Vitronectin and its receptors.

The extracellular matrix protein vitronectin is recognized as an adhesive substrate by cells expressing at least one of four known vitronectin receptors: integrins alpha v beta 1, alpha v beta 3, alpha v beta 5 or alpha IIb beta 3. Cell interaction with vitronectin may induce spreading and migration and have an effect on cell growth and differentiation in specific processes, such as tumor growth and metastasis, wound healing, bone resorption and viral infection.

Comparison of in vitro and in vivo approaches to studying brain colonization by breast cancer cells.

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

[Total knee replacement in haemophilic arthropathy. A clinical and radiological evaluation of 30 patients]. / Kniegelenksendoprothetik bei hämophiler Arthropathie. Klinische und radiologische Verlaufskontrolle mit 30 Patienten.

UNLABELLED: Purpose of this retrospective study was to evaluate our own results after total knee replacement in patients with haemophilia. Patients, material, method: 30 patients with haemophilia who underwent total knee replacement between 1987 and 2005 were included. We used the clinical and radiological Knee Society Score. Furthermore, the Petterson and the Arnold and Hilgartner score were applied. RESULTS: The mean age at the time of surgery was 43.2 (27-66). At the time of follow-up examination the mean age was 51.6 (30-82) years. The mean follow-up was 7.1 (2-20) years. Preoperative, he mean Arnold and Hilgartner score was 4.17 (±0.59) and the mean Petterson-Score was 9±2.29. Compared to the preoperative deficiency in knee function (KSS-Score 88.17±33.58) an improvement with 166.67 (±22.73) points was seen. 1 patient showed an aseptic loosening after 11 years. DISCUSSION: Total knee replacement in patients with haemophilia improves knee function and quality of life. The results of our study represent results in earlier published studies. Compared to a non-haemophilic normal population the rate of perioperative complications was not increased.

Stu2 promotes mitotic spindle elongation in anaphase.

During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins.

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction between L1-Ig6 and alphaIIbbeta3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3 > alphavbeta1 > alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Involvement of integrin alpha V gene expression in human melanoma tumorigenicity.

Human melanoma originates in the skin and can lead to wide-spread metastatic disease. Analysis of melanoma biopsy material has shown that the vitronectin receptor, integrin alpha v beta 3, is a specific marker of the most malignant cells, i.e., vertically invasive primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymic nude mice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo.

Melanoma-associated gangliosides in the fish genus Xiphophorus.

Gangliosides from benign and malignant melanomas and from normal skin of the fish genus Xiphophorus were isolated and analyzed by thin-layer chromatography. Individual ganglioside components were characterized by mapping according to their sialic acid content and by cleavage with neuraminidases. In all three tissues examined, sulfatide and the gangliosides NeuAc-GalCer (GM4), II3NeuAc-LacCer (GM3), II3NeuAc-GgOse3Cer (GM2), and II3(NeuAc)2-LacCer (GD3) were found. Ganglioside GD3 yielded a positive reaction, following immunoadsorption with mouse monoclonal antibody R24 on thin-layer plates. Two alkali-labile disialoganglioside species were specifically recognized by mouse monoclonal antibody D1.1, thus indicating the presence of O-acetyl-neuraminic acid residues. One of them, a major ganglioside component of the malignant melanoma, was identified as O-acetyl-GD3, since it could be converted to the R24-positive GD3 ganglioside after alkaline saponification. The other one appears to be restricted to the malignant tumor and represents a novel melanoma-associated ganglioside derivative. It was characterized as O-acetyl(NeuAc)2-nLc4Cer by exoglycosidase cleavage, by proving its neutral carbohydrate backbone as type II-chain lacto-series oligosaccharide using mouse monoclonal antibody 1B2, and by its cross-reaction with antibody R24 following alkaline treatment. Using antibody R24 and cryopreserved tissue sections of both benign and malignant amelanotic melanomas from albino fishes, it was demonstrated that one of the main melanoma-associated gangliosides, GD3, was exposed predominantly in the malignant tumor. Thus, the chemical nature and even the immunohistochemical localization of the gangliosides in fish melanomas proved to be very similar to those of the known gangliosides in the phylogenetically distant human melanomas.

Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions.

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

ADP-ribosylation and de-ADP-ribosylation of the rho protein by Clostridium botulinum exoenzyme C3. Regulation by EDTA, guanine nucleotides and pH.

Pretreatment of rho protein purified from pig brain cytosol with EDTA (3 mM) for 10 min at 30 degrees C inhibited its ADP-ribosylation by Clostridium botulinum C3 ADP-ribosyltransferase by more than 90%. The EDTA effect was not caused by alteration of C3. GDP or GDP beta S present during the pretreatment period completely prevented the decrease in ADP-ribosylation with half-maximal and maximal effects at 3 and 300 microM, respectively. GTP or GTP gamma S were less efficacious in preventing the decrease in ADP-ribosylation, but were more potent (half-maximal and maximal effects at 0.1 and 3 microM, respectively). [32P]ADP-ribose incorporated in pig brain rho by C3 was de-ADP-ribosylated by the enzyme in the presence of nicotinamide and at low pH. Concomitantly, [32P]NAD was formed. The pH optima for ADP-ribosylation and de-ADP-ribosylation were pH 7.5 and 5.5, respectively. De-ADP-ribosylation was most efficient with nicotinamide, less effective with 3-acetylpyridine and not observed with 3-aminopyridine, 4-aminopyridine, 4-acetylpyridine and isonicotinic acid. As observed for the ADP-ribosylation, the de-ADP-ribosylation by C3 was maximal with the GDP-bound form of rho and blocked after EDTA treatment.

Merging and scoring molecular interactions utilising existing community standards: tools, use-cases and a case study.

The evidence that two molecules interact in a living cell is often inferred from multiple different experiments. Experimental data is captured in multiple repositories, but there is no simple way to assess the evidence of an interaction occurring in a cellular environment. Merging and scoring of data are commonly required operations after querying for the details of specific molecular interactions, to remove redundancy and assess the strength of accompanying experimental evidence. We have developed both a merging algorithm and a scoring system for molecular interactions based on the proteomics standard initiative-molecular interaction standards. In this manuscript, we introduce these two algorithms and provide community access to the tool suite, describe examples of how these tools are useful to selectively present molecular interaction data and demonstrate a case where the algorithms were successfully used to identify a systematic error in an existing dataset.

Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product.

The 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 was purified from porcine brain cytosol by acetone precipitation, CM-Sephadex, octyl-Sepharose and TSK phenyl-5PW HPLC chromatography to apparent homogeneity. ADP-ribosylation of the protein was increased by guanine nucleotides (GTP, GDP, GTP gamma S, each 100 microM) but not by GMP, ATP or ATP gamma S. The purified 22 kDa protein bound maximally 0.9 mol [35S]GTP gamma S and hydrolyzed GTP with the rate 0.007 mol per mol protein. Amino acid sequences were obtained from two tryptic peptides, selected from an in situ digestion of Immobilon electrotransferred, gel purified ADP-ribosylated substrate. The two sequences obtained, cover 23 residues from the corresponding sequences in human rho.

ADP-ribosylation of Rho proteins inhibits sperm motility.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

Enhancement of Clostridium botulinum C3-catalysed ADP-ribosylation of recombinant rhoA by sodium dodecyl sulfate.

The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by Clostridium botulinum C3 exoenzyme (C3) was studied. SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic proteins maximally at 0.01% whereas higher concentrations of the detergent (> 0.01%) inhibited the ADP-ribosylation. In contrast, ADP-ribosylation of human platelet membranes and of recombinant rhoB was inhibited by the detergent. The Km for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 microM. Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affected by the amphiphilic wasp venom mastoparan, in the presence of SDS (0.01%) mastoparan (100 microM) inhibited the ADP-ribosylation. C3-associated NAD-glycohydrolase activity was maximally and half-maximally inhibited by 0.1 and 0.013% SDS, respectively. Inhibition of NAD-glycohydrolase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment. SDS (0.01%) completely inhibited the [3H]GTP binding of rhoA whereas the release of previously bound nucleotide was not affected. The data indicate that changes in the lipophilicity of rhoA protein largely affect its ability to serve as a substrate for C3-like ADP-ribosyltransferases.

