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Biomedical subjects

H Winkler

Publications and source records attributed to H Winkler.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vitro release of vancomycin and tobramycin from impregnated human and bovine bone grafts.

In order to combine the effects of bone repair and eradication of infection, with both Gram-positive and Gram-negative pathogens, the behaviour of a compound of bone graft and antibiotics was investigated. Samples of human and bovine bone, cancellous and cortical, were processed and incubated with vancomycin and tobramycin, respectively. The compound was placed in 5% human albumin and the surrounding liquid was exchanged completely every 24 h. Concentrations of antibiotics in the fluid were measured over < or = 28 days using high pressure liquid chromatography and a bioassay. All tested combinations eluted mainly in the initial phase with a logarithmic decrease over the testing period. The concentration of antibiotics in the albumin was well above the MIC for common pathogens throughout the investigation in all tested specimens. The highest initial concentrations were measured in the compound of bovine bone together with vancomycin (24395.8 +/- 1138.9 mg/L), decreasing to 9.02 +/- 1.3 mg/L after 11 exchanges. Human and bovine bone did not have significantly different properties. The storage capability of cortical bone was generally lower than that of cancellous bone. Tobramycin concentrations were significantly lower in the initial phase; however, it eluted more steadily and over a longer period, so that from day 6 onwards, its concentration was greater than that of vancomycin. After 28 days, the tobramycin concentration was 18.09 +/- 2.46 mg/L (bovine cancellous bone). In conclusion, bone, if processed adequately, is an excellent carrier for vancomycin and tobramycin. Cortical bone is as suitable as cancellous bone. The pharmacokinetics of human and bovine bone are comparable. Using an antibiotic-graft compound, eradication of pathogens and grafting of bony defects may be accomplished in a one-stage procedure.

Animals↗
PubMed2000

Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients.

We have determined the levels of secretoneurin (SN), a novel 33-amino acid neuropeptide belonging to the class of chromogranins, in the serum and urine of healthy subjects and patients suffering from various tumors. SN serum levels averaged 22.1+/-1.1 fmol/mL. They were 5-fold higher in younger children and then declined continuously. SN levels were positively correlated with serum creatinine, suggesting an influence of renal function on the clearance of SN from the serum. In the urine 80.0 fmol/mL SN was present. In patients with endocrine tumors like gut carcinoids, endocrine pancreatic tumors, oat cell lung carcinomas, and pheochromocytomas, SN serum levels were elevated up to 45-fold. Patients suffering from neuroblastomas, insulinomas, pituitary adenomas including acromegaly, and solid nonendocrine tumors had concentrations in the normal range. In human serum, SN-immunoreactivity was confined to the free peptide SN; neither larger intermediate-sized forms nor the precursor secretogranin II were detected. An efficient removal of the small molecule SN from the serum by the kidney explains why SN serum levels are lower when compared to chromogranin A, which is present as large molecule in serum.

Adolescent↗
PubMed2000

Elevated levels of serum secretoneurin in patients with therapy resistant carcinoma of the prostate.

PURPOSE: The majority of prostate cancers show some degree of neuroendocrine differentiation. It was previously demonstrated that chromogranin A, a constituent of large dense core vesicles of neuroendocrine cells, is frequently elevated in patients with metastatic prostate cancer. We evaluate the expression of secretoneurin, which is generated by proteolytic processing of secretogranin II (chromogranin C), in patients with prostate disease. MATERIALS AND METHODS: Secretoneurin was measured in sera of 16 healthy men whose blood was drawn for prostate cancer screening (controls), and in 9 patients with prostatitis, 19 with benign prostate hyperplasia and 54 with prostate cancer detected by radioimmunoassay. Therapy resistant disease (clinical stage D3) was noted in 20 prostate cancer cases. Serum prostate specific antigen was measured in all patients and controls. In addition, chromogranin A, prostate acid phosphatase and interleukin-6 were determined in patients with D3 prostate cancer. Molecular properties of secretoneurin immunoreactivity were analyzed by gel filtration chromatography followed by radioimmunoassay. RESULTS: Mean secretoneurin was 58.9+/-8 fmol./ml. in patients with therapy resistant prostate cancer. Levels were significantly higher than those measured in sera from controls and patients with prostatitis, benign prostatic hyperplasia and pT2 or pT3 prostate cancer. There was a statistically significant correlation between secretoneurin and chromogranin A in patients with endocrine therapy failure (r = 0.543, p<0.05). There was no correlation between serum secretoneurin and prostate specific antigen, prostate acid phosphatase or interleukin-6. Gel filtration chromatography analysis of sera of 3 patients with D3 prostate cancer revealed a peak of secretoneurin immunoreactivity where intact secretoneurin elutes, thus showing that the processed peptide is circulating in the blood. CONCLUSIONS: Secretoneurin is elevated in sera of patients with endocrine therapy refractory prostate cancer. Our results support the concept that neuroendocrine differentiation is associated with prostate cancer progression.

