Giovanni Sichel

In vitro scavenger activity of some flavonoids and melanins against O2−dot

The scavenger activity against O2-. of some flavonoids and melanins (synthetic melanins and melanins isolated from animal tissues, vegetable seeds, and mushroom spores) has been studied by ESR spectrometry. All these substances, except flavon and flavanone, diminish the signal of O2-. generated in vitro by a system containing H2O2 and acetone in an alkaline medium. It is shown that the presence of hydroxyl groups in the B ring of flavonoids is essential for their scavenger activity. Moreover, the presence of a hydroxyl at C-3 enhances the scavenger ability of flavonoids. Generally, aglycons are more active than their glycosides. It seems plausible that the antioxidant property of these substances comes from their scavenger activity against O2-(.). It is also pointed out that the scavenger activity shown by melanins, is strictly correlated with their nature of stable free radical.

The gene for SP-40,40 human homolog of rat sulfated glycoprotein 2, rat clusterin, and rat testosterone-repressed prostate message 2, maps to chromosome 8

Sulfated glycoprotein 2 (SGP-2) is a rat glycoprotein that is particularly abundant in seminal fluid, where it is found associated with the acrosome and the tail of mature spermatozoa; for this reason it has been suggested that it has an important role in spermatogenesis. On the basis of nucleotide sequence homology, it has been proposed that the orthologous human gene is that coding for serum protein-40,40 (SP-40,40), a serum protein also called complement lysis inhibitor (CLI), SP-40,40 has been shown to act as a control mechanism of the complement cascade: in fact, it prevents the binding of a C5b-C7 complex to the membrane of the target cell and in this way inhibits complement-mediated cytolysis. SGP-2 and SP-40,40 seem then to be part of different biological systems. Furthermore it has been shown that another protein, testosterone-repressed prostate message 2 (TRPM-2), shares sequence homology with SGP-2 and SP-40,40. TRPM-2 is expressed at high levels and in a temporally precisely defined manner in dying cells, an observation that would suggest its involvement in the cascade of events leading to cell death. We have used a large panel of 24 mouse/human hybrid cell lines and a cDNA for SGP-2, which is also highly homologous to that for rat clusterin, to map the chromosomal location of the orthologous human gene. The mapping data and the Southern analysis presented in this paper, in addition to the data available from the literature, strongly suggest that in the human genome there is a single locus homologous to the probe used and that it codes for the protein which has been called, in different species, SP-40,40, SGP-2, clusterin, and TRPM-2. The chromosomal mapping of the locus for this multiname protein should facilitate its cloning and a better understanding of the apparently many biological functions of its product.

Eumelanins as free radicals trap and superoxide dismutase activities in Amphibia

Abstract 1. 1. Electron paramagnetic resonance (EPR) studies concerning the interaction between purified melanins and superoxide anion are reported; in addition the superoxide dismutase activities in the liver and brain of frog and rat were measured. Liver melanins considerably inhibit the EPR signal ascribed to superoxide anion. 2. 2. The rat and frog brain show similar values of Cu, Zn SOD and Mn SOD. On the contrary, in frog liver, the specific activity of Cu, Zn SOD was significantly lower when compared to rat; no Mn SOD activity has been found in the frog liver.

Melanosynthesis in the kupffer cells of amphibia

Abstract 1. 1. Radioactive melanins have been isolated from liver sections of Rama esculenta L. after incubation in survival with either [ 14 C]tyrosine or [ 14 C]DOPA. 2. 2. In both cases incorporation values are at least two fold when compared with inactivated controls. 3. 3. The histoautoradiograms show that labelled precursors are specifically incorporated in Kupffer cells. 4. 4. It can be concluded that melanins occurring in the pigmented clusters of Amphibian liver originate from melanosynthetic activity of Kupffer cells and not from a phagocytosis process.

Characterisation of Kupffer cells in some Amphibia

A study on the Kupffer cells (KCs) of Amphibia was undertaken in order to compare these cells with those of endothermic animals. Liver tissue and isolated and cultured KCs were studied by light microscopy and by transmission and scanning electron microscopy. We have shown that amphibian KCs can be divided into 2 principal types: ‘small’ and ‘large’. Both cell types possess the distinctive KC morphology. They show nonspecific esterase activity, weak endogenous peroxidase activity in the nuclear envelope and in the rough endoplasmic reticulum, and the ability to engulf naturally present cell debris or experimentally administered zymosan or latex particles. The principal difference between the small and the large cells consists in the substantial quantity of inclusion bodies that exist only in the latter. We conclude that amphibian KCs, apart from their ability to build melanosomes and synthesise melanins, are very similar to mammalian KCs.

