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Biomedical subjects

J Knolle

Publications and source records attributed to J Knolle.

At least 19 recordsLinked to original sources

alpha(v)beta(3) Antagonists based on a central thiophene scaffold.

A series of novel, highly potent alpha(v)beta(3) antagonists based on a thiophene scaffold and containing an acylguanidine as an Arg-mimetic is described. A number of structural features, such as cyclic versus open guanidine and a variety of lipophilic side chains, carbamates, sulfonamides and beta-amino acids were explored with respect to inhibition of alpha(v)beta(3) mediated cell adhesion and selectivity versus alpha(IIb)beta(3) binding. In addition, compound 19 was found to be active in the TPTX model of osteoporosis.

Animals↗

Effect of selective or combined inhibition of integrins alpha(IIb)beta(3) and alpha(v)beta(3) on thrombosis and neointima after oversized porcine coronary angioplasty.

BACKGROUND: Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective alpha(IIb)beta(3) antagonist (lamifiban), a selective alpha(v)beta(3) antagonist (VO514), and a combined alpha(IIb)beta(3)/alpha(v)beta(3) antagonist (G3580). METHODS AND RESULTS: In vitro, both alpha(v)beta(3) inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective alpha(IIb)beta(3) inhibition had no effect. Intravenous infusions of either alpha(IIb)beta(3) inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective alpha(v)beta(3) inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were administered for 14 days after oversized balloon angioplasty injury. After PTCA, there was regional upregulation of integrin alpha(v)beta(3) in the developing neointima, as assessed by immunohistochemistry. Six hours after PTCA, obstruction of lumen by thrombus was reduced significantly by alpha(IIb)beta(3) inhibition compared with either control or alpha(v)beta(3) inhibition (mean control, 18.7%; VO514, 18.5%; lamifiban, 6.4%; G3580, 7.9%). Twenty-eight days after PTCA, there was a significant reduction of neointima with inhibitors of either integrin (mean intima/media ratio: control, 3.08; VO514, 1.33; lamifiban, 0.97; G3580, 1.32). CONCLUSIONS: We conclude that both integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) participate in neointima development after experimental angioplasty.

Acetates↗

Flow cytometric analysis of DNA content and proliferation and immunohistochemical staining of Ki-67 in non-small cell lung cancer.

BACKGROUND: The aim of our study was to examine the significance of tumour DNA-content and proliferation in lung cancer. METHODS: The DNA content and S-phase fraction (SPF) was determined by flow cytometry in 125 resected tumours of patients with non-small cell lung cancer. In 40 cases we compared the SPF with immunohistochemical staining of the Ki-67 protein using MIB-1 antibody. RESULTS: DNA aneuploidy was detected in 84.8% (106/125). Cell cycle analysis for the determination of proliferation activity was only possible in 69 (55.2%) cases. An SPF of 0-8% as a sign of low proliferation was found in 27 specimens. In advanced tumours at stage III and IV the proportion of tumours with SPF 9-16% was significantly (p<0.05) increased as compared to tumours at stage I and II. There was a significant correlation (p=0.012, ascent: 0.045) between SPF and MIB-1. Patients with aneuploid tumours had a relative risk of 1.4 to die earlier than patients with diploid tumours. Patients with SPF of 9-16% in the tumour tended to decreased survival (5-year survival rate: 29%) in correlation to patients with a percentage of SPF 0-8% (5-year survival rate: 38%, p=0.5). These differences were significant (p=0.048) in patients with adenocarcinomas only. In the multivariate COX-regression model age (p=0.03) and stage (p=0.0001) were significant prognostic factors, ploidy state (p=0.33) was of no prognostic significance. CONCLUSIONS: Flow cytometry seems to be a useful method for understanding the clinical behaviour of lung cancer. Especially the SPF in adenocarcinomas may be used as a prognostic indicator.

Analysis of Variance↗

An antisense oligonucleotide targeting the alphaV integrin gene inhibits adhesion and induces apoptosis in breast cancer cells.

