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Biomedical subjects

R D'Ovidio

Publications and source records attributed to R D'Ovidio.

At least 19 recordsLinked to original sources
PubMed2004

The glucose oxidase of Penicillium variabile P16: gene cloning, sequencing and expression.

AIMS: Isolation and characterization of the glucose oxidase (GOX)-encoding gene from a Penicillium variabile strain (P16) having a high level of GOX activity and comparison of its expression with that of another strain of P. variabile (NRRL 1048) characterized by low GOX activity. METHODS AND RESULTS: The gene, isolated by PCR consisted of 1818 bp encoding 605 amino acid residues. Gene expression was analysed by Northern blotting and compared with that of P. variabile NRRL 1048. The higher GOX activity of strain P16 appeared likely because of de novo mRNA synthesis. Southern blotting analyses of the genomic DNA showed that the hybridization pattern of the two strains differed for the size of hybridizing fragment detected by the probe and slightly for their signal intensity. CONCLUSIONS: The GOX-encoding gene of P. variabile P16 was isolated and characterized to identify the molecular bases of its high level of expression and in view of improving enzyme production by developing a process based on heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: GOX-encoding genes can be subjected to high difference in their expression levels. The P16 strain of P. variable producing large amount of GOX as well as its encoding gene might be exploited for industrial applications.

Amino Acid Sequence↗
PubMed2003

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers.

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.

Blotting, Southern↗
PubMed2003

Heterologous expression and purification of native and mutated low molecular mass glutenin subunits from durum wheat.

Wheat technological properties are correlated with the size of glutenin polymers, consisting of high and low molecular mass glutenin subunits, linked together by disulphide bonds. In order to unravel glutenin polymer structure, we considered three LMW-GS genes, which differ in the number of cysteine residues and in the repetitive domain length. The three LMW-GS genes have been expressed in Escherichia coli, and purified with a yield of 40-100 mg/l of culture volume, depending on protein type. Single polypeptides are being used in re-oxidation and micro-mixographic experiments, in order to detect the influence of the differential structural characteristics on glutenin polymer formation.

Base Sequence↗
PubMed2003

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species.

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.

Base Sequence↗
PubMed2002

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Amino Acid Sequence↗
PubMed2001

The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins capable of inhibiting fungal endopolygalacturonases (PGs). Plants have evolved different PGIPs with specific recognition abilities against the many PGs produced by fungi. The genes encoding PGIPs are organized into families, and different members of each family may encode proteins with nearly identical characteristics but different specificities and regulation. PGIPs are typically induced by pathogen infection and stress-related signals. The recognition ability of PGIPs resides in their LRR (leucine-rich repeat) structure, where solvent-exposed residues in the beta-strand/beta-turn motifs of the LRRs are determinants of specificity. Manipulation of the primary structure of PGIPs is expected to generate more efficient PGIPs with novel recognition specificities to protect crop plants against pathogens.

Amino Acid Sequence↗
PubMed1998

Developmentally and wound-regulated expression of the gene encoding a cell wall copper amine oxidase in chickpea seedlings.

A chickpea cDNA encoding a cell wall copper amine oxidase (CuAO) was cloned and characterised. The 2010 bp open reading frame encodes a protein of 76.5 kDa which shares significant primary structure homology with other known CuAOs. Southern blot analysis indicates that in chickpea CuAO is encoded by a single gene or a small gene family. This cDNA was essential for studying the role of CuAO during seedling development and wound healing in chickpea seedlings. CuAO transcript level and activity were modulated during seedling development in parallel with cell maturation. Moreover, mechanical wounding induced a rapid increase of CuAO mRNA accumulation and enzyme activity which remained high during the wound-healing process. Aminoguanidine, a specific CuAO inhibitor, decreased the deposition of lignin-suberin barrier along the lesion. CuAO may be a limiting factor in H2O2 production in the cell wall of chickpea seedlings and its expression seems to integrate with the remodelling of plant cell wall occurring during ontogenesis and wound healing.

Amine Oxidase (Copper-Containing)↗
PubMed1998

A simple clinical model for the diagnosis of deep-vein thrombosis combined with impedance plethysmography: potential for an improvement in the diagnostic process.

OBJECTIVES: We recently demonstrated the utility of a clinical model combined with ultrasonography to assist the diagnostic approach in patients with suspected deep-vein thrombosis (DVT). In this study we also sought to demonstrate that the model is useful with impedance plethysmography, a less accurate and less utilized diagnostic test. The original clinical model is slightly cumbersome to use; thus at the completion of the study we attempted to develop a simpler scoring system with a goal of maintaining accuracy. DESIGN: An open, nonrandomized, multicentre trial. SETTING: Three centres, two in Canada, and one in Italy. SUBJECTS: Ambulatory patients with suspected deep-vein thrombosis. INTERVENTIONS: All patients were assessed clinically to determine the probability for deep-vein thrombosis prior to performing impedance plethysmography and venography. We compared the accuracy of impedance plethysmography between the three pretest probability categories of high, moderate and low. All of the above were performed and interpreted by independent observers. When the study was completed, we revised the clinical model by first performing a simple regression analysis then a multiple logistic regression analysis; a scoring system was devised using the latter. RESULTS: Impedance plethysmography is significantly more sensitive and less specific for all DVT in patients with high pretest probability for deep-vein thrombosis (P = 0.001). The post- test probability (positive predictive value) for deep-vein thrombosis with an abnormal impedance plethysmography result was significantly different (P = 0.0001) between the three pretest probability categories. Multiple regression analysis has provided a new model with only nine variables and a simple scoring system. The retrospective application of the revised clinical model, which is simpler to use, suggests it will provide similar results as the original clinical model when combined with impedance plethysmography. The combination of impedance plethysmography and the clinical model suggests patients are likely to have false positive results if they have a low or moderate pretest probability for deep-vein thrombosis and false negative results if the pretest probability is high. The combination of a low pretest probability and a normal impedance plethysmography result may exclude the need for serial testing, and represented more than 50% of our patient population. CONCLUSIONS: The use of the clinical model in conjunction with impedance plethysmography would decrease the number of false positive and negative diagnoses and could markedly decrease the need for serial impedance plethysmography. Combining the clinical model with impedance plethysmography could overcome the fact that impedance plethysmography is clearly less accurate than venous ultrasound imaging. The use of the revised clinical model may increase acceptability and utility, but prospective testing is required before widespread use.

