Complementary hydropathy identifies a cellular prion protein receptor.

Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.

TP53 PIN3 and MDM2 SNP309 polymorphisms as genetic modifiers in the Li-Fraumeni syndrome: impact on age at first diagnosis.

BACKGROUND: Li-Fraumeni and Li-Fraumeni-like syndromes (LFS/LFL), characterised by the development of multiple early onset cancers with heterogeneous tumour patterns, are associated with germline TP53 mutations. Polymorphisms in the TP53 pathway (TP53 PEX4 at codon 72, rs1042522; MDM2 SNP309, rs2279744) have modifier effects on germline TP53 mutations that may account for the individual and familial diversity of tumour patterns. METHODS AND RESULTS: Four polymorphisms were analysed in a series of 135 Brazilian LFS/LFL cancer patients (32 TP53 mutation carriers and 103 wild-type subjects). We report for the first time that another polymorphism in the TP53 gene, TP53 PIN3 (rs17878362), has a strong modifier effect on germline TP53 mutations. This polymorphism, which consists of a 16 bp duplication in intron 3 (A1, non-duplicated allele; A2, duplicated allele), is associated with a difference of 19.0 years in the mean age at the first diagnosis in TP53 mutation carriers (n = 25, A1A1: 28.0 years; n = 7, A1A2: 47.0 years; p = 0.01). In addition, cancer occurrence before the age of 35 years is exclusively observed in A1A1 homozygotes. In this series, the effect of TP53 PEX4 and MDM2 SNP309 on age at diagnosis was similar to the one reported in other series and was smaller than the one of TP53 PIN3 (TP53 PIN3: difference of 19.0 years; TP53 PEX4: 8.3 years; MDM2 SNP309: 12.5 years). CONCLUSION: These results suggest that TP53 PIN3 is another polymorphism in the TP53 pathway that may have a modifier effect on germline TP53 mutations and may contribute to the phenotypic diversity of germline TP53 mutations associated with LFS/LFL patients.

Presence of laminin receptors in Staphylococcus aureus.

A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state.

EJ-ras oncogene-induced malignant transformation is characterized by a series of changes in cell surface carbohydrates and cell-cell and cell-matrix interactions. Here, we show that EJ-ras-transformed NIH-3T3 fibroblasts acquired a migratory phenotype on laminin-1 surfaces. Such a phenotype was accompanied by overexpression of: (a) functional alpha6beta1, but not other laminin binding beta1-integrins; and (b) glycoconjugates on the cell surface bearing large oligosaccharides recognized by leukoagglutinin from Phaseolus vulgaris (L-PHA). The internal pool of pre-beta1-integrins was differently regulated in EJ-ras-transformed cells compared with nontransfected fibroblasts. Conversion of pre-beta1- into mature beta1-integrins was faster in EJ-ras-transformed cells, a process associated with the overexpression of the alpha6-chain. Overexpression of L-PHA-reactive oligosaccharides is dependent on the activity of N-acetylglucosaminyltransferase V, which is increased in transformed cells [J. W. Dennis et al., Science (Washington DC), 236: 582-585, 1987]. We show that beta1-integrins were the major carriers of L-PHA-reactive oligosaccharides on the cell surface. This glycosylation pattern, however, was not necessary for either the cell surface expression of beta1-integrins or their functional activity in the migratory response to laminin-1. Moreover, EJ-ras-transformed fibroblasts aggregated spontaneously. These effects were not observed in c-jun-transfected fibroblasts, which were unable to migrate on laminin, did not overexpress either beta1-integrins or L-PHA-reactive oligosaccharides, and did not self-aggregate.

Independent prognostic value of laminin receptor expression in breast cancer survival.

