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Biomedical subjects

M De Luca

Publications and source records attributed to M De Luca.

At least 91 records · Page 5Linked to original sources
PubMed1996

n-3 fatty acids do not enhance LDL susceptibility to oxidation in hypertriacylglycerolemic hemodialyzed subjects.

Recent data suggest that treatment with n-3 fatty acids could enhance the susceptibility of plasma low-density-lipoprotein (LDL) to oxidation. Twelve hypertriacylglycerolemic, hemodialyzed patients were treated with 2.5 g n-3 fatty acids/d for 2 mo. Treatment was then withdrawn for 2 mo (washout phase). Plasma total cholesterol and LDL cholesterol increased significantly (9% and 28%) and plasma triacylglycerols decreased significantly after the n-3 phase compared with baseline and washout values. LDL susceptibility to oxidation was tested by oxidation of LDL particles with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). No significant changes were observed for the lag phase and the peroxidation rate. The vitamin E content of LDL also did not change significantly. The results thus suggest that a daily dosage of 2.5 g n-3 fatty acids does not enhance LDL susceptibility to oxidation, while retaining its hypotriacylglycerolemic effect.

Amidines↗
PubMed1996

Resistance to alpha interferon therapy in HCV chronic liver disease: role of hepatic fibrosis.

The response rate to interferon in HCV chronic liver disease is insufficient to date and the causes of this failure are not fully understood. Hepatic fibrosis hinders the blood-hepatocyte exchange of substances and we hypothesized that this process may also reduce the efficacy of interferon. Serum levels of connective tissue metabolites are related, to some extent, to the amount of extracellular matrix in the liver. Therefore, the usefulness was evaluated of serum tests of connective tissue metabolism compared to standard biochemical and histological parameters in predicting the probability of primary response to interferon. Sixty-eight patients with HCV chronic liver disease were treated with alpha-interferon for 1 year. At multivariate analysis time 0, the serum level of the P1 fragment of laminin was found to be the only factor independently associated with the response to treatment. As is well known, higher serum concentrations of the P1 fragment of laminin are associated with active basement membrane turnover and derangement of the hepatic structure. Therefore, this process seems to reduce the probability of response to interferon and, if confirmed, evaluation of serum the P1 fragment of laminin may be a useful test to predict the response to interferon and to define the therapeutic strategy, especially as far as the dose of interferon is concerned.

Antiviral Agents↗
PubMed1996

Allele frequency distributions at seven DNA hypervariable loci in a population sample from Calabria (southern Italy).

Genotype and allele frequencies at seven Variable Number of Tandem Repeats (VNTR) loci currently used for forensic purposes have been estimated in a population sample from Calabria (south Italy). DNA target regions relevant to four microsatellites (THO.1; REN.4; D12S67; DYS19) and three minisatellites (D1S80; 3'APOB; TPO.10) were amplified by Polymerase Chain Reaction (PCR) and analysed by electrophoresis and ethidium bromide or silver staining. For all loci, the observed genotypes were found to be in agreement with those expected by the Hardy-Weinberg equilibrium. Data on allele frequencies were in line with those found in sample groups from northern or central Italy, tested for some of the above polymorphisms.

Adolescent↗
PubMed1995

A novel cobra venom metalloproteinase, mocarhagin, cleaves a 10-amino acid peptide from the mature N terminus of P-selectin glycoprotein ligand receptor, PSGL-1, and abolishes P-selectin binding.

Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDY decreases DFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.

Amino Acid Sequence↗
PubMed1995

Jararhagin and jaracetin: novel snake venom inhibitors of the integrin collagen receptor, alpha 2 beta 1.

Two novel proteins, jararhagin and jaracetin, were purified from Bothrops jararaca viper venom. Jararhagin is a 55-kDa member of the metalloprotease-disintegrin family. Jaracetin is a 60-kDa dimer representing a differently processed form of jararhagin. Like botrocetin, a previously described viper venom protein, jararhagin and jaracetin modulated binding of von Willebrand Factor to the glycoprotein Ib-IX complex on platelets through a specific interaction with the von Willebrand Factor A1 domain. Both jararhagin and jaracetin, but not botrocetin, also blocked alpha 2 beta 1-dependent platelet adhesion to collagen, a receptor interaction mediated through a homologous A domain on the integrin alpha 2 subunit.

Animals↗
PubMed1995

Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing.

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.

3T3 Cells↗
PubMed1995

HIV-1 spreads from lymphocytes to normal human keratinocytes suitable for autologous and allogenic transplantation.