Adult↗
PubMed1999

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Actins↗
PubMed1999

Relative amounts and molecular forms of NESP55 in various bovine tissues.

NESP55 (neuroendocrine secretory protein with Mr 55,000) comprises a novel chromogranin-like protein, which is paternally imprinted at the genomic level. We used antisera raised against GAIPIRRH, a peptide present at the C-terminus of this protein, and against TC-14, a peptide located in the N-terminal half of NESP55. Radioimmunoassay, gel-filtration chromatography and immunoblotting were used to determine the levels and the molecular forms of NESP55 in different bovine organs. The tissues with the highest levels of GAIPIRRH immunoreactivity were, in decreasing order: the adrenal medulla, the anterior pituitary, the posterior pituitary, various brain regions, and the intestine. The degree of proteolytic processing revealed differences among the tissues analyzed. The lowest processing was detected in the anterior pituitary and in the brain where only a peak corresponding to the intact precursor was present. This was also true for cerebrospinal fluid (CSF). In the posterior pituitary and in the intestine, the free peptide GAIPIRRH was the predominant molecular form. GAIPIRRH-IR, as in the CSF, is present in serum mainly as an intact precursor. A relatively high concentration of GAIPIRRH-IR was found in the kidney medulla, probably due to an endocytotic re-uptake of this molecule from the tubuli after filtration in the glomeruli. The present study is consistent with the concept that NESP55, like the other chromogranins, becomes proteolytically processed. The function of this new chromogranin-like protein, therefore, might be to represent a precursor of biologically active peptides.

Animals↗
PubMed1999

Regulation of NF-kappaB RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase Czeta in primary endothelial cells.

The activity of the transcription factor NF-kappaB is thought to be regulated mainly through cytoplasmic retention by IkappaB molecules. Here we present evidence of a second mechanism of regulation acting on NF-kappaB after release from IkappaB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-kappaB through a pathway involving activation of protein kinase Czeta (PKCzeta) and p21(ras). We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCzeta or p21(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.

Amino Acid Sequence↗
PubMed1999

The processing of secretogranin II in the peripheral nervous system: release of secretoneurin from porcine sympathetic nerve terminals.

The distribution of secretoneurin (SN), a peptide derived from secretogranin II (SgII), in the coeliac ganglion, the splenic nerve and the spleen was examined by immunohistochemistry. In the ganglion, SN immunoreactivity (IR) was unevenly distributed. Positive nerve terminals densely surrounded some postganglionic perikarya in which also intense SN-IR was present. In the crushed splenic nerves, intense immunoreactivities appeared proximal (but to a less extent also distal) to the crush of the nerve. Analysis by cytofluorimetric scanning (CFS) demonstrated that SN-IR and neuropeptide Y immunoreactivity (NPY-IR) were predominant in the axons proximal to the crush representing anterogradely transported components. Using radioimmunoassay (RIA) we demonstrated that upon electrical stimulation (10 Hz, 1 min) of the splenic nerve, significant amounts of SN-IR (64.2+/-2.3 fmol) were released together with NA (4. 1x106+/-0.2 fmol) and NPY (330.0+/-7.2 fmol) from the isolated perfused porcine spleen. To evaluate the processing of SgII in sympathetic neurons, boiled tissue extracts (coeliac ganglia and splenic nerve) and boiled spleen perfusate (used as a suitable source for vesicle derived peptides) were analysed by gel filtration chromatography followed by SN-RIA. In all cases immunoreactivity was present solely as SN, indicating that SgII was fully processed to the free peptide. The evidence that SN is transported to the nerve terminals and is released from the porcine spleen upon nerve stimulation, suggests that it may modulate adrenergic neurotransmission and may also play a role in the neuroimmune communication.

Animals↗
PubMed1999

Pig splenic nerve: peptides derived from chromogranins by proteolytic processing during axonal transport.

We have investigated the proteolytic processing of chromogranin A, chromogranin B and NESP55 (a novel chromogranin-like protein) during axonal transport using pig splenic nerve as a model. We have also studied the presence of chromogranin-derived peptides in the perfusate during electrical stimulation of this nerve. High-performance gel filtration chromatography followed by radioimmunoassay (RIA) revealed that chromogranins are proteolytically processed to varying degrees during axonal transport. For chromogranin A and NESP55, the precursor is still present in the proximal part of the nerve, whereas in the distal part and nerve terminals, intermediate-sized peptides and the free peptides GE-25 and GAIPIRRH dominate, respectively. For chromogranin B, the precursor has already been processed to an intermediate-sized peptide in the proximal part of the nerve, which is also present in the distal parts together with the free peptide PE-11. For chromogranin B and NESP55, only the free peptides PE-11 and GAIPIRRH, or in the case of chromogranin A, the free peptide GE-25 plus an intermediate-sized one, are released from the terminals into the splenic perfusate. These results demonstrate that chromogranins are processed to smaller peptides during axonal transport.