We studied the possible prognostic role of laminin, estrogen (ER), and progesterone (PR) receptors and other pathological factors in relation to the disease-free interval and overall survival of female breast carcinoma patients. Multivariate analyses of clinical and pathological data with respect to the above survival time variables were performed by Cox regression. The statistical dependence of prognosis on ER, PR, and tumor size was based on the discriminant cutoff value that could best distinguish between survival curves. Axillary nodal status was the most significant independent factor in the prediction of both disease-free interval and overall survival of these patients. Use of the information on laminin receptor expression, PR concentration, tumor size, lymphocytic infiltrate, and tumor necrosis improved significantly the prediction of the risk of recurrence. Patients with tumors expressing laminin receptors had 40% less risk of recurrence (P = 0.0209) than those with no expression. On the other hand, four covariates were independently predictive of the risk of death: axillary nodal status, lymphocytic infiltrate, PR and ER concentration. There was a marginally significant (10% level) interaction between tumor size and lymphocytic infiltrate with respect to the prediction of the risk of recurrence. The above sets of variables were used to classify patients into risk groups for the prediction of recurrence and death.

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein.

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.

Imbalance of antioxidant defense in mice lacking cellular prion protein.

Prion diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from its normal cellular isoform, PrPC, to the infectious scrapie isoform, PrP(Sc). In spite of many studies, the physiological function of PrPC remains unknown. Recent work shows that PrPC binds Cu2+, internalizing it into the cytoplasm. Since many antioxidant enzymes depend on Cu2+ (e.g., Cu/ZnSOD), their function could be affected in prion diseases. Here we investigate a possible relationship between PrP(C) and the cellular antioxidant systems in different structures isolated from PrPC knockout and wild-type mice by determining oxidative damage in protein and lipids and activity of antioxidant enzymes (CAT, SOD) and stress-adaptive enzymes (ODC). Our results show that, in the absence of PrPC, there is an increased oxidation of lipid and protein in all structures investigated. Decreased SOD activity and changes in CAT/ODC activities were also observed. Taking into account these results, we suggest that the physiological function of PrP(C) is related to cellular antioxidant defenses. Therefore, during development of prion diseases, the whole organism becomes more sensitive to ROS injury, leading to a progressive oxidative disruption of tissues and vital organs, especially the central nervous system.

Loss of heterozygosity affecting the APC and MCC genetic loci in patients with primary breast carcinomas.

Tumor suppressor genes APC and MCC were identified recently, and their chromosomal location was ascribed to chromosome 5q21. Mutations in the APC gene give rise to familial adenomatous polyposis and occur in many perhaps even the majority, of sporadic colon cancers. Loss of heterozygosity has been described in other human tumors such as lung and esophageal cancers. Here we show loss of heterozygosity (LOH) in 87 patients with breast cancer for the APC and/or MCC loci using a polymerase chain reaction-LOH assay. LOH affected loci in APC exons 11 and 15 in 9 of 35 (25%) and 4 of 34 (11%) heterozygous patients, respectively. LOH at the MCC exon 10 locus occurred in 7 of 40 (17%) informative samples. These data suggest that allelic deletion of APC and/or MCC is probably involved in the pathogenesis and/or progression of a subset of breast cancers.

Are synthetic peptides suitable for the detection of continuous epitopes only?

Present-day methods for the definition of antibody binding sites on antigenic polypeptides show a strong bias toward identification of sequential epitopes. We have employed anti-sequence monoclonal antibodies raised against short synthetic peptides to screen a random-primed cDNA library constructed in an expression vector. Sequence data analysis performed on three clones thus isolated showed that the clones did not cross-hybridize and that the antigenic peptide sequence was found in none of them. Our findings suggest therefore that sequential epitopes may be preferentially identified because of an inherent bias in commonly used epitope mapping protocols which masks available conformational determinants.

Alternative spliced transcripts as cancer markers.

Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.

Upregulation of collagen messenger RNA expression occurs immediately after lung damage.

