗
PubMed1994

Probing alpha-helical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain rotamer conformation.

The relationship between alpha-helical secondary structure and the fluorescence properties of an intrinsic tryptophan residue were investigated. A monomeric alpha-helix forming peptide and a dimeric coiled-coil forming peptide containing a central tryptophan residue were synthesized. The fluorescence parameters of the tryptophan residue were determined for these model systems at a range of fractional alpha-helical contents. The steady-state emission maximum was independent of the fractional alpha-helical content. A minimum of three exponential decay times was required to fully describe the time-resolved fluorescence data. Changes were observed in the decay times and more significantly, in their relative contributions that could be correlated with alpha-helix content. The results were also shown to be consistent with a model in which the decay times were independent of both alpha-helix content and emission wavelength. In this model the relative contributions of the decay time components were directly proportional to the alpha-helix content. Data were also analyzed according to a continuous distribution of exponential decay time model, employing global analysis techniques. The recovered distributions had "widths" that were both poorly defined and independent of peptide conformation. We propose that the three decay times are associated with the three ground-state chi 1 rotamers of the tryptophan residue and that the changes in the relative contributions of the decay times are the result of conformational constraints, imposed by the alpha-helical main-chain, on the chi 1 rotamer populations.

Amino Acid Sequence↗
PubMed1994

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment.

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH2, I; cyclo[Lys26-Asp30]-hPTH-(20-34)-NH2, II; cyclo[Glu22-Lys26]-hPTH-(20-34)-NH2, III; cyclo[Lys27-Asp30]- hPTH-(20-34)-NH2, IV; and [Leu27]-hPTH-(20-34)-NH2 V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in alpha-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of alpha-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV. This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV. The data suggest that in the region bounded approximately by residues 24-32, an amphiphilic alpha-helix is important for correct functional binding to the PTH receptor.

Amino Acid Sequence↗
PubMed1993

Protein kinase C-activating domains of parathyroid hormone-related protein.

N-terminal fragments of PTH-related protein (PTHrP), PTHrP-(1-34), and PTHrP-(1-40) stimulated both adenylyl cyclase and a mechanism that increases membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. There were two peaks in the PKC response to the N-terminal PTHrP fragments: one peak was obtained with picomolar and the other with nanomolar PTHrP concentrations. The PKC-stimulating picomolar concentrations of the PTHrP fragments did not detectably stimulate adenylyl cyclase, but the nanomolar concentrations did. Since a similar two-peak response of PKC activity was obtained with PTHrP-(28-34), the single, N-terminal PKC activation domain of the PTHrP is in the same 28-34 region of the molecule as that of PTH despite this region having different primary amino acid sequences in the two hormones. Unlike PTH, PTHrP has a second PKC activation domain, as indicated by the ability of picomolar concentrations of the PTHrP-(107-111) fragment to stimulate maximally membrane-associated PKC activity in the osteosarcoma cells.

Adenylyl Cyclases↗
PubMed1993

Relationship between apoptosis and the cell cycle in lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1.

The mechanism of switching between the cell cycle and active cell death (apoptosis) was investigated in cytokine-dependent CTLL cells. These cells proliferate in the presence of interleukin 2 (IL2), but accumulate in early G1 and undergo apoptosis in its absence. In the absence of IL2 the cells also become sensitive to glucocorticoid-induced apoptosis. Using specific inhibitors of protein kinase C and tyrosine kinases we established that two signals are required to fully repress cell death and stimulate G1 progression. One of these signals activates protein kinase C (PKC) which represses cell death and the other activates a tyrosine kinase which confers glucocorticoid resistance and permits cell cycle progression. Thus, phorbol esters can activate PKC and maintain cell viability in the absence of IL2, but the cells cannot proliferate. Moreover, the cells remain sensitive to glucocorticoid-induced apoptosis unless the tyrosine kinase-mediated signal is also given. There is a correlation between the presence of AP1 DNA-binding activity and the repression of the cell death pathway. The c-jun gene is expressed constitutively and both IL2 and phorbol esters induce the expression of c-fos to generate a functional AP1 capable of repressing cell death. However, only interleukin 2 can initiate the tyrosine kinase-mediated modification that confers dexamethasone resistance and permits G1 progression. In the absence of IL2 glucocorticoids stimulate AP1 degradation and induce apoptosis.