Animals↗
PubMed1999

Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy.

Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6 x histidine) N-terminal tags. After reconstitution with 57Fe(II), the Mössbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, > 90% of the iron in hTH1 had Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift delta (1.8-77 K) = 1.26-1.24 mm s-1 and quadrupole splitting delta EQ = 2.68 mm s-1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (delta = 1.07 mm s-1 and delta EQ = 2.89 mm s-1 at 77 K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (delta = 1.24 mm s-1 and delta EQ = 2.87 mm s-1 at 77 K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mössbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.

Absorptiometry, Photon↗
PubMed1999

Proteolytic processing, axonal transport and differential distribution of chromogranins A and B, and secretogranin II (secretoneurin) in rat sciatic nerve and spinal cord.

The chromogranin family comprises chromogranin A and B, and secretogranin II. The present study has focused on the axonal transport of chromogranins/secretogranin II and their detailed distribution in peripheral nerves and the spinal cord. With radioimmunoassay (RIA) and column chromatography, we first studied the processing of chromogranin B and secretogranin II during axonal transport. No larger precursors of these peptides were detected in the sciatic nerves, indicating that they are already processed to a high degree early during axonal transport. We also analysed nerve segments above and below a crush, using RIA, in order to compare these accumulation data with those obtained by the cytofluorimetric-scanning (CFS) technique. For the latter technique, the amounts of accumulation distal to the crush (presumably representing recycling and retrogradely transported peptides) were 30-40% of the amounts in the proximal accumulation for chromogranin A and secretoneurin, in contrast to chromogranin B, which showed 15% recycling. With the RIA, the corresponding values for secretoneurin and PE-11 (antibody against chromogranin B) were 42% and 14%, respectively. Therefore, the data obtained by CFS were in excellent agreement with those obtained by RIA. In crushed sciatic nerves, chromogranin A was present in large axons as well as in small- and medium-sized axons. Chromogranin B was mainly restricted to large axons, while secretoneurin was localized to bundles of small axons. This differential distribution was also found in the spinal roots and in the peripheral terminals. Chromogranin A was present in both ventral and dorsal roots, and chromogranin B was detected in ventral roots and in large sensory axons in the dorsal roots. Secretoneurin was dominant in the dorsal root. Double-labelling studies with antibodies against choline acetyltransferase/vesicular acetylcholine transporter, or against tyrosine hydroxylase, confirmed that chromogranin A was distributed in cholinergic, sensory, as well as adrenergic neurons. Chromogranin B was mainly present in cholinergic motor neurons and large sensory neurons, and secretoneurin was restricted to adrenergic and sensory neurons. The present study demonstrates that chromogranins A and B, and secretoneurin are transported with fast axonal transport in the peripheral nerves, with different amounts of recycling, and that they are differentially distributed in different types of neurons in the peripheral nervous system and the spinal cord, suggesting that each of them may play a special role in subsets of neurons.

Animals↗
PubMed1999

Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve.

Subcellular fractionation of bovine splenic nerves, which consist mainly of sympathetic nerve fibers, has been useful for characterizing cellular organelles en route to the terminal. In the present study we have characterized the subcellular distribution of both secretory and membrane proteins. A newly discovered chromogranin-like protein, NESP55, was found in large dense-core vesicles. The endogenous processing of NESP55 was comparable to that of chromogranins but more limited than that of secretogranin II and chromogranin B. For membrane proteins three major types of distribution were found. The amine carrier VMAT2 was confined to large dense-core vesicles. VAMP or synaptobrevin was present both in large dense-core vesicles and in lighter vesicles, whereas SNAP-25, syntaxin, and two types (N and L) of Ca2+ channels were found in a special population of lighter vesicles but were not present in large dense-core vesicles or at the most in very low concentrations. The plasma membrane norepinephrine transporter was apparently present in a separate type of vesicle, but this requires further study. These results further characterize vesicles en route to the terminal and establish for the first time that peptides involved in exocytosis (syntaxin, SNAP-25, and N- and L-type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense-core vesicles indicates that the formation of small dense-core vesicles in the terminals requires a reuse of membrane components of large dense-core vesicles.

Amines↗
PubMed1998

Regulated and endothelial cell-specific expression of Fas ligand: an in vitro model for a strategy aiming at inhibiting xenograft rejection.

