Biomedical subjects

D Parente

Publications and source records attributed to D Parente.

9 recordsLinked to original sources

PubMed2001

Implicit and explicit memory in junior high and college students.

This study compared the performance of 25 seventh and eighth grade boys and girls on tests of implicit and explicit memory to that of 34 men and women in college. The latter performed significantly better on the explicit memory test. Young adolescents showed a significant priming effect on the implicit memory test; college students did not. Findings suggest a ceiling effect for college students. The results support the distinction between implicit and explicit memory.

Adult↗

PubMed2000

Capillary electrophoresis of supercoiled DNA molecules: parameters governing the resolution of topoisomers and their separation from open forms.

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis. This technique is expected to be applied in the research of novel anticancer molecules targeting the activity of topoisomerase I. The separation of a plasmid mixture containing fully supercoiled molecules, single topoisomers, and their relaxed and open circular forms was tested in an electric field of 200 V/cm using Tris/borate buffer with the addition of magnesium ions at low concentrations and various sieving polymers. The resulting separation is quite simple to achieve and is clearly comparable to that obtained in agarose gels run at low voltage, but with an improved resolution, a higher quantitativity, and a higher speed of analysis. We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a good resolution of topoisomers. (II) Cellulose derivatives can be used as sieving polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.

Base Sequence↗

PubMed2000

Influence of aerobic and stretching exercise on anxiety and sensation-seeking mood state.

This study randomly assigned participants to either an aerobic, non-aerobic, or lecture group to assess whether one bout of exercise would alter mood state. Regardless of experimental condition, changes in mood state were found on the Sensation Seeking and Anxiety State Tests, where sensation-seeking scores increased and anxiety state decreased from pre- to posttest.

Adolescent↗

PubMed1998

Overproduction of soluble, extracellular cytotoxin alpha-sarcin in Escherichia coli.

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.

Amino Acid Sequence↗

PubMed1996

Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein.

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.

Amino Acid Sequence↗

PubMed1992

POEMS syndrome: follow-up study of a case.

We report here the case of a 20-year-old man with POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M proteins, skin changes). This rare syndrome followed a 3-year history of a syndrome that mimics a chronic inflammatory demyelinating polyneuropathy (CIDP). Treatment with cyclophosphamide induced regression of the syndrome and improved peripheral nerve conduction.

Adult↗

PubMed1991

Prochymosin expression in Bacillus subtilis.

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.

Amino Acid Sequence↗

PubMedDate not supplied

Role of cross-linking agents in determining the biochemical and pharmacokinetic properties of Mgr6-clavin immunotoxins.

Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the gp185(HER-2) extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and purification parameters were analyzed in an effort to optimize the antitumor activity and stability of the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling procedures were used and both disulfide and thioether linkages were obtained. Unhindered and hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT) were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT). To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT. To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins were purified and characterized using a single-step chromatographic procedure. Specificity and cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation with glutathione is proportional to the presence of methyl groups and increases exponentially with the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more slowly; i.e., the disulfide bearing a methyl group doubled the beta-phase half-life (from 3.5 to 7.1 h) compared with that of the unhindered, while a geminal dimethyl protection increased the elimination phase to 24 h. The thioether linkage showed its intrinsic stability with a beta-phase half-life of 46 h. The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl steric hindered disulfide suggest clinical usefulness.

Animals↗

PubMedDate not supplied

Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin.

We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of the Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type 1 ribosome-inactivating protein (RIP) from Aspergillus clavatus. ErbB2 is a tyrosine kinase receptor which is overexpressed in most adenocarcinomas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by inactivating ribosomes. A recombinant DNA construct containing the cDNA of the single chain Fv fragment (scFv) of the MGR6 antibody fused to the clavin cDNA, was expressed at high levels in Escherichia coli as an insoluble fusion protein containing an N-terminal affinity tag of six consecutive histidine residues. Inclusion bodies were denatured and the recombinant fusion protein was purified under denaturing conditions by single-step purification using immobilised metal ion affinity chromatography (IMAC). The purified immunotoxin was renatured at high yield and histidine tag removed by digestion with enterokinase. The purity of the immunotoxin obtained after refolding was confirmed by SDS-PAGE, RP-HPLC, GPC-HPLC and N-terminal sequence analysis. Cell-free protein synthesis inhibition and binding assays showed that both clavin and scFv(MGR6) maintained their properties after refolding.

Antibodies, Monoclonal↗