The aim of this study was to show the anti-adhesive potential of an antisense oligodeoxynucleotide (ODN) approach when designed to suppress the cellular function of the alphaV integrin subunit in breast cancer cells. The alphaV integrins play major roles in favouring breast cancer spreading. In this study, we inhibited alphaV subunit synthesis in the human breast carcinoma cell line, MDA-MB231, by a partially phosphorothioated antisense oligodeoxynucleotide (5543-ODN). The alphaV antisense 5543-ODN reduced alphaV, but not actin, mRNA transcription and protein expression by 55% and 65% respectively (1 microM, 72 h). Control sense and mismatch reagents were inactive. The antisense, but not the sense and mismatch, 5543-ODN induced dose- and time-dependent inhibition of MDA-MB231 adhesion to serum, vitronectin, fibrinogen and fibronectin substrates but was inactive on adhesion to laminin. Thus, the alphaV integrin was located in adhesion structures, which were disrupted by treatment with the alphaV antisense 5543-ODN. Antisense treated cells also showed evidence of programmed cell death with the appearance of apoptotic bodies. MDA-MB231 cells express a mutant form of the pro-apoptotic factor p53; however, no changes in the expression of p53 were observed by Western blotting. Immunofluorescence did reveal an increased nuclear translocation of p53 suggesting activation of the protein, but such a translocation did not lead to significant changes in either the expression of the cyclin dependent kinase inhibitor, p21(WAF1/CIP1) the cell survival factor Bcl-2 or the pro-apoptotic factor Bax.

Antigens, CD↗

Significance of proliferative activity and DNA ploidy in pancreatic cancer and chronic pancreatitis.

BACKGROUND: Precise preoperative assessment of diagnosis and prognosis in patients with pancreatic tumors would facilitate improvement of treatment strategies. In this context, we evaluated the significance of the proliferative index and of static DNA cytophotometry in the diagnosis and prognosis of pancreatic tumors. METHODS: Consecutive surgical specimens from 26 patients with ductal pancreatic cancers and eight patients with chronic pancreatitis were investigated by: 1. Staging; 2. Conventional histological and cytological grading; 3. MIB-1 (Ki-67 labeling) proliferating index; and 4. Static DNA cytophotometry. RESULTS: All patients with chronic pancreatitis had a normal MIB-1 labeling index and a euploid DNA content. In contrast, patients with pancreatic cancers rarely had a normal labeling index (1 of 26 patients) or a euploid DNA content (6 of 26 patients). Staging significantly correlated with survival time. However, it did not correlate with cytological criteria. Cytological criteria, such as conventional grading, MIB-1 proliferating index, and DNA ploidy, were not significantly correlated with survival time. Conventional grading was significantly correlated (p < 0.02) with proliferating index, but not with DNA ploidy. CONCLUSION: Proliferating index and DNA ploidy are relevant cytological markers that can help to discriminate between chronic pancreatitis and pancreatic cancer. The prognostic significance of these markers in pancreatic cancer patients, however, seems to be less relevant than tumor stage and of limited relevance for the individual cancer patient.

Adult↗

Malignant myoepithelioma of the vulva resembling a rhabdoid tumour.

AIMS: We report an example of malignant myoepithelioma of the vulva, which has not been hitherto described. We discuss the differential diagnosis and briefly review the literature. METHODS AND RESULTS: The lesion was found in an 81-year-old woman as an indolent 40 mm tumour. The neoplastic cells showed a myoid, spindled, epithelioid and plasmacytoid phenotype. Hyalinization of extracellular material and myxoid changes were present. There was a partly solid and microcystic pattern and a tight cohesiveness of cells was lacking. The circumscribed multinodular tumour somewhat resembled an extrarenal rhabdoid tumour, having large tumour cells with prominent nucleoli and large amounts of acidophilic cytoplasm. Immunohistochemically, the tumour cells were immunoreactive for cytokeratin, vimentin, muscle-specific actin, alpha-smooth muscle actin, and S100 protein, but not for desmin, epithelial membrane antigen, factor VIII-related antigen, CD34 and CD31. CONCLUSIONS: The histological and cytomorphological appearance of the tumour well as the immunohistochemical findings suggest the diagnosis of malignant myoepithelioma, possibly derived from minor vestibulary glands or ectopic breast tissue. Differential diagnoses are, in particular, extrarenal rhabdoid tumour and 'proximal type' epithelioid sarcoma. Differentiation is important, because the tumours show a different behaviour and prognosis.