Normal human keratinocytes can reconstitute in vitro cohesive sheets of epithelium suitable for grafting onto patients. Despite the widespread use of autografts and allografts, no data are yet available on productive infection by human immunodeficiency virus (HIV-1) of human keratinocytes. To address this point, we challenged keratinocytes at the second passage of culture with HTLV-IIIB virus by cell-free and cell-mediated inoculum. Viral entry was not achieved by cell-free inoculum, thus demonstrating that cultured keratinocytes do not provide the membrane requirements for viral binding and/or internalization. By contrast, the cell-mediated inoculum overcame specific receptor constraints, leading to viral integration and productive infection. The p24gag viral protein was transiently released in the culture supernatant, although at low level. The viral progeny produced by infected keratinocytes was rescued and amplified by co-culture experiments performed with the HIV-1 high sensitive CEM-SS human T-cell line. Viral integration, p24gag production, and secondary transmission to lymphoid cells was further confirmed with keratinocytes infected at the fourth passage of culture. Taken together, our results demonstrate that cultured keratinocytes can be infected by HTLV-IIIB virus, which can be maintained in semi-latent form for several passages after inoculum and rescued to full replication by a proper target. The in vitro demonstration of lympho-epithelial HIV-1 spreadings warns against the use of inappropriately screened biopsies for the preparation of skin grafts.

Acquired Immunodeficiency Syndrome↗
PubMed1995

The molecular mechanism of platelet adhesion.

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which vWF or the GP Ib-V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of vWF by the GP Ib-V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.

Animals↗
PubMed1995

Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity.

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

14-3-3 Proteins↗
PubMed1995

DYS19, D12S67, and D1S80 polymorphisms in population samples from southern Italy and Greece.

Genotype and allele frequencies of the DYS19, D12S67, and D1S80 highly polymorphic loci were determined in population samples from southern Italy (103 subjects) and Greece (84 subjects) using the amplified fragment length polymorphism (AFLP) technique (polymerase chain reaction followed by native PAGE and silver staining). Five, eleven, and eighteen alleles were found at the DYS19, D12S67, and D1S80 loci, respectively. PIC values ranged from 0.55 (DYS19 locus in Italians) to 0.79 (D12S67 locus in Italians). The distribution of D12S67 and D1S80 genotypes conformed to Hardy-Weinberg equilibrium, as confirmed by three statistics. Heterogeneity G tests, carried out on allele frequency distributions, showed a significant difference between the samples at the DYS19 locus, whereas no difference was found with regard to the other polymorphisms. Using data from the literature, we widened the comparison to other European groups analyzed for the same markers. All the polymorphisms were found to distinguish between populations of the same main ethnic group. In particular, D1S80 allele frequencies distinguished the Finns from other European groups (Spanish, German, Italian, and Greek samples). The reduced assay time, the high polymorphism level, and the ability to distinguish between populations indicate that these markers have potential value in population genetic studies.

Alleles↗
PubMed1995

Recombinant tissue plasminogen activator (rt-PA) as first-line therapy for declotting of haemodialysis access.

One of the most common complication in haemodialysis patients is thrombosis of the arteriovenous fistula (AVF). Thirty-five patients with a total of 42 thromboses of the angioaccess were infused via a small needle: (i) into the feeding artery (50% of the cases); (ii) into a AVF venous segment of the arteriovenous fistula (42.8%); (iii) directly into the thrombus (7.1%), by rt-PA. After an initial pulse of 5-10 mg, according to body weight, the drug was continuously infused by a pump with the speed automatically programmed in 30 Brescia-Cimino autologous AV fistulae and 12 polytetrafluoroethylene (PTFE) grafts. A complete thrombolysis with return of bruit and thrill was obtained in 71.4% of the cases using a mean drug dose of 21 mg and an infusion time of 3.8 h. All the successful cases underwent haemodialysis via AVF on the same day. No bleeding occurred at remote sites. Local bleeding occurred in 16% of the cases; in no case was it so severe as to require the suspension of the therapy or blood transfusions. The median cumulative duration of patency after thrombolysis was 32.4 months. Respectively 21, 12 and two patients had a functioning angioaccess after 3.6, 32.4 and 36 months from the lytic approach. Failure of the treatment was not related to the patients' gender or age, AVF age, route of administration of the drug, type of vessel (natural or artificial), or delay between the discovery of the fistula occlusion and the start of the therapy. In unsuccessful cases an organic lesion of the vessels was documented by angiography or echo colour Doppler. In summary, rt-PA local infusion provides a useful means of preservation of AV fistulae and may be used as the therapy of first choice in dialysis patients without active bleeding or high bleeding risk.

