Adriano Podestà

Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine

1. The specificity of a monoclonal IgG1 raised against a 5-methylcytidine-keyhole limpet hemocyanin conjugate was investigated by inhibition experiments with soluble competing antigens. 2. A competitive enzyme immunoassay has been set up, with the antigen immobilized on polystyrene microtitration wells. 3. The analysis of the cross-reaction profile allowed the topography of the antigen-antibody interaction to be described. 4. The binding properties of the monoclonal antibody are discussed in terms of both analytical applications and working limitations in the immunochemical study of gene methylation.

A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells

In the developing chicken embryo, active DNA demethylation requires both RNA and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550, 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in the 5- and 12-day-old embryos the highest specific activity of 5-methylcytosine DNA glycosylase is found in the brain, the eyes and the skin. In situ hybridization with antisense CpG-rich RNA tightly associated to the DNA demethylation complex shows a restricted expression pattern only in proliferating tissues such as the neuroepithelia of the brain in 5-day-old embryos. The RNA is absent in differentiated tissues like the skeletal and heart muscle, liver and the crystallin-producing cells in the lens. The CpG-rich RNA is transcribed in a developmental stage-specific rather than in a cell-specific manner. In contrast transcripts of DNA methyltransferase are found in dividing and quiescent cells. In situ hybridization with a probe of a RNA helicase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA. The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. The results indicate that proliferating cells have on the average 15% more methylated cytosines than non-dividing cells. This represents roughly 3x10(6) more methylation sites per haploid genome.

In vitro effects of different formulations of bovine collagen on cultured human skin

Irritant effects and cytotoxicity of three different products based on collagen were investigated: a sponge formulation and a thin film composed by Type I collagen from bovine Achilles tendon, and a membrane prepared from bovine derma. The test system was a three-dimensional human skin model, developed by Advanced Tissue Science, La Jolla, CA, USA. Squares of dermal tissue (11 x 11 mm) were cultured in suitable media and exposed to the products under study. Dimethyl sulphoxide was used as the chemical control of tissue responsiveness to irritating substances. After 24 and 48 h the prostaglandin E2 (PGE2) concentration and the lactate dehydrogenase (LDH) activity in the culture medium were measured, as indexes of early inflammatory response and cell membrane breakdown, respectively. In addition, cell morphology was examined by light microscopy. The highest PGE2 concentrations were observed after cell exposure to the collagen sponges. The intensity of the inflammatory response changed accordingly to the collagen dose in use. However, it was never followed by an increased rate of cell death, as revealed by LDH activity measurement and microscopy. These findings suggest that hydrolysis of exogenous collagen starts shortly after it is kept in contact with tissues and evokes a local inflammatory response whose intensity depends on the pharmaceutical formulation in use.

Time-Resolved Fluoroimmunoassay

A method is reported to set up a standard competitive TR-FIA. A simple and inexpensive way to prepare reagents and carry out operations is presented as well, with the aim to make it possible to perform a very sensitive analytical procedure in a personalized way within a nondedicated biochemistry laboratory. This protocol is general and can be easily modified with consideration to the analytical target. Once the antibody is available, both its labeling with diethylenetriaminepentaacetic acid dianhydride and Eu3+, and the setting-up of the assay with measurement of europium ion time-resolved fluorescence in a home-made enhancement solution become feasible.

Immunological and Biochemical Evidence that Blepharismin is not a Prosthetic Group

Abstract A polyclonal, multispecific antiserum was raised against a whole 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate-extract of nonirradiated Blepharisma japonicum cells. It was used to reveal the composition of solutions that were hypothesized to contain the photoreceptor of the ciliate. A Bio-Gel A 1.5 m fine column chromatography of the extract allowed recovery of a single elution peak isolated by recording the 580 nm light absorbance. Fused-rocket immunoelectrophoresis of this material revealed a large number of >300 kDa coeluted proteins. Blepharismin-rich material with a molecular mass of approximately 50 kDa, consisting of at least nine proteins was obtained when the same extract underwent preparative isoelectric focusing before column chromatography separation. Purification of the pigment obtained from light-exposed cells gave blepharismin-rich material with a molecular weight of approximately 200 kDa. Comparison of the materials obtained under the same conditions, either from the dark-kept or light-irradiated cells, by means of pore-gradient electrophoresis confirmed that proteins present in the two preparations were different. It revealed only a very small amount, if any, of proteins in the chromatography fractions with the highest absorbance at 600 nm. Results are discussed on the basis of the hypothesis that a specific blepharismin-binding protein does not exist in the protozoan.

Highly specific polyclonal antisera against estriol: Cross-reactivity restriction following affinity chromatography

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.

Tumor-promoting effects of cannabinoid receptor type 1 in human melanoma cells.

The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma.

A SOURCE OF FALSE-NEGATIVE RESULTS IN CLENBUTEROL ANALYSIS IN TISSUES OF VEAL CALVES

The possibility of false-negative results in clenbuterol analysis was investigated in bovine tissues. An extraction procedure currently in use was adapted to process 100 specimens of different tissues each time. Its efficiency and accuracy were investigated radiometrically by means of a series of different molar concentration of the tritiated drug. In samples not submitted to extensive delipidation, unreliability of the analysis was evident. The measurement of tissue clenbuterol content, by a competitive ELISA, gave results numerically similar to those existing in literature, but with an accuracy high enough to minimize the frequency of false-negative results.

Shared reaction in solid-phase immunoassay for estriol determination

In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.

Specificity in protein-ligand interactions: a model from estrone-antiserum binding

The specificity of protein-ligand interactions has been investigated using the binding of structurally related steroids to a polyclonal anti-estrone antiserum as a model. The cross-reaction profile of the native antiserum was compared with profiles obtained for the same antiserum preparation following affinity separation on stationary phases carrying structures which mimic parts of the antigen molecule: the coupling bridge, the A ring of the estrogen and the carrier protein. This fractionation produced antibody mixtures with different specificities from that observed for the pre-affinity antiserum. The changes in specificity observed and, more importantly, the direction of each variation detected, suggested the basis for a description of the nature of molecular recognition of small ligands by a protein as discrete rather than continuous. This methodology has revealed some recognition mechanisms.