ADP-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and compound 48/80.

The amphiphilic agents melittin, mast cell degranulating peptide and compound 48/80 inhibit the ADP-ribosylation of the small GTP-binding proteins rho by Clostridium botulinum exoenzyme C3. Half-maximal and maximal inhibition (greater than 90%) of ADP-ribosylation occurred at about 8 and 25 micrograms/ml for compound 48/80, at 10 and 45 microM for mast cell degranulating peptide and at 15 and 50 microM for melittin, respectively. In addition, these compounds increase the steady state GTP hydrolysis and the association and dissociation rate of GTP-binding of rho proteins through an increase of GDP/GTP exchange. The data suggest that the amphiphilic agents tested interact with small GTP-binding proteins of the rho protein family.

Rethinking physiologic stability: touch and intracranial pressure.

The purpose of this research was to examine the contingent nature of physiologic stability with respect to the impact of nursing and parental care touch on intracranial pressure (ICP) in children. Data were reanalyzed from those previously collected in eight children in a pediatric intensive care unit who had intracranial hypertension from a variety of causes and whose ICP was invasively monitored. One hundred forty-nine clusters of spontaneous touch/talking were available for analysis after those occurring close in time to procedures and drugs affecting ICP were dropped. Twenty-three episodes of investigator touch (without talking) were also analyzed. ICP stability was defined as any tracing over a defined time period in which the peak-to-trough amplitude did not exceed twice the calculated resting variability. Such an approach allowed classification and counting of stable versus unstable baselines, and stable versus unstable responses to touch. Therefore, ICP stability was examined by comparing the stability of the ICP tracing the last minute prior to a cluster of nonprocedural touch (baseline) with the first minute after the cluster. Clusters of spontaneous touch were nearly always associated with talking to the child and rarely were followed by change in level of ICP greater than that child's ICP variability at rest. Investigator stroking without talking never was followed by a significant change in level of ICP. There was a contingent relationship between stability of the ICP tracing prior to a cluster of touching/talking such that the probability of ICP becoming more stable when the touch/talk occurred on an unstable baseline was twice that of touch/talk occurring on an stable baseline.

Day-to-day demands of Parkinson's disease.

This interpretive study explored the experience of having Parkinson's disease in middle life. The researcher examined the day-to-day demands experienced by 16 people (9 men and 7 women), ages 42-59 years, who had been diagnosed with Parkinson's disease for a range of 1-16 years. Demands directly attributable to the illness included acknowledging symptoms and seeking help, balancing emotional responses, dealing with a changing body, gaining formal and practical knowledge, and dealing with unpredictability. The illness also created demands related to changing roles, sense of identity, and relationships. This study advances understanding of the difficulties and challenges experienced by those who live with Parkinson's disease and has implications for nursing practice with this population.

Continuity challenges of Parkinson's disease in middle life.

This interpretive study explored the experience of having Parkinson's disease (PD) in middle life. The researcher examined how persons with PD (N = 16) coped with challenges to their sense of self and a future of limited horizons. Participants were ages 42-59 years (mean age = 48 years) and had been diagnosed for a range of 1-16 years. In-depth interviews, focusing on day to day experiences, coping episodes and future hopes and concerns, were conducted on 3 occasions. Findings indicated that participants maintained an intact identity by sustaining a sense of continuity with their pre-Parkinson's self. They dealt with the progressive nature of their illness of by keeping open a range of possibilities for the future. This study advances understanding of the challenges experienced by those with PD and suggests ways in which nurses may be able to be more effective in supporting individuals who are living with this disease.