Diagnosis, Differential↗
PubMed1998

Characterization of a low-molecular-weight glutenin subunit gene from bread wheat and the corresponding protein that represents a major subunit of the glutenin polymer.

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

Amino Acid Sequence↗
PubMed1995

Accuracy of clinical assessment of deep-vein thrombosis.

The clinical diagnosis of deep-vein thrombosis is generally thought to be unreliable. From experience, we hypothesised that this widely held view might be incorrect. We developed a clinical model and prospectively tested its ability in three tertiary care centres to stratify symptomatic outpatients with suspected deep-vein thrombosis into groups with high, moderate, or low probability groups of deep-vein thrombosis. We evaluated our clinical model in combination with venous ultrasonography to determine the potential for an improved and simplified diagnostic approach in patients with suspected deep-vein thrombosis. All patients were clinically assessed to determine the probability for deep-vein thrombosis before they had ultrasonography and venography. All tests were performed and interpreted by independent observers. In 529 patients, the clinical model predicted prevalence of deep-vein thrombosis in the three categories: 85% in the high pretest probability category, 33% in the moderate, and 5% in the low category. There was no statistical difference in the performance of the model in the three centres. The model demonstrated excellent interobserver reliability (Kappa = 0.85). There were important differences with ultrasonography between the high and low pretest probability groups for both positive predictive values (100% (95% CI, 94-100%) vs (63% [35-85%], respectively). Thus, use of the clinical model combined with ultrasonography would decrease the number of false positive and negative diagnosis if venography were done when the ultrasound result and pretest probability were discordant. The diagnostic process could be simplified by excluding those patients with low pretest probability and normal ultrasound results from serial testing.

Decision Trees↗
PubMed1995

FokI DNA repeats in the genome of Vicia faba: species specificity, structure, redundancy modulation, and nuclear organization.

Tandemly repeated DNA sequences about 60 bp in length, which may be isolated by digestion with FokI restriction endonuclease, were studied by means of molecular and cytological hybridization in Vicia faba and other Vicia species. The results obtained can be summarized as follows: (i) FokI repeats are almost species specific to V. faba, since they hybridize to a minimum extent to genomic DNA of only two out of five related species; (ii) these tandemly repeated elements display variability in structure even within one and the same array, where different repeats may share not more than 71% homology; (iii) their redundancy in the genome of V. faba is remarkably high and varies largely between land races (copy numbers per haploid, 1C, genome range from 21.51 x 10(6) to 5.39 x 10(6)); (iv) FokI repeats are clustered in differing amounts in each subtelocentric pair of the chromosome complement and are missing or present in a nondetectable amount in the submetacentric pair; (vi) chromosome regions that bear these repeats associate closely to varying degrees in interphase nuclei. These results are discussed in relation to possible functional roles that tandemly repeated DNA sequences such as the FokI elements might play.

Base Sequence↗
PubMed1994

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein.

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endo-PGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.

Amino Acid Sequence↗
PubMed1993

Single-seed PCR of LMW glutenin genes to distinguish between durum wheat cultivars with good and poor technological properties.

Polymerase chain reaction (PCR) was used to amplify low-molecular-weight (LMW) glutenin sequences from genomic DNA extracted from a single germinating seed of several durum wheat genotypes. Electrophoretic analysis of PCR reactions showed the presence of amplified products characteristic of durum wheat cultivars with good and poor technological properties. This PCR-based approach is proposed as a very efficient and safe alternative to standard procedures for selecting durum wheat genotypes with good qualitative characteristics.

Base Sequence↗
PubMed1991

Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba.

Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.

Base Sequence↗
PubMed1991

Negative and positive regulation of a novel proline-rich protein mRNA by fungal elicitor and wounding.

The structure and expression of a cDNA clone (PvPRP1) isolated from a cDNA library prepared from bean (Phaseolus vulgaris) cells treated with fungal elicitor have been characterized. Sequence analysis of the 1.1 kb insert revealed a complete open reading frame which encodes a 32 kDa protein. The protein resembles other proline-rich proteins in plants but possesses several unique features: (i) the N-terminal half of the protein is proline rich and contains three identical repeats of Pro-Val-His-Pro-Pro-Val-Lys-Pro-Pro-Val and six related repeats; (ii) the proline-rich region contains two tracts of six histidine residues; and (iii) the C-terminal half is low in proline and lacks repeats. Genomic blotting experiments suggest the presence of a single PvPRP1 gene as well as more distantly related genes within the bean genome. A dramatic decrease in PvPRP1 mRNA levels occurs within 2 h of elicitor treatment of cell cultures. The PvPRP1 mRNA is present at a moderate level in hypocotyls. Upon wounding, the mRNA level initially decreases over 5 h and then accumulates over 25 h to levels which are higher than the basal level in unwounded hypocotyls. Based on the similarity to other proline-rich proteins with repeated motifs, including the presence of a putative signal peptide, it is likely that the PvPRP1 protein is targetted to the cell wall. The expression of the PvPRP1 gene appears to be integrated with the remodeling of the plant cell wall during the defense response.

Amino Acid Sequence↗