Adaptor Protein Complex 1↗
PubMed1993

DNA content and PCNA immunoreactivity in oral precancerous and cancerous lesions.

Thirty-three dysplastic lesions showing varying degrees of atypia located in the oral cavity and 83 squamous cell carcinomas located in the oral cavity and tongue (n = 56) or in the lips (n = 27) were analysed by means of proliferating cell nuclear antigen (PCNA) immunoreactivity and image cytometric DNA measurements. The results show that in dysplastic lesions increasing cellular atypia correlated to elevated proliferative activity and aneuploidy occurring in the basal cell layers. In highly differentiated squamous carcinomas increased PCNA immunoreactivity and aneuploidy was preferentially observed focally (grade 1 tumours) or in the invasive zones (grade 2 tumours). In contrast, more poorly differentiated carcinomas (grade 3 and 4 tumours) showed strongly elevated proliferative activity and aneuploidy throughout the entire tumour mass.

Aged↗
PubMed1992

Structural elements of human parathyroid hormone and their possible relation to biological activities.

Human parathyroid hormone (hPTH) and several deletion analogues were examined for the presence of secondary structure using circular dichroism spectroscopy. The spectra of hPTH and the deletion analogues 8-84, 34-53, 53-84, 1-34, 13-34, 1-19, and 20-34, in neutral, aqueous buffer, gave no evidence for extensive secondary structure. An alpha-helical-like spectral contribution was found to arise from a region within peptide 13-34. This spectral contribution was speculated to arise from partial stability of a helix consisting of residues 17-29. Molecular dynamics simulations of peptide 1-34 suggested that this peptide tends to fold with a bend defined by residues 10-14, with the amino-terminal and carboxyl-terminal residues tending to be in more extended forms and the other residues in helical-like conformations. The addition of trifluoroethanol promoted the formation of alpha-helix, mainly in the 1-34 region. The putative helix comprised of residues 17-29 was stabilized by the addition of 10-20% TFE, while a second putative helix proximal to the amino terminus, and comprised of residues 3-11, was stabilized by slightly higher concentrations of TFE. An amphiphilic sequence was identified within the 20-34 fragment. The development of alpha-helix on binding this fragment, and other analogues containing this sequence, to palmitoyloleoylphosphatidylserine vesicles provided experimental evidence for the potential role of this amphiphilic sequence in binding to membranes or to a membrane receptor. The relationships between these alpha-helical regions in 1-34, either potentiated by trifluoroethanol or lipid vesicles, are discussed in terms of different receptor-binding regions within hPTH.

Amino Acid Sequence↗
PubMed1992

Preparation and biological activities of potential vasopressin photoaffinity labels.

Several potential photoaffinity analogues of the peptide hormone vasopressin (VP) were prepared by classical solid-phase peptide synthesis using two different pathways. Peptide sequences were built by introduction of (a) Nar-protected aminophenylalanine or (b) nitrophenylalanine in the photolabeling position. Conversion to the azido peptide was completed in pathway a after cleavage and before purification and in pathway b from small quantities of purified nitrophenylalanine-containing precursor peptides. V1 receptor binding properties were measured using membranes prepared from rat liver cells. The binding potential of agonistic VP structures was abolished by the introduction of an azido or a nitro group into the aromatic side chain at position 3. Cyclo desamino-beta,beta-dialkyl-Cys1-type VP antagonist structures were prepared with the photoactivable moiety in position 2 and an iodination residue in position 9. One particular compound, [Dmpa1, Phe(N3)2, Val4, Lys8,D-Tyr9]VP (8), containing beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, had excellent binding properties, both in the radioiodinated (Kd = 4.8 +/- 1.9 x 10(-10) M) and noniodinated form (Kd = 6.4 +/- 0.98 x 10(-10) M). The analogues with long-chain beta-alkylation (diethyl and pentamethylene) and the linear antagonist photolabel showed significantly less affinity. Optimal binding properties were obtained within a very narrow range of hydrophobicity; greater or lesser hydrophobicity was correlated to less potent binding. The precursor analogues, containing nitrophenylalanine, displayed a structure-activity relationship similar to that of the azido peptides. The most potent analogues will be used for receptor labeling studies. A linear antagonist structure having a photosensitive group in position 1, has also been prepared, but this compound displayed much less affinity than the cyclic antagonists. The most potent compounds were also highly selective for the V1 receptor and did not recognize the V2 receptor from other preparations.

