Ernesto Geremia

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging.

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.

Mitochondrial DNA, RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [3H]thymidine into DNA and of [14C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [3H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.

Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain.

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.

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.

Anti-rhinovirus activity of 3-methylthio-5-aryl-4-isothiazolecarbonitrile derivatives

A series of 3-methylthio-5-aryl-4-isothiazolecarbonitriles has been evaluated as anti rhinovirus agents against a panel of 17 representative human rhinovirus (HRV) serotypes, belonging to both A and B groups. No anti rhinovirus activity was detected for 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (IS-2). Isothiazole derivatives with bulky substituents (O-Bn or O-But groups) on the para position of the phenyl ring were the most effective compounds of this series. In fact, a reduction in virus-induced cytopathogenicity was demonstrated for the O-Bn substituted IS-50 compound against the majority (88%) of the rhinoviruses tested, whereas the compound with an O-Ts group (IS-44) was found to be a specific inhibitor of group B serotypes, exhibiting the lowest IC(50) against HRVs type 2, 85 and 89. Our studies on the mechanism of action of IS-44 demonstrated that it prevents the thermal inactivation of HRV 2 infectivity, probably due to a conformational shift in the viral capsid and a decrease in affinity for the cellular receptor, resulting in an inhibition of attachment of the virions.

Synthesis of new 3,4,5-trisubstituted isothiazoles as effective inhibitory agents of enteroviruses.

The synthesis and evaluation of 3,4,5-trisubstituted isothiazoles as antiviral agents led to the discovery of several compounds effective in vitro against enteroviruses polio 1 and ECHO 9. Structure-activity relationship studies revealed that a short thioalkyl chain in the 3-position and a methyl ester group in the 4-position are the structural components that, to a large extent, contribute to the positive biological profile in terms of both selectivity and low cytotoxicity. Under one-step growth conditions, methyl 3-methylthio-5-phenyl-4-isothiazolecarboxilate caused the greatest activity if added within 1 h after poliovirus adsorption. These data suggest interference with early events of viral replication.

Synthesis and antiviral activity of a new series of 4-isothiazolecarbonitriles

A series of 4-isothiazolecarbonitriles was synthesized and screened for in vitro antiviral activity. The effect of various substituents on the phenyl ring, as well as the substitution of the phenyl for other aromatic and heteroaromatic rings, was examined to establish the requirements for optimum activity. The most active member of the series, 3methylthio-5-phenyl-4-isothiazolecarbonitrile, exhibited a high level of activity against enteroviruses polio 1 and ECHO 9. Preliminary studies on its mechanism of action indicated that this compound had an effect on an early event in the replication of poliovirus type 1.

Effects of Undernutrition on Nucleic Acid Synthesis in Neuronal and Glial Cells from Different Regions of Developing Rat Brain

Rats were undernourished by being given half their normal diet from the 10th day of pregnancy, 10-, 15- and 30-day-old rats were studied. Incorporation of labelled precursors into brain DNA and RNA was carried out in vitro with slices from cerebral cortex, brain stem and cerebellum. Neuronal and glial cells were subsequently isolated and analyzed for specific radioactivity. The proliferation and differentiation of all brain cells were affected by undernutrition. Glial cells in particular and the small neuronal cells appeared most vulnerable probably because of their intense postnatal development.

Antipoliovirus activity of isothiazole derivatives: mode of action of 5,5′-diphenyl-3,3′-diisothiazole disulfide (DID)

SummaryIn this report we describe the antiviral activity of 5,5′-diphenyl-3,3′-diisothiazole disulfide (DID) and discuss its mode of action. DID selectively inhibits the replication of poliovirus type 1 (therapeutic index=255) by affecting some early process of the virus growth cycle. The compound does not interfere with adsorption and internalization of virus to HEp-2 cells, nor with uncoating of the viral RNA. However, no viral RNA synthesis occurs after 2h post-infection in the presence of 50µM DID.Thus, we investigated some molecular events in poliovirus replication occurring between uncoating and viral RNA synthesis. In our experimental design, we studied the activity of RNA polymerase complex isolated from HEp-2 infected cells in the presence or absence of DID. Our results showed that the RNA polymerase complex was formed in the presence of DID. On the contrary, DID markedly inhibited poliovirus RNA synthesis in a cell-free system using RNA polymerase complex isolated from infected cells.These findings indicate that DID may exert its antiviral activity by preventing viral RNA chain elongation via the inhibition of replicase activity and/or interfering with viral RNA polymerase complex.

Inhibitory effects of 3-imino-5-phenyl-3H-1,2-dithiole on poliovirus type 1 replication in vitro

The effect of 3-imino-5-phenyl-3H-1,2-dithiole (PDTI) on different steps of the replicative cycle of poliovirus type 1 in HEp-2 cells was studied. This compound inhibited the replication of poliovirus type 1 as shown by cytopathic effect and virus yield reduction. This inhibitory action was not due to a virucidal effect, nor did the cells to have been pretreated. Under one-step growth conditions 3-imino-5-phenyl-3H-1,2-dithiole caused the greatest inhibition if added within 1 h after poliovirus adsorption. [5-3H]uridine incorporation into RNA showed that PDTI reduced poliovirus RNA synthesis. In fact, in the presence of PDTI viral RNA synthesis was shut off completely at 2 h post infection, and at 4 h post infection viral RNA synthesis was reduced by 50%. The compound may have an inhibitory effect on the early transcriptional and/or replicative functions of the poliovirus genome.