Biomedical subjects
Publications and source records attributed to M Giorgi.
The authors, after having completed a wide bibliographic research on this subject, examine the etiopathogenesis of Brodie's syndrome, and distinguish a congenital from an acquired form. The different therapeutic possibilities, orthodontic or surgical, have been examined and discussed.
Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF. Using antibodies to the beta chains of fibronectin (beta 1) and vitronectin (beta 3) receptors it was found that ECs adherent to vWF show clustering of both receptors. The beta 1 receptor antibodies are arranged along stress fibers at sites of extracellular matrix contact while the beta 3 receptor antibodies were sharply confined at adhesion plaques. ECs release and organize endogenous fibronectin early during adhesion to vWF. Upon blocking protein synthesis and secretion, ECs can equally adhere and spread on vWF but, while the beta 3 receptors are regularly organized, the beta 1 receptors remain diffuse. This suggests that the organization of the beta 1 receptors depend on the release of fibronectin and/or other matrix proteins operated by the same cell. Antibodies to the beta 3 receptors fully block EC adhesion to vWF and detach ECs seeded on this substratum. In contrast, antibodies to the beta 1 receptors are poorly active. Overall these results fit with an accessory role of beta 1 receptors and indicate a leading role for the beta 3 receptors in EC interaction with vWF. To identify the EC binding domain on vWF we used monoclonal antibodies produced against a peptide representing the residues Glu1737-Ser1750 of the mature vWF and thought to be important in mediating its binding to the platelet receptor glycoprotein IIb-IIIa. We found that the antibody that recognizes the residues 1,744-1,746, containing the Arg-Gly-Asp sequence, completely inhibit EC adhesion to vWF whereas a second antibody recognizing the adjacent residues 1,740-1,742 (Arg-Gly-Asp-free) is inactive. Both antibodies do not interfere with EC adhesion to vitronectin. This defines the molecular domain on vWF that is specifically recognized by ECs and reaffirms the direct role of the Arg-Gly-Asp sequence as the integrin receptor recognition site also in the vWF molecule.
A cell-specific isozyme of calmodulin (CaM)-dependent phosphodiesterase that exhibits micromolar affinity for cAMP has been purified 900-fold from mouse testis by DEAE chromatography, gel filtration, affinity chromatography with CaM-Sepharose 4B, and isoelectric focusing. The highly purified enzyme is stimulated 5-6-fold by CaM in the presence of Ca2+ and hydrolyzes both cAMP and cGMP with anomalous substrate dependence, i.e. high and low affinity components (Km 2 and 20 microM) are observed either in the presence or absence of CaM. Each of the substrates acts as a noncompetitive inhibitor of the other, suggesting the presence of two distinct catalytic sites on the enzyme. Hydrodynamic studies suggest that the testis phosphodiesterase is an asymmetric monomer of 68-70 kDa that forms a dimer after interaction with Ca2+ and CaM; the tetrameric complex exhibits an apparent molecular size of 180 kDa. These enzymatic and biophysical properties differ in many respects from those of the brain isozyme, suggesting that they are different proteins. Nevertheless, common epitopes do exist, since the testis enzyme interacted with rabbit antibodies raised against bovine brain CaM-dependent phosphodiesterase. The major peptide of 68 kDa was strongly reactive on immunoblots, and was distinguished unambiguously from the 60-kDa species from mouse brain. A comparison of the immunoreactive fragments produced by limited proteolysis with staphylococcal V-8 protease indicated several similarities in the domains of these polypeptides. Thus, although differing in several important physical and biochemical parameters, the testis enzyme appears immunologically related to CaM-dependent phosphodiesterase from brain. On the basis of these data, we conclude that common elements of the structural genes for these isozymes have been conserved, whereas certain biological properties, including substrate specificity, have diverged substantially.
Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.
We report that a 2.4-kilobase (kb) pim-1 transcript is expressed in the germ cells of mouse testis. Analysis of purified populations of spermatogenic cell types indicates that the 2.4-kb transcript is selectively expressed in haploid postmeiotic early spermatids. The evidence for a developmentally regulated expression of pim-1 in haploid spermatids suggests a possible developmental role for this protooncogene product. The 2.4-kb pim-1 transcript present in postmeiotic cells differs in size from the 2.8-kb transcript usually detected in somatic tissues. Similar testis-specific transcripts have been seen for mos and abl genes. These data suggest specificity in transcription or processing of certain genes in haploid male germ cells. We have also analyzed other representative protooncogenes, including examples of protein kinases, the ras family, and the "nuclear" protooncogenes. The results indicate that additional protooncogenes are preferentially expressed in either meiotic pachytene cells or postmeiotic early spermatids. These findings suggest a differential regulation of gene expression in these two developmental stages of germ cells. In particular, analysis of expression of the three members of the ras gene family indicates a distinct temporal differential regulation in the expression of the Harvey, Kirsten, and N-ras genes in these germ cells.
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Alpha-fetoprotein (AFP) and cholinesterase levels in amniotic fluid were determined and the efficiency of these laboratory tests in the prenatal diagnosis of neural tube defects was examined. Using the AFP test with cut-off levels correlated to gestational age, we have detected 8 cases of neural tube defects and one case of abdominal wall defect in about 1,200 pregnancies; false-negative values were absent. Acetylcholinesterase (AChE) and butyrylcholinesterase activities were measured and the electrophoretic pattern of AChE was examined in 100 amniotic fluid samples. The diagnosis of neural tube defects was always confirmed. There were no diagnostic problems due to blood-contaminated amniotic fluid samples. The results obtained using different quantitative methods for the determination of cholinesterase activity, as well as the potential use of these tests in routine examinations, are discussed.
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Tanax or T-61, a euthanasia solution commonly used in veterinary medicine, has been often involved in suicide attempts (humans) and malicious intoxications (animals). For forensic reasons, the identification of one or more of the three components (embutramide, mebenzonium iodide, and tetracaine hydrochloride) of Tanax is needed to confirm the hypothesis of intoxication. This study was performed with new high-performance liquid chromatographic and gas chromatographic-mass spectrometric methods to identify embutramide in biological matrices (blood, liver, kidney) from different animal species. The good sensitivity and specificity of both methods recommend their use in toxicological analysis in both human and veterinary medicine.
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