Affinity Labels↗
PubMed1992

The protein kinase-C activation domain of the parathyroid hormone.

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.

Animals↗
PubMed1991

Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli.

Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.

Amino Acid Sequence↗
PubMed1989

[Observations on post-traumatic osteitis].

The following study reports the frequency, the promoting factors, the therapy and her results of the 32 patients with posttraumatic osteitis we treated between 1984 and 1986 in the Surgical University Clinic Tübingen. Most of the primary injuries were caused by traffic-accidents; especially dangerous were those with motor-bikes, which led frequently via open fractures of the shank to osteitis. We saw the highest infection-rates after plate-osteosyntheses. The infects became obvious in most cases either about one month after the accident or a year later coinciding with the increased use of the limb. The most frequent bacterium was Staph. aureus both in the mono-infections and in the mixed-infections forming a third of the group. We always performed an operative therapy with the intention to stabilize the bone and to clean the infection-site. The strict performance of this management led to infect-suppression in all cases with the need of only one amputation.

Adult↗
PubMed1988

Synthesis and immunological evaluation of N-terminal, noncrossreactive tachykinin antigens.

The N-terminal hexa- or pentapeptide sequences of the three mammalian tachykinins substance P, neurokinin A, and neurokinin B have been synthesized by the conventional solid-phase procedure with 6-aminocaproyl-S-(acetamidomethyl)cysteine as a C-terminal spacer and attachment function. A fourth sequence, with an additional N-terminal 6-aminocaproyl residue on the substance P-hapten sequence, was cyclized N- to C-terminally. For this purpose, a four-level protection scheme has been applied: BOC-TFA for N-terminal protection and cleavage; TFA-stable but HF-labile anchoring function and side-chain protection; S-acetamidomethyl for semipermanent thiol protection. The side chain amino function of Lys was protected with NO2Z, stable against HF but readily cleaved with hydrogenation. The hapten sequences were coupled to maleimidated BSA, after the Acm group was removed by mercury/hydrogen sulfide treatment. Mice immunized with the three linear hapten sequences produced sera that were specific in enzyme-linked immunosorbant assay for the presented hapten and the respective tachykinin but displayed no crossreactivity at all toward the other haptens nor to one of the other tachykinins. It is concluded that this approach produced antisera, specific and selective for its respective mammalian tachykinins.

Animals↗
PubMed1986

[Results of conservative treatment of distal radius fractures].

The results of conservative treatment of fractures of the distal radius in 303 patients over a period of two years are demonstrated. 226 patients could be followed-up. The healing of the fracture was satisfactory in 78%, while the results regarding the various age groups showed considerable differences. Along with the demonstration of the functional and anatomical results in relation to age, sex, and localization the technique of fracture treatment as well as follow-up treatment are discussed.

Adolescent↗
PubMed1985

[Significance of computer tomography for operative tactics in esophageal cancer].

In patients with tumor stage T1-CT is useful in the detection of enlarged lymph nodes. In 93% of the cases with tumor stage T2-CT gives exact information about tumor extension and infiltration. In addition 70% of the patients with positive lymph nodes can be evaluated. In T3 tumor infiltration in nearby organs can be demonstrated in 94% and lymph node metastases in 79% of the cases. The therapeutic consequences are: In tumor stage T2 and T3 subtotal esophagectomy with retrosternal stomach replacement by cervical anastomosis should be aspired for reasons of postoperative irradation of the esophageal bed.

Esophageal Neoplasms↗