Adult↗
PubMed1994

Psoriatic lesions in patients with chronic liver disease are distinct from psoriasis vulgaris lesions, as judged on basis of integrin adhesion receptors.

Psoriatic lesions are relatively frequent in patients with chronic liver disease. Furthermore, therapy with interferons tends to exacerbate the symptoms. The pathogenesis of psoriatic lesions is unclear. An important question is whether such lesions may be linked to the underlying chronic liver disease in these patients, or whether they are incidental manifestations of psoriasis vulgaris. We collected biopsy specimens from involved and uninvolved skin areas of chronic liver disease patients with psoriatic manifestations, as well as from psoriasis vulgaris patients, and investigated the patterns of integrin adhesion receptors by means of immunohistochemical methods. Integrin expression is known to be characteristically altered in psoriasis vulgaris. We found some of these changes in chronic liver disease psoriatic lesions-namely pericellular redistribution and suprabasal expression of the basement membrane receptor alpha 6 beta 4 and of the intercellular integrins alpha 2 beta 1 and alpha 3 beta 1. However, psoriasis vulgaris causes two other typical changes: One is the induction of the prototype fibronectin receptor alpha 5 beta 1, and the other is the alteration of integrin expression in areas of the epidermis that are macroscopically normal. These two changes were not found in chronic liver disease psoriasis biopsy specimens in 14 patients investigated. Thus integrin expression may be useful in differentiating chronic liver disease psoriatic lesions from psoriasis vulgaris lesions. Even though the two types of lesions are indistinguishable on inspection or by their histological features, they may be caused by distinct pathogenetic mechanisms. It remains to be seen whether the underlying chronic liver disease has a role, albeit indirect, in such mechanisms.

Aged↗
PubMed1994

Role of integrins in cell adhesion and polarity in normal keratinocytes and human skin pathologies.

In vitro, normal human keratinocytes reconstitute a differentiated stratified epidermis, maintaining the same gene expression pattern as its in vivo counterpart and are suitable for permanent grafting onto patients. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are also mediated by integrin receptors that are sorted to defined plasma membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized at the basal surface of basal cells and codistributes with laminin and nicein/kalinin; the alpha 2 beta 1 and alpha 3 beta 1 integrins are enriched laterally and play crucial roles in cell-cell interaction and proper colony morphology. During wound healing, proliferating and migrating keratinocytes express on their plasma membrane alpha v beta 5 and alpha 5 beta 1, which allow keratinocyte attachment and migration over the provisional matrix present in the wound. TGF beta, which is an autocrine and paracrine mediator in wound healing, specifically increases the synthesis and expression of alpha v beta 5 and alpha 5 beta 1, induces the de novo expression of alpha v beta 6, and determines the loss of integrin polarization. In hyperproliferative skin diseases, such as skin cancer or psoriasis vulgaris, and in normal keratinocytes forced into more frequent cell cycles, the polarized expression of integrins is lost, and alpha 5 beta 1 becomes costitutively expressed on the plasma membrane. In addition, the alpha 6 beta 4 integrin becomes associated with focal contacts. Nerve growth factor (NGF) is a potent autocrine stimulator of keratinocyte growth and induces melanocyte migration toward the leading edge of a healing wound.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1993

Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues.

We have identified and cloned a new member of the trk gene family, termed trkE, which generates a 3.9-kilobase (kb) transcript in normal human keratinocytes and in a variety of normal human tissues, but not in liver. Albeit at low level, trkE transcript is expressed also by PC12 cells. The open reading frame codes for a polypeptide of 876 amino acids exhibiting the classic features of cell surface tyrosine protein kinases. trkE catalytic domain is 41% identical to trkA and shows several features unique to the trk gene family. Its extracellular domain does not show significant homology to any known proteins. trkE is the first member of this gene family found abundantly and widely expressed in normal human tissues. Several lines of evidence suggest that NGF is also the ligand for trkE; (i) normal human keratinocytes bind NGF with high affinity, (ii) NGF stimulates keratinocyte growth in an autocrine fashion, (iii) NGF exerts its biological effect on keratinocytes through the stimulation of a trk-specific tyrosine kinase, and (iv) keratinocytes lack trkA but do express large amount of trkE. trkE might also be the NGF receptor by other human peripheral tissues, such as pancreatic islets, and might represent a non-neuronal receptor for this ligand.

Amino Acid Sequence↗
PubMed1993

Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.

Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.

3T3 Cells↗