Aged↗

Oligodeoxynucleotide targeted to the alphav gene inhibits alphav integrin synthesis, impairs osteoclast function, and activates intracellular signals to apoptosis.

The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Animals↗

Structure of HOE/BAY 793 complexed to human immunodeficiency virus (HIV-1) protease in two different crystal forms--structure/function relationship and influence of crystal packing.

Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C2-symmetric dimer, cleaving the gag and gag-pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C2-symmetrical inhibitors in the form of substrate-based transition-state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central unit [Budt, K.-H., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. & Winkler, I. (1990) European Patent application EP0428,849; Budt, K.-H., Hansen, J., Knolle, J., Meichsner, C., Ruppert, D., Paessens, A. & Stowasser B. (1993) IXth International Conference on AIDS; Budt, K.-H., Peyman, A., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. (1995) Bioorg. Med. Chem. 3, 559-571.] The structure of this inhibitor bound to HIV-1 protease, in two different crystal forms, has been solved at 0.24-nm resolution using X-ray crystallography. In both forms, the details of the inhibitor-protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active-site aspartates. The main binding interactions comprise several specific H-bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HIV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV-1 protease, and must be considered in the future design of drugs against HIV protease and in structure-based drug design in general.

Crystallography, X-Ray↗

HIV protease inhibitor HOE/BAY 793, structure-activity relationships in a series of C2-symmetric diols.

A detailed structure-activity relationship of C2-symmetric diol inhibitors of HIV-1 protease leads to inhibitor 6 (HOE/BAY 793) which is outstanding in the inhibition of the enzyme and in the inhibition of viral replication in HIV infected cell culture (IC50: 0.3 nM; EC50: 3 nM). There are well defined steric requirements for the design of the side chains P1-P3 of the inhibitors. In addition, all three side chains need to be lipophilic. While the enzyme tolerates hydrophilic substituents in some cases, drastic reductions in anti-HIV activity are observed in cell culture, most likely due to insufficient cell penetration.

Carbon↗

[Lipid island (xanthelasma) of the duodenal mucosa--a rarely found diagnosis and differential diagnosis].

A case report ist given on a 54-year-old patient with the so called lipid island (xanthelasma) in the duodenal mucosa. Xanthelasmas are very rarely located in the duodenal mucosa. The diagnosis and differential diagnosis of this entity are reported. The relevance of histochemistry and immunohistochemistry for differentiation of signet ring carcinoma cells in the mucosa is discussed.

Biopsy↗

[Shock and coagulation disorders in systemic mastocytosis].

A 41-year-old man was admitted in circulatory shock of unknown aetiology (systolic pressure 50 mm Hg) and marked reddening of the upper part of the body as well as maculopapular rash over the whole body. After 1500 ml of colloidal solution had been infused the blood pressure rose to a level at which the patient's condition was no longer at risk. He reported having had a similar attack of flushing and circulatory collapse during the last few years, each time after drinking 3-41 of beer. Laboratory tests showed thromboplastin time 56%, partial thromboplastin time 130 s and thrombin time > 180 s. Three hours after admission the coagulation times had further deteriorated, but had become normal within 20 hours. After rest and after a provocation (hot bath) the serum concentrations were: heparin 0.21 U/ml and 0.85 U/ml; histamine 1.9 micrograms/ml and 2.3 micrograms/ml; serotonin 23 micrograms/ml and 38 micrograms/ml. Histological examination of an iliac crest bone marrow biopsy revealed dense collections of mast cells, as seen in systemic mastocytosis. A skin biopsy was diagnostic of urticaria pigmentosa.

Adult↗

The adenosquamous lung carcinoma: clinical and pathological characteristics.

