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P Costantino

Publications and source records attributed to P Costantino.

At least 19 recordsLinked to original sources
PubMed2001

The plant oncogene rolD encodes a functional ornithine cyclodeaminase.

The plant oncogene rolD stimulates the reproductive phase transition in plants. We define here the function of its gene product. We show that the RolD protein bears sequence homology with ornithine cyclodeaminase, an uncommon enzyme of specialized-niche eubacteria and archaea that catalyzes the unusual NAD(+)-dependent conversion of ornithine to proline. To confirm the prediction of the bioinformatic analysis, the RolD protein was expressed in Escherichia coli and purified. An ornithine-dependent NAD(+) reduction that can be ascribed only to ornithine cyclodeaminase (OCD) activity was detected both in bacterial extracts containing RolD and in assays on the purified RolD protein. Furthermore, OCD activity was observed in soluble extracts from plants overexpressing rolD. The role of rolD in plant pathogenesis and its effect on plant reproductive development are discussed in light of the newly demonstrated enzymatic activity of its gene product.

Amino Acid Sequence↗
PubMed2001

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Amino Acid Sequence↗
PubMed2001

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency.

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.

Asteraceae↗
PubMed2000

A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III.

The design of chimeric proteins is a major field of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.

Amino Acid Motifs↗
PubMed2000

Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination.

We describe here the Arabidopsis gene DAG1, encoding a zinc finger transcription factor of the Dof family, and show that it is involved in the control of seed germination. By a reverse genetics approach, we isolated an Arabidopsis mutant line with one T-DNA insertion in DAG1. Seeds from homozygous knockout dag1-1 plants do not develop dormancy and germinate also in the absence of light. Segregation analysis indicates that the effect of the mutation is maternal. Accordingly, in situ mRNA hybridizations reveal expression of DAG1 in the vascular tissue of the flower and maturing fruit but not in the seed.

Arabidopsis↗
PubMed2000

Prevention of cerebrospinal fluid rhinorrhea in neurotologic surgery.

OBJECTIVE: To determine the efficacy and safety of quick-setting hydroxyapatite cement in eliminating cerebrospinal fluid (CSF) rhinorrhea following neurotologic surgery. STUDY DESIGN: A prospective study of 40 consecutive patients undergoing neurotologic surgery in whom the dura was opened. SETTING: All patients were treated as hospital inpatients at a tertiary referral center. PATIENTS: 25 men and 15 women between the ages of 20 and 72 years (mean age 51 years) underwent neurotologic surgery at the parent institution. INTERVENTION: Various neurotologic procedures were performed for the resection of 25 acoustic tumors, 5 meningiomas, 3 glomus tumors, 2 vestibular nerve sections, 2 chordomas, 1 epidermoid tumor, and 1 meningoencephelocele, and for 2 patients referred to our institution with known CSF leaks following acoustic tumor surgery. A new form of quick-setting hydroxyapatite cement, which that hardens within 3 to 5 minutes was used to seal the air cell tracts of the temporal bone in all cases. MAIN OUTCOME MEASURE: The presence of CSF rhinorrhea postoperatively. RESULTS: CSF rhinorrhea occurred in 2 patients following acoustic tumor surgery, the first through an occult air cell tract at the margin of the drilled internal auditory canal, and the second via an oval window fistula 1 month after a translabyrinthine approach. CONCLUSIONS: This form of hydroxyapatite cement appears safe, reliable, effective, and economical for the prevention of CSF rhinorrhea following neurotologic surgery. CSF rhinorrhea cannot be eliminated unless our ability to identify all potential air cell tract communications improves.

Adult↗
PubMed2000

Physicochemical characterisation of the pertussis vaccine.

The characterisation of an acellular pertussis vaccine composed of a genetically modified pertussis toxin, filamentous haemagglutinin and pertactin is described. The three antigens are submitted to a mild treatment with formaldehyde in the presence of lysine before their use in vaccine formulation. Characterisation is performed by amino acid analysis, SDS-PAGE, analytical size exclusion chromatography and, in the case of pertactin, isoelectrofocusing. The effect of some variables on pertactin formaldehyde treatment has been studied by means of isoelectrofocusing and mouse immunogenicity.

Amino Acids↗
PubMed2000

Physicochemical characterisation of the oligosaccharide component of vaccines.

