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Biomedical subjects

R Capparelli

Publications and source records attributed to R Capparelli.

At least 19 recordsLinked to original sources
PubMed2006

Cloning and expression of two plant proteins: similar antimicrobial activity of native and recombinant form.

Antimicrobial peptides and proteins are being studied with increasing interest because of their broad range antimicrobial activity. Among plant antimicrobial proteins, the wheat seed polypeptides, puroindoline a and puroindoline b, are particularly interesting because of their established antibacterial activity. In this paper we describe different strategies used to clone His tagged and GST tagged puroindolines obtaining 1.5 mg recombinant protein from 1 l culture. The antimicrobial activity of recombinant and native puroindolines was comparable.

Anti-Bacterial Agents↗
PubMed2006

Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally.

A bacteriophage lytic for Escherichia coli O157:H7 was isolated from bovine manure. Following in-vivo selection, the phage acquired the capacity to persist in the circulatory system of mice for at least 38 days. When mice were infected experimentally with E. coli O157:H7 (10(7) CFU/mouse), simultaneous injection of the mice with phage (10(8) PFU/mouse) cleared E. coli O157:H7 from the mice within 48 h.

Animals↗
PubMed2004

Real Time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindoline genes and proteins.

Developing seeds from Triticum aestivum (wheat) cultivars were collected after flowering and analysed for puroindoline a and b gene expression by Real Time RT-PCR. Mature seeds were investigated for the presence and the amount of starch-associated puroindoline a and b proteins by flow cytometry. Puroindoline a gene and protein were found to have a predominant role in controlling wheat kernel hardness.

Flow Cytometry↗
PubMed2003

Puroindoline A-gene expression is involved in association of puroindolines to starch.

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars ( Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.

Base Sequence↗
PubMed2003

Selection of bacteriophage-resistant mutants of Streptococcus thermophilus.

Phage-resistant mutants have been isolated from Streptococcus thermophilus. Selection was carried out using anti-phage antibodies or Hoechst 33258-labelled phages. Two mutants out of eight tested displayed reduced acidifying capacity. Selection of the bacteria that extruded more rapidly the fluorochrome 5-6-carboxyfluorescein diacetate (CFDA) restored the acidifying capacity of these two mutants to the level of the parental strains. Mutants displaying phage resistance and good acidifying capacity were obtained in 4-5 days. New phages that are able to overcome the protection mechanisms of the existing bacteria arise continually in the dairy environment. The procedures described here permit to replace promptly the starter culture susceptible to newly emerged phages with a resistant one.

Adsorption↗
PubMed1999

Intravenous or intranasal administration of gliadin is able to down-regulate the specific immune response in mice.

The mucosal lesion in coeliac disease (CD) represents an immunologically mediated injury triggered by gliadin and is restricted by a particular assortment of major histocompatibility complex (MHC) class II genes. Therefore, immunomodulatory strategies to tolerize gliadin-specific, class II-restricted T-cell responses could represent an alternative to current treatments of CD, which are based on a gluten-free diet. In this study, BALB/c mice derived from a gluten-free diet colony were tolerized by either intranasal (i.n.) or intravenous (i.v.) administration of single or multiple doses of gliadin. While a single dose failed to induce tolerance, a significant decrease in gliadin-specific T-cell proliferation was detected (P < 0.001) after multiple i.n. or i.v. administrations. No significant difference in antibody titre was detected for antigen-specific immunoglobulin G (IgG) or the IgG1 subclass, but a lower IgG2a-specific titre was observed. Both interferon-gamma (IFN-gamma) and interleukin (IL)-2 expression, measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), were reduced on antigen administration, both i.v. and i.n. Neither regimen showed a regulatory effect on IL-4 production. As T helper 1 (Th1) cytokines seem to be important in the pathogenesis of CD, our data therefore highlight the potential of i.n. and i.v. routes for the design of useful immunomodulatory strategies for CD.

