Apoptotic DNA fragmentation, and its in vitro prevention by nicotinamide, in lymphocytes from HIV-1-seropositive patients and in HIV-1-infected MT-4 cells.

Apoptosis seems to play an important role in the decline of CD4+ T-cells in patients infected with HIV-1. Moreover, extensive interest in apoptosis comes from the observation that it correlates both with the progression and the severity of HIV-1 infection. A cross-sectional study was made to evaluate whether such correlation may also extend to the early phases of ex vivo apoptosis, after 20 h of culture. DNA fragmentation, a parameter associated with apoptosis, was evaluated with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) technique, which preferentially labels apoptosis in comparison to necrosis. The results obtained indicate that a negative correlation exists between the proportion of lymphocytes exhibiting DNA strand breaks and the absolute number of CD4+ T-cells per microliter. DNA fragmentation was significantly higher in patients with AIDS or advanced HIV-1 infection as compared to asymptomatic patients or seronegative individuals. No significant difference was found in relation to antiretroviral therapy. Furthermore, the addition of nicotinamide to the cultures significantly reduced DNA fragmentation of both in vitro HIV-1-infected MT-4 cells and lymphocytes from six HIV-1-seropositive individuals. The results of this study confirm that DNA fragmentation, as an early marker of apoptosis, correlates with the severity of HIV-1 infection and suggest that nicotinamide may be involved in the modulation of HIV-1-related apoptosis.

Investigation of the potential role of membrane CD38 in protection against cell death induced by HIV-1.

The aim of the present study was to assess the possibility that mCD38, a bifunctional ectoenzyme with NAD+ hydrolase and ADP ribose cyclase activities, exerts a protective role in the development of acute, non-syncytial cell death from HIV-1. This hypothesis was tested in a panel of human T-cell lines with defined membrane CD4 (mCD4) expression. A negative correlation was found between the levels of mCD38 expression and the rate of acute cell death from HIV-1 observed 96 h after infection. The negligible rate of cell death from HIV-1 detected in some cell lines (H9 and Supt-1) is apparently unrelated to the level of mCD4 expression, whereas the association with high levels of mCD38 is confirmed. In H9 and Supt-1 cells, the fraction of cells positive to HIV-1 p24 is lower than in the mCD38low cell lines (MT-4, MT-2, C8166). This suggests that high CD38 expression is correlated to resistance to HIV-1 infection, resulting in a lower rate of cell death. A further finding supporting the work hypothesis is that the addition of nicotinamide, a reaction product of CD38, confers to MT-4 cells (mCD38low) partial protection against acute cell death from HIV-1. This indicates that nicotinamide may be at least partially responsible for the correlation observed between high levels of mCD38 and negligible rates of acute cell death from HIV-1.

The whole blood oxygen affinity in normal human newborns: II) P50, n value and related parameters.

The cord blood from 82 normal newborns (48 males and 34 females) has been extensively investigated, to check p50, cooperativity and related parameters. The most important results are the following: Neonatal p50 is slightly above 20 torr, in good agreement with the literature data, but this value refers to the p50 "CO-free" (T50); otherwise, the original p50 is slightly less than 22 torr, a value which is not indicative enough, because neonatal blood contains a consistent excess of Hb-CO. Either this CO-excess and the low p50 value seem to be mainly depending on the low binding capacity of Hb-F with DPG, even if further studies are still necessary at regard. The empirical factor correcting adult p50 to T50 is -0.27, a value which must be increased to -0.36 for neonatal blood. Multiple gas analyses of the samples indicate that oxygen transport is a very complicate equilibrium involving pH, CO2, bicarbonate balance and other factors. A complete statistical analysis of the data has been carried out and the main result indicates that oxygen assumption favours CO2 releasing and vice versa, a phenomenon already well known and globally called "Bohr effect".

The three polymorphic chains of human Hb-F studied in normal Italian newborns.