Glycoconjugate vaccines are being developed against Haemophilus influenzae (Hib) and meningococcal Type A and C micro-organisms; they consist of oligosaccharides of intermediate chain length conjugated to the carrier protein CRM (a non-toxic diphtheria toxin mutant). The oligosaccharides can be quantified using specific composition analyses and their structure and identity (and pattern of acetylation) evaluated by use of NMR spectroscopy. The average molecular-size (degree of polymerisation) can be determined using colorimetric assays, qualified by analysis of authentic standards. The molecular-size distribution of these anionic oligosaccharides can be achieved using ion exchange chromatography or application of the rapid and sensitive analytical HPAEC-PAD system (high performance anion-exchange chromatography with pulsed amperometric detection). Preparative ion exchange chromatography permits the isolation of purified oligomers, which can be well-characterised using the methods described above. Molecular size can be confirmed by use of mass spectrometry. These vaccines are semi-synthetic products and therefore their preparation involves several steps of chemical reaction, the detailed physicochemical characterisation of the oligosaccharide-components permits the consistent production of these well-defined glycoconjugate vaccines.

Chemical Phenomena↗
PubMed1999

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.

We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.

Bacterial Capsules↗
PubMed1999

Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines.

We have developed a chromatographic method suitable for the fractionation of polysaccharides having a negatively charged group. The method permits the removal of all those polysaccharide fragments having a short sequence and which are likely unsuitable for conjugate vaccine construction. The selected polysaccharide fragments can be used to produce glycoconjugate vaccines containing a restricted saccharide polydispersion. We have applied this chromatographic method to three different antigens, Haemophilus influenzae type b and Neisseria meningitidis group A and group C polysaccharides. The method is easily adapted for manufacturing purposes.

Antigens, Bacterial↗
PubMed1999

The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants.

The Dof proteins are a large family of plant transcription factors that share a single highly conserved zinc finger. The tobacco Dof protein NtBBF1 was identified by its ability to bind to regulatory domain B in the promoter of the rolB oncogene. In this study, we show that the ACT T TA target sequence of NtBBF1 in domain B is necessary for tissue-specific expression of rolB. beta-Glucuronidase (GUS) activity of tobacco plants containing a rolB promoter-GUS fusion with a mutated NtBBF1 target sequence within domain B is almost completely suppressed in apical meristems and is severely abated in the vascular system. The ACT T TA motif is shown here also to be one of the cis-regulatory elements involved in auxin induction of rolB. The pattern of NtBBF1 expression in plants is remarkably similar to that of rolB, except in mesophyll cells of mature leaves, in which only NtBBF1 expression could be detected. Ectopic expression of rolB in mesophyll cells was achieved by particle gun delivery if the NtBBF1 binding sequence was intact. These data provide evidence that in the plant, a Dof protein DNA binding sequence acts as a transcriptional regulatory motif, and they point to NtBBF1 as the protein involved in mediating tissue-specific and auxin-inducible expression of rolB.

Binding Sites↗
PubMed1998

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b.

A competitive ELISA method is described for the measurement of total antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HibCPS) in human sera. The competitive method showed an excellent correlation to the radioantigen binding assay (RABA, or Farr assay) and improved correlation of sera with low titers with respect to the more conventional noncompetitive method. Overestimation of samples in the low concentration range was no longer observed with the competitive ELISA method. The free HibCPS competition allowed us to eliminate the day-to-day background variation typical of some sera; thus, only values representing the true anti-HibCPS response were determined. The use of precoated microplates, which could be stored up to 8 months, greatly improved the speed of the procedure. An overall correlation coefficient of 0. 9660 was found when 407 serum samples with a wide variety of anti-HibCPS antibody levels were tested with the competitive ELISA and RABA. The regression line was very close to the ideal line, with a slope of 1.0045 and an intercept of -0.1996. A subset of 96 serum samples representative of all pre- and postimmunization samples was used to compare the competitive ELISA with a previously described ELISA method. The competitive method performed in two laboratories in different countries showed a better correlation with the RABA. The correlation factors were 0.9770 and 0.9816, respectively, while a factor of 0.9547 was found with the previously described noncompetitive procedure, which was better for this method than previously reported (r = 0.917). Therefore, the competitive ELISA is proposed for the assay of anti-HibCPS titers in sera from vaccinated subjects.

Adult↗
PubMed1996

The plant oncogene rolD stimulates flowering in transgenic tobacco plants.