BACKGROUND: Immunologically privileged sites have been shown to express Fas ligand (FasL) and may protect themselves by inducing apoptosis of infiltrating inflammatory cells. We asked whether the Fas/FasL interaction could be used to protect a xenograft from rejection. We proposed that endothelial cells that are resistant to Fas-mediated killing could be considered as a vehicle for expression of recombinant FasL. METHODS: Based on the tetracycline-regulated expression system, constructs were designed that allow endothelial cell-specific and regulated expression of FasL by placing the tetracycline-dependent transactivator under control of the murine intercellular adhesion molecule-2 promoter. RESULTS: Primary bovine endothelial cells transfected with FasL efficiently killed Fas-expressing cells in a regulated manner. Not only Fas-positive cell lines but also human peripheral blood lymphocytes underwent apoptosis upon exposure to FasL-transfected endothelial cells. CONCLUSION: This in vitro model may provide tools for the generation of transgenic animals to be used as donors for vascularized xenograft transplantation.

Animals↗
PubMed1998

Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis.

We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized xenografts. Our previous studies have shown that inhibition of NF-kappaB by adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of proinflammatory genes in EC. However, I kappaB alpha sensitizes EC to TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase (MnSOD), and cellular inhibitor of apoptosis 2. We report here that adenovirus mediated expression of a dominant negative C-terminal truncation mutant of p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as efficiently as does I kappaB alpha. However, contrary to I kappaB alpha, p65RHD does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally well. We present evidence that this difference in sensitization of EC to apoptosis is due to the ability of p65RHD, but not I kappaB alpha, to inhibit the constitutive expression of c-myc, a gene involved in the regulation of TNF-alpha-mediated apoptosis. These data demonstrate that it is possible to block the expression of proinflammatory genes during EC activation by targeting NF-kappaB, without sensitizing EC to apoptosis and establishes the role of c-myc in controlling induction of apoptosis during EC activation. Finally, these data provide the basis for a potential approach to suppress EC activation in vivo in transgenic pigs to be used as donors for xenotransplantation.

Adenoviridae↗
PubMed1998

Levels and molecular forms of chromogranins in human childhood neuroblastomas and ganglioneuromas.

The chromogranins are a class of acidic proteins found in large secretory granules of neuroendocrine tissues and tumors derived from them. We measured the relative amounts and characterized the molecular forms of two members of this family, i.e. chromogranin A and secretogranin II, in 14 neuroblastomas and five ganglioneuromas. In all the tumors investigated significant amounts of chromogranin A and secretogranin II were found. Neuroblastomas contained two times and ganglioneuromas 45 times more secretogranin II compared to chromogranin A. Both proteins were processed in these tumors to a great extent to smaller peptides, only limited amounts of intact chromogranin A or secretogranin II were present. In general, proteolytic processing of secretogranin II to the small neuropeptide secretoneurin was more complete than that of chromogranin A to the peptide GE-25. Proteolytic processing of both chromogranins as well as the total amounts of these proteins were unrelated to tumor staging.

Adrenal Gland Neoplasms↗
PubMed1998

Formation and sequence analysis of secretoneurin, a neuropeptide derived from secretogranin II, in mammalian, bird, reptile, amphibian and fish brains.

Secretoneurin is a recently-characterized neuropeptide derived from secretogranin II, a protein belonging to the class of chromogranins. We investigated the phylogeny of this peptide by immunoblotting and gel-filtration high performance liquid chromatography followed by radioimmunoassay of brain extracts of various species including chicken, lizard, frog and fish. In addition the amino acid sequence of secretoneurin from pig, hamster, rabbit, guinea-pig and chicken was established by reverse transcriptase polymerase chain reaction. Secretoneurin is strongly conserved during evolution, it is not only expressed in various mammalian species but found also in the brain of birds, reptiles, amphibians and fish. In all these species a significant or near complete processing of secretogranin II to secretoneurin was observed. These data provide significant evidence for the neuropeptide nature of the novel functional peptide.

Amino Acid Sequence↗
PubMed1998

Unexpected candidemia complicating ureteroscopy and urinary stenting.

Two elderly patients with obstructive renal calculi who developed Candida albicans bloodstream infection within 12 h following ureteroscopy and ureteral stenting are described. Both patients were treated with prolonged courses of broad-spectrum antibiotics and were found to have urine cultures positive for Candida albicans prior to the urologic procedures. One patient also developed bilateral candidal endophthalmitis. The clinical presentation was indistinguishable from bacteremia complicating manipulation of the urinary tract. The patients were successfully treated with systemic antifungal therapy. Candiduria may present a risk for dissemination during invasive, relatively simple urologic procedures.

Aged↗
PubMed1998

Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency.

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A-->C (H20P) and a 1016 T-->C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A-->G (H389R) and 1549 C-->G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).

Anemia, Hemolytic, Congenital Nonspherocytic↗