Both the known polymorphisms of human Hb-F have been investigated here. Two similar groups of full-term newborns have been considered: pure F-globin has been studied by suitable fingerprinting techniques in the first group, while isoelectrofocusing techniques have been employed in the second. The results clearly indicate that G/A gamma polymorphism can be correctly determined with both the procedures, while the T/I gamma polymorphism cannot, because IEF is not sensitive enough towards low quantities of A gamma T chains. Moreover, in the first group of newborns, the A gamma T gene frequency (P) is 0.23, while the total G/A gamma ratio of both groups is about 7/3, the two data being in full agreement with those of the literature. Furthermore, additional reports show that the main functional parameters of the whole neonatal blood are not significantly different either in the presence or in the absence of A gamma T chains.

G gamma and a gamma globin chain synthesis in bone marrow and peripheral blood of beta-thalassaemia homozygotes.

G gamma, A gamma and beta globin chain synthesis has been investigated in the peripheral blood and bone marrow from eight beta-thalassaemia homozygotes. In five out of eight cases total gamma chain synthesis was higher in the peripheral blood than in the bone marrow; in seven out of eight cases A gamma chain synthesis was markedly higher in the marrow than in the peripheral blood. These data suggest that ineffective erythropoiesis selects F-cells synthesizing the largest amounts of G gamma chains, while A gamma producing cells are preferentially destroyed in the marrow.

gamma chain composition in five Italian newborns heterozygous for Hb F Malta G gamma-117 His leads to Arg.

The percentage of Hb F Malta G gamma-177 His replaced by Arg and the gamma chain composition have been evaluated at birth and at different times after birth in five Italian newborns heterozygous for this variant. The percentage of Hb F Malta ranged at birth from 24% to 31% of the total Hb F, while the average G gamma/A gamma chain ratio was about 7/3, overlapping the values observed in normal newborns. T gamma chains were detected in three out of five newborns, with a percentage of about 10% of the total Hb F. After birth the Hb F Malta declined faster than the total Hb F; thus, the Hb F decrease during the first 45 d of life is mainly due to the switch-off of the G gamma locus containing the Hb F Malta gene.

Human T gamma globin chain is a variant of A gamma chain (A gamma Sardinia).

Isoelectric focusing, cellulose acetate electrophoresis, and carboxymethylcellulose chromatography in the presence of Nonidet P-40 allow the separation of pure gamma chains into two fractions. Amino acid analysis of their cyanogen bromide fragment 3 (gamma CB3) identifies these fractions as the separated G gamma (Gly-136) and A gamma (Ala-136) globin chains. Fingerprint and amino acid analyses of the gamma Tp9 tryptic peptide from the purified A gamma and G gamma fractions from two different patients demonstrate that the commonly occurring gamma Sardinia variant (gamma 75 isoleucine leads to threonine), also known as T gamma chain, has alanine in position 136. From this analysis we suggest that the T gamma gene is an allele of the A gamma locus (A gamma Sardinia) rather than a third gamma locus.

[Biochemical and clinical studies of 2 new Italian cases of Hb G-Ferrara heterozygosis]. / Indagini biochimiche e cliniche su due nuovi casi italiani di eterozigosi per l'Hb G-Ferrara.

The Authors report the data concerning a family coming from Ficarolo (Rovigo). The father and one son were found hematologically healthy, but resulted carriers of Hb G-Ferrara. This is an unstable rare pigment with a replacement in the beta 57 (E-1) helicoidal position. The type and site of the mutation explain the "in vitro" molecular instability; on the other hand, this latter doesn't seem to cause any hematological disorder, even if an increased met-Hb production was found when affected blood was incubated at 37 degrees C for two hours in sterile conditions.

Significance of a new type of human fetal hemoglobin carrying a replacement isoleucine replaced by threonine at position 75 )E 19) of the gamma chain.

A new type of hemoglobin F, in which isoleucine in position 75 (E 19) of the gamma chain is replaced by a threonine residue, has been found in 29 out of 32 homozygotes for beta thalassemia. The amount of this hemoglobin ranges from traces to 40% of the total Hb F. The same gamma75 Thr chain is also present in the Hb F of 40% of normal newborns and premature infants examined, of one 14-week-old fetus and in one out of 3 patients with aplastic anemia and raised levels of Hb F. Our results strongly suggest that the synthesis of this new chain is under the control of a gamma gene nonallelic with those coding for Agamma and Ggamma chains.