The Agrobacterium rhizogenes T-DNA oncogene rolD under the control of its own 5' regulatory region was transferred to day-neutral tobacco plants. The main trait induced by rolD in transgenic plants is a striking precocity in flower setting and a strong enhancement of the flowering potential. In rolD plants, early flowering is followed by the very rapid growth of numerous lateral inflorescences. The analysis of several morphological and histological parameters suggests that some characteristic morphological abnormalities observed in rolD plants can be accounted for by their early reproductive phase transition and points to the involvement in the transition of a greater portion of the plant body than is the case for untransformed tobacco. The in vitro morphogenic potential of tissues from rolD plants was also tested. Superficial thin cell layer explants from rolD plants show an earlier and much enhanced flower organogenesis, compared to controls, both on flowering and on hormone-free medium.

Cells, Cultured↗
PubMed1996

A rolB regulatory factor belongs to a new class of single zinc finger plant proteins.

A protein which binds to the regulatory domain B necessary for expression of the plant oncogene rolB in (root) meristems contains a single zinc finger of a novel type conserved in dicots and monocots. Band shift analysis revealed the presence in tobacco nuclei of a protein selectively binding to domain B, a tetramer of which was used to isolate a cDNA (NtBBF1, Nicotiana tabacum rolB domain B Factor 1) from a tobacco expression library. The corresponding genomic clone was also isolated. The protein encoded by NtBBF1 contains a single C2C2 zinc finger, and its target sequence in domain B was identified by means of mutagenized oligonucleotides. The DNA-binding capability of the zinc finger was assessed by means of a fusion of this latter with the glutathione-S-transferase protein, that was shown to bind the same target sequence as NtBBF1. A number of other tobacco cDNAs encoding different proteins with a domain (BBF domain) encompassing the zinc finger identical to NtBBF1 were also isolated. Furthermore, a cDNA encoding a protein with an almost identical single zinc finger was isolated from Arabidopsis. A very closely related zinc finger has very recently been identified in maize transcription factors and termed the Dof domain. It is proposed that the tobacco, Arabidopsis and maize BBF/Dof domain proteins are members of a new broad family of plant transcription factors acting through a single zinc finger widely utilized in the plant kingdom.

Amino Acid Sequence↗
PubMed1995

Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide.

Vaccine development against Group B Neisseria meningitidis is complicated by the nature of the capsular polysaccharide, which is alpha 2-8-linked poly-sialic acid, identical in structure to the poly-sialic acid found in many mammalian tissues during development. To test the feasibility of a vaccine based on this polysaccharide, we synthesized several conjugates of meningococcal B polysaccharide linked to a carrier protein (tetanus toxoid or diphtheria CRM197), via an adipic acid dihydrazide (ADH) spacer. All conjugates induced a strong immune response. However, most of the antibodies were not directed against the Meningococcus B polysaccharide and could not be inhibited by the purified polysaccharide alone. Further investigations showed that the antibodies recognized an epitope composed by the junction between the spacer and the polysaccharide and protein, that is not present in the native polysaccharide and is generated during the coupling reaction. This epitope becomes immunodominant with respect to the poorly immunogenic polysaccharide. While the majority of the immune response is directed against the above epitope, the conjugates induced also an immune response against the Meningococcus B polysaccharide. The anti-Meningococcus B antibodies elicited are of the IgM and IgG class and are inhibitable by the polysaccharide. Moreover, they are bactericidal, thus suggesting that they would induce protection against disease.

Adipates↗
PubMed1995

Comparison of fentanyl with clonidine as adjuvants for epidural analgesia with 0.125% bupivacaine in the first stage of labor: a preliminary report.

48 primiparae received epidural analgesia in labor with 10 ml of 0.125% bupivacaine with epinephrine 1:800 000, and then were divided in 4 equal groups (n = 12) to receive one of the following: 5 ml saline (B); 100 mug of fentanyl (BF); 150 microg of clonidine (BC); 75 microg of clonidine and 50 microg of fentanyl (BCF). All the patients had satisfactory analgesia. Onset was similar in the 4 groups but the duration of analgesia was significantly prolonged by the addition of either 100 microg of fentanyl or 150 microg of clonidine (respectively 89.8 min and 92.5 min vs 62.5 min) (P < 0.0001). The addition of both clonidine (75 microg) and fentanyl (50 microg) produced a considerably prolonged analgesia (177.5 min) (P < 0.0001). No episodes of bradycardia were observed. Hypotension, reversed by i.v. ephedrine, occurred in 2 patients of BCF group and in 1 patient of BF and BC groups. Only patients receiving fentanyl had pruritus. Both fentanyl and clonidine produced sedation, but both incidence and severity were greater with the mixture. No differences in neonatal outcome assessed by Apgar scores and NACS, were observed.

Journal Article↗