BACKGROUND: Mortality of ARDS still exceeds 50%. Though pulmonary fibrosis is a marker of severe prognosis in the evolution of ARDS, its onset is not yet established. Cardiopulmonary bypass (CPB), usually utilized in patients with a previously normal lung, can cause ARDS and often causes alveolar damage, the earliest lesion observed in ARDS, thus providing a unique opportunity to study the molecular mechanisms of fibrogenesis. OBJECTIVE: To measure immediately after CPB, at the onset of alveolar damage, the expression of messenger RNAs (mRNAs) for collagen type I. METHODS: Pre-CPB and post-CPB lung biopsy specimens were obtained from patients submitted to myocardial revascularization for coronary artery disease. Alveolar damage was characterized by comparison between before and after specimens and quantified by point counting of polymorphonuclear cells (PMN). Type I collagen mRNAs were quantified by scanning densitometry of Northern blot autoradiographs, corrected for RNA loading by 18S ribosomal RNA hybridization. RESULTS: Alveolar damage was characterized by lung interstitial edema and by polymorphonuclear cell infiltration after CPB (PMN pre-CPB 0.010+/-0.004xPMN post-CPB 0.052+/-0.022; n=7; p=0.0017, t test). Type I collagen mRNA increased 91.1+/-68.2% (Ln pre-CPBxLn post-CPB; n=15; p<0.00001, t test) immediately after CPB (mean CPB time, 108.8+/-37.2 min). CONCLUSION: Fibrogenesis, as measured at the molecular level, is a very early event following diffuse alveolar damage, attributable mainly to resident fibroblast activation.

Normal inhibitory avoidance learning and anxiety, but increased locomotor activity in mice devoid of PrP(C).

Prions are the causative agents of transmissible spongiform encephalopathies. The transmissible agent (PrP(Sc)) is an abnormal form of PrP(C), a normal neuronal protein. The physiological role of PrP(C) remains unclear. In the present report, we evaluated behavioral parameters in Prnp(0/0) mice devoid of PrP(C). Prnp(0/0) mice showed normal short- and long-term retention of a step-down inhibitory avoidance task and normal behavior in an elevated plus maze test of anxiety. During a 5-min exploration of an open field, Prnp(0/0) mice showed normal number of rearings, defecation, and latency to initiate locomotion, but a significant increase in the number of crossings. The results suggest that Prnp(0/0) mice show normal fear-motivated memory, anxiety and exploratory behavior, and a slight increase in locomotor activity during exploration of a novel environment.

Cellular prion protein binds laminin and mediates neuritogenesis.

Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.

Changes in cortical and hippocampal ectonucleotidase activities in mice lacking cellular prion protein.

Animals lacking cellular prion protein (PrP(c)) expression are more susceptible to seizures. Adenosine is an endogenous anticonvulsant agent and it levels in the synaptic cleft are regulated by ectonucleotidases. We evaluated ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult PrP(c) null mice and wild-type mice (genetic background 129/Sv X C57BL/6J). There was an increase (47%) in adenosine triphosphate (ATP) hydrolysis in hippocampal synaptosomes of PrP(c) knockout mice as compared with the wild-type animals. In cortical synaptosomes, ATP hydrolysis was similar in both PrP(c) mice and controls. However, there was a significant decrease in adenosine diphosphate (ADP) hydrolysis in both hippocampal (-39%) and cortical (-25%) synaptosomes in PrP(c) null animals compared to wild-type mice. Changes in brain ectonucleotidases activities related to modifications in the PrP(c) expression may contribute, at least in part, to the higher sensitivity to seizures of PrP(c) null mice.

Oligosaccharide residues of Loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity.

Loxosceles spp. (brown spider) envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravascular coagulation in some cases. Using lectin-immunolabeling, lectin-affinity chromatography, glycosidase and proteinase K treatments we were able to identify several venom N-glycosylated proteins with high-mannose oligosaccharide structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminoglycan residues. Working with enzymatically or chemically deglycosylated venom we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the venom were sugar-independent when compared to glycosylated venom. Nevertheless, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high-mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reduction of approximately 2 kDa in its molecular weight and a reduction of the gelatinolytic effect to a residual activity of 28% when compared to the glycosylated molecule, indicating a post-translational glycosylation-dependent gelatinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity when compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of brown spider venom and opens the possibility for a carbohydrate-based therapy.