Administration, Intranasal↗
PubMed1998

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry.

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.

Animals↗
PubMed1997

DNA content differences in laboratory mouse strains determined by flow cytometry.

The nuclear DNA content of seven mouse laboratory strains has been measured by flow cytometry. The differences observed between strains as well as those between sexes within the strain were all statistically significant. The highest DNA content (approximately 6.4 pg/female nucleus) was found in the Balb/c strain; the lowest (approximately 5.7 pg/male nucleus) in the C3H/he strain. The difference between sexes varied from 1.6% (in CD-1 mice) to 6.3% (in nude mice). The interest of these results is twofold. First, the mouse can now be used to study the adaptive significance of genome size variation, so far studied only in plants. Second, DNA content analysis can become a quick method for mouse strain identification.

Animals↗
PubMed1997

Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry.

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 microm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 microm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 microm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 microm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).

Antibodies, Viral↗
PubMed1996

Detection of antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry.

An assay has been developed to detect antibodies to Staphylococcus aureus in water buffalo milk by flow cytometry. The method was the protein A-deficient strain Wood 46 of S aureus incubated with milk samples and fluorescein-labelled rabbit anti-water buffalo antiserum. The assay can detect antibodies when the pathogen is not detectable by bacterial tests and can determine the antibody titre directly on undiluted samples.

Animals↗
PubMed1993

In vitro biosynthesis of lactase in suckling and adult rabbits. Regulatory mechanisms involved in the decline of the lactase activity.

Steady state forms, levels and the in vitro biosynthesis of lactase-phlorizin hydrolase (LPH) proteins have been studied in proximal and middle intestine of suckling and adult rabbits. In most adult tissues the lactase activity and the LPH protein content were low and the synthesis rate of the 200 kDa lactase precursor was reduced in comparison to suckling tissues. In a few tissues with low enzymatic activity the LPH protein content was relatively high, and high lactase synthesis occurred. In addition, the ratio (labeled lactase)/(lactase protein) was lower in the middle jejunum of the adult rabbit than in the proximal region. Both decreased synthesis of LPH precursor and increased turnover or inactivation of the enzyme may cause the decline of the lactase activity.

Animals↗
PubMed1992

Isolation of mouse Lyt2- mutant cells using magnetic particles.

A procedure is described for the selection of mouse Lyt2- mutant cells. The procedure is based upon repeated cycles of selection with rat monoclonal antibodies to the Lyt2 antigen and magnetic particles coated with goat anti-mouse IgG. Stable Lyt2- mutant clones were derived from cells previously mutagenized with X rays and, at a lower frequency, also from non-mutagenized cells. The procedure can be used to rescue Lyt2- cells when the ratio of Lyt2+:Lyt2- cells is 10(4):1.

Animals↗
PubMed1990

Goat-mouse hybridomas secreting goat immunoglobulins.

Monoclonal antibodies specific for an allotypic marker of goat IgG2 were used to select goat-mouse hybrid cells secreting goat IgG2. Four of these hybrid cell clones continued to synthesize goat IgG2 (5-15 micrograms/ml) for over eight months. They will be used to study goat IgG gene regulation.

Animals↗
PubMed1990

Identification of goat allotypic determinants and generation of goat-mouse hybridomas secreting goat IgG2.

Five allotypic determinants controlled by independent genes have been identified in goat. Of these determinants, four have been detected with alloimmune antisera and one with monoclonal antibodies. The specificities A1, C1 and D1 are lipoproteins; B1 is possibly an alpha 2 macroglobulin and E1 and IgG2. The specificity B1 is not expressed until the age of 3-4 months. The gene controlling the specificity E1 is present at about the same frequency (0.38-0.41) in goat, sheep, cattle and water buffalo. Stable hybridomas secreting goat IgG2 have been obtained by the fusion of goat peripheral lymphocytes with mouse myeloma cells.

Animals↗