The adenosquamous carcinoma is a rare combined tumour of non-small cell lung cancer (NSCLC). The survival prognosis of surgically treated patients with adenosquamous carcinoma and patients with squamous cell carcinoma, large cell carcinoma or adenocarcinoma were compared during a study. Two hundred and seventyfive patients who had been treated surgically because of primary lung cancer in the Department of Thoracic-Surgery at the Martin-Luther-University Halle-Wittenberg between 1980 and 1989 were evaluated. The five year survival study of 172 patients who underwent resection because of squamous cell carcinoma was 45%, the one of patients with adenocarcinoma (n = 84) was 27%. 26% was the five year survival rate of the patients (n = 9) with large cell carcinoma. Of 13 patients (4%) with adenosquamous carcinoma none survived five years after surgical treatment. The two year survival rate was 28%. The presented results demonstrate the poor survival prognosis of patients suffering from adenosquamous carcinoma and ask for an adjuvant therapy.

Adenocarcinoma↗

Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies.

The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions. A 1000-fold molar excess of each of the unlabeled ligands quenched the specific labeling suggesting that the agonist and the antagonist compete for overlapping binding site(s). Covalent coupling of the receptor to bradykinin or HOE140, followed by Western blotting and immunoprinting with specific anti-ligand antibodies confirmed that the major ligand-binding form of the receptor is of 69 kDa. Anti-idiotypic antibodies which bear the internal image of bradykinin (Haasemann, M., Buschko, J., Faussner, A., Roscher, A.A., Hoebeke, J., Burch, R.M., and Müller-Esterl, W. (1991) J. Immunol. 147, 3882-3892) immunoprecipitated the 125I-labeled receptor as a major band of 68 kDa and a minor band of 47 kDa indicative of partial proteolysis. Chemical deglycosylation of the 125I-labeled receptor shifted the apparent molecular mass from 69 to 44 kDa demonstrating that the receptor is heavily glycosylated. Two-dimensional electrophoresis of the affinity-purified receptor revealed overlapping spots of 69 kDa and of pI 6.8-7.1 pointing to a microheterogeneity of the carbohydrate moiety. Elucidation of the key structural features of the B2 receptor protein will aid in understanding the structure-function relationships governing this prototypic peptide receptor.

Amino Acid Sequence↗

Integrin and Arg-Gly-Asp dependence of cell adhesion to the native and unfolded triple helix of collagen type VI.

Pepsin-solubilized collagen VI in triple-helical and heat-denatured, unfolded form was shown to promote Mg(2+)- and Mn(2+)-dependent attachment and spreading of various cell lines. On the triple-helical substrate no inhibition of cell adhesion was observed with several synthetic RGD peptides except in the case of A375 melanoma cells. In contrast, adhesion to the unfolded substrate was highly sensitive to RGD inhibition. Nine synthetic peptides were designed according to 10 RGD sequences present in the triple-helical sequence of human collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. Only one peptide, corresponding to the C-terminal end of alpha 3(VI) chain, showed substantial inhibitory activity, whereas several peptides were active in direct adhesion assays when used as albumin conjugates. Inhibition tests with antibodies to integrin subunits, affinity chromatography, and ligand binding with purified integrins (alpha 1 beta 1, alpha 2 beta 1, alpha V beta 3, and alpha IIb beta 3) were used to identify collagen VI receptors. Binding to the triple-helical substrate is mediated by alpha 1 beta 1 and alpha 2 beta 1 integrins. Binding of both integrins to collagen VI was weaker than that to collagens I and/or IV. Recognition of the denatured substrate is mediated by beta 1 and beta 3 integrins. Activity was shown for alpha 5 beta 1 and alpha V beta 3 and weakly for alpha IIb beta 3 but not all alpha subunits possibly involved were identified. Distinct sets of receptors were also involved in A375 cell binding to triple-helical (beta 1-mediated) and denatured (beta 3-mediated) collagen VI, even though in this case both interactions could be efficiently inhibited by RGD peptides.

Amino Acid Sequence↗