Detection and characterization of metalloproteinases with gelatinolytic, fibronectinolytic and fibrinogenolytic activities in brown spider (Loxosceles intermedia) venom.

By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inability to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving action whereas serine-protease inhibitors, thiol-protease inhibitor and acid-protease inhibitor showed little or no effect on the proteolytic activity of the venom indicating involvement of a metalloproteinase. Zymogram analysis of venom detected a 35 kDa molecule with gelatinolytic activity. The metalloproteinase nature was further supported by its sensitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decreased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose affinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogenolytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,10 phenanthroline and APMA sensitivity on this proteolytic effect confirmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. intermedia venom with different specificities one gelatinolytic and another, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom.

TP53 mutations in upper aerodigestive squamous cell carcinomas from a group of Brazilian patients.

BACKGROUND: Loss of function of the tumor suppressor gene TP53 contributes to the development of several tumors. PATIENTS AND METHODS: We screened DNA samples from 47 patients with upper respiratory system squamous cell carcinomas for the presence of TP53 mutations. Exons 4 to 8 of the TP53 gene were amplified by the polymerase chain reaction, and mutations were identified by single-strand conformation polymorphism analysis. RESULTS: The TP53 mutations were demonstrated in 23 cases (49%). Mutations were distributed as follows: exon 4, 5 cases; exon 5, 4 cases; exon 6, 6 cases; exon 7, 4 cases; and exon 8, 4 cases. Demographic variables, tumor site, stage, family history of cancer, and tobacco smoking were not predictors of TP53 mutations. There was an increasing number of mutations in the more undifferentiated tumors (P = 0.0594). CONCLUSIONS: These findings suggest that TP53 mutations are associated with tumor differentiation, but not with the risk of lymph node metastasis in the group of patients analyzed.

Participation of glycosylation sites in the binding of Staphylococcus aureus to laminin.

1. Microbial pathogenicity is in many instances associated with the ability to adhere to host surfaces or to extracellular matrix components. 2. Laminin is a major glycoprotein of basement membranes which can promote specific bacterial adhesion. Staphylococcus aureus is a pathogenic bacterium which presents a laminin receptor of about 50-kDa molecular mass (Lopes JD, Reis M & Brentani RR (1985). Science, 229: 275-277). 3. Adhesion inhibition assays of [125iodine]-labeled bacteria to laminin demonstrate that the receptor binding site is contained in the pepsin-derived (P1) laminin fragment. 4. Cell adhesion to laminin is unaffected by periodate oxidation of sugars on the surface of bacteria or by removal of divalent cations by ethylenediaminetetraacetic acid (EDTA). In contrast, bacterial adhesion is reduced when laminin is deglycosylated with N-glycosidase F or when bacteria are submitted to controlled trypsin digestion. 5. Laminin binding to the S. aureus 50-kDa band in immunoblotting assays has confirmed all of these results obtained in cell adhesion experiments.

Biology of metastasis.

1. Metastasis, i.e., dissemination of tumor cells throughout the organism, is the cause of death for most cancer patients. We discuss here the role of the interaction between basement membrane laminin and specific cell receptors in the process. We further show that such interaction is mediated by receptors not only in cancer cells but also in normal granulocytes, in a parasitic protozoan and even in an invasive prokaryote. 2. Metastatic potential has been correlated with the number of receptors/cell and preliminary results with a monoclonal antibody against the S. aureus laminin receptor suggest that the binding site at the receptor level might be conserved. This conclusion is supported by the facts that this monoclonal antibody can recognize the eukaryotic receptor and inhibit laminin binding.

Cellular adhesion to laminin involves a chondroitin sulfate proteoglycan.

Cell-extracellular matrix interactions are intimately involved in the regulation of many cellular processes such as embryonic development or tumor cell growth and metastasis. In our previous work we were able to detect a 90/100-kDa laminin binding chondroitin sulfate proteoglycan. A search for this molecule in different cell lines showed that it is only found in cells that adhere to laminin.