The extracutaneous pigmentary system: evidence for the melanosynthesis in Amphibia and Reptilia liver

Abstract 1. 1. In vitro incorporations of [ 14 C] l -tyrosine and [ 14 C] l -DOPA into purified melanin, extracted from frog and turtle liver after incubation of surviving tissue and purified melanosomes were measured. 2. 2. The results show an incorporation of labelled precursors in melanin both in incubating tissue slices and incubating isolated melanosomes and that the radioactivity detected in purified melanin was not due to an adsorption or binding phenomenon of labelled tyrosine. 3. 3. We conclude that melanins occurring in the pigment cells of Amphibia and Reptilia liver originate from the melanosynthetic activity of Kupffer cells and that these pigment cells are to be considered as belonging to the extracutaneous pigmentary system.

Genomics and transcription analysis of human TFIID.

TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAFIIs (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAFIIs role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAFII genes (TAFII250, TAFII135, TAFII100, TAFII80, TAFII55, TAFII43, TAFII31, TAFII28, TAFII20/15) and of two previously unknown sequences related to TAFII250 and TAFII31, respectively. Except for those encoding TAFII250 and TAFII31, these genes are present in a single copy and, with the exclusion of those for TAFII43 and TAFII28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAFII transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAFII20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAFII isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAFII mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.

Genes for human general transcription initiation factors TFIIIB, TFIIIB-associated proteins, TFIIIC2 and PTF/SNAPC: functional and positional candidates for tumour predisposition or inherited genetic diseases?

TFIIIB, TFIIIC2, and PTF/SNAPC are heteromultimeric general transcription factors (GTFs) needed for expression of genes encoding small cytoplasmic (scRNAs) and small nuclear RNAs (snRNAs). Their activity is stimulated by viral oncogenes, such as SV40 large T antigen and Adenovirus E1A, and is repressed by specific transcription factors (STFs) acting as anti-oncogenes, such as p53 and pRb. GTFs role as final targets of critical signal transduction pathways, that control cell proliferation and differentiation, and their involvement in gene expression regulation suggest that the genes encoding them are potential proto-oncogenes or anti-oncogenes or may be otherwise involved in the pathogenesis of inherited genetic diseases. To test our hypothesis through the positional candidate gene approach, we have determined the physical localization in the human genome of the 11 genes, encoding the subunits of these GTFs, and of three genes for proteins associated with TFIIIB (GTF3BAPs). Our data, obtained by chromosomal in situ hybridization, radiation hybrids and somatic cell hybrids analysis, demonstrate that these genes are present in the human genome as single copy sequences and that some cluster to the same cytogenetic band, alone or in combination with class II GTFs. Intriguingly, some of them are localized within chromosomal regions where recurrent, cytogenetically detectable mutations are seen in specific neoplasias, such as neuroblastoma, uterine leyomioma, mucoepidermoid carcinoma of the salivary glands and hemangiopericytoma, or where mutations causing inherited genetic diseases map, such as Peutz-Jeghers syndrome. Their molecular function and genomic position make these GTF genes interesting candidates for causal involvement in oncogenesis or in the pathogenesis of inherited genetic diseases.

Seasonal dependence of ESR features of frog melanins

Abstract 1. 1. Isolated melanins taken from the liver and skin of Rana esculenta L. have been studied by ESR spectroscopy in both cold and warm periods of the year. 2. 2. The following differences have been observed: the ESR spectra of isolated melanins from the winter frogs are asymmetric if recorded at 100 mW of microwave power, whereas in the summer frogs they are symmetric. 3. 3. More over, the spin density is less in winter frogs and higher in summer frogs. 4. 4. That seems to indicate that the polymer undergoes structural modifications which can probably be correlated to the difference in metabolic states between the cold months and the warm ones.

Genetic characterization of general transcription factors TFIIF and TFIIB of Homo sapiens sapiens

Analysis of loci GTF2F1 and GTF2B, encoding Rap 74 (a subunit of TFIIF) and TFIIB, respectively, showed that they are present in a single copy in the human genome and are localized at 19p13.3 and 1p22, respectively. By using as probe a cDNA for Rap 30 (the other subunit of TFIIF), we localized the GTF2F2 locus to 13q14; the same probe also detected a cross-hybridizing sequence at 4q31 whose functional importance remains to be elucidated. These data and those previously published by our group demonstrate that genes coding for class II general transcription factors with reported sequence similarity to bacterial sigma proteins are scattered in different regions of the human genome, with no evidence of clustering. This dispersion and the identification of homologs of both TBP and TFIIB in Archaea suggest an early evolutionary origin of the general transcription apparatus of contemporary eukaryotes.