Membrane interactions involved in the induction of acid labile interferon alpha

Human normal peripheral blood mononuclear cells (PBMC) produce acid-labile interferon (IFN) alpha when stimulated in vitro with HIV-infected cells fixed with glutaraldehyde. The cells responsible for IFN production are mainly B lymphocytes. The present study was aimed to further elucidate the cellular source of this IFN and to analyze the membrane interactions involved in the induction process. To this purpose PBMC were stimulated with inducers of acid labile IFN alpha in the presence or absence of a panel of monoclonal antibodies (MoAbs) against antigens of the lymphocyte membrane, namely HLA Class I and II and CD4. The results indicate that both HLA Class II and CD4 antigens are involved in the induction process. Conversely B cell lines seem capable of producing conventional alpha IFN but they fail to produce acid labile IFN alpha even in the presence of cooperating CD4 positive T cell lines. Furthermore PBMC cultured for more than 20 hours prior to stimulation lose the ability to produce acid labile IFN alpha, while remaining fully capable of producing conventional IFN alpha and gamma. It remains to be established whether this phenomenon reflects the disappearance of some membrane structure necessary for acid labile IFN alpha induction, or whether it is due to some early appearing functional alteration of B cells.     

Role of glycosilation in the susceptibility of "acid labile" interferon alpha to acid treatment.

Mononuclear cells from blood of healthy donors produce acid-labile interferon (IFN) alpha when stimulated with HIV-infected cells. A large proportion of this IFN appears to be glycosilated, as treatment with neuraminidase causes a shift of the isoelectric point (IP) from pH = 5.2-5.4 to pH = 5.8-6.2. To assess the role of glycosilation in determining the instability of antiviral activity after exposure to acid (pH lower than 4) peripheral blood mononuclear cells (PBMC) were induced to produce IFN with HIV-infected cells in the presence of tunicamycin, an inhibitor of glycosilation. The IFN produced under such experimental conditions (tu-IFN) was acid-stable. Tu-IFN was compared to a standard acid-labile IFN by affinity chromatography on Con A-sepharose. The elution pattern showed that tu-IFN does not bind to the gel, whereas the acid-labile IFN is eluted in two fractions, one unbound, which is stable at pH2, and one bound, which retains the initial acid-lability. These results suggest that acid-labile IFN alpha is largely glycosilated, and that the presence of glycosilated molecules contribute to render the IFN molecule unstable at acidic pH. It is to be determined whether some glycosilated molecule present in the IFN preparation, or glycosilation of the IFN molecule per se, is responsible for acid-lability of the antiviral activity.     

Variability on morphological and ecological seed traits of Limonium avei (De Not.) Brullo & Erben (Plumbaginaceae)

Limonium avei is an annual species occurring in the salt‐marshes and in limited surfaces of rocky areas around the Mediterranean coasts. Seed lots from five populations of this species, along a latitudinal gradient, were analyzed using an image analysis system to detect differences in seed morphology among populations. Germination requirements at constant (5–25°C) and alternating temperatures (25/10°C), both in light and in darkness, were evaluated for all populations, as well as the effect of the calyx removal on final seed germination and its rate. Morpho‐colorimetric analysis clearly identified seeds from different populations, habitats and substrates without misattributions among them. The calyx slowed the germination process, influencing both final germination and rate with respect to naked seeds. Seeds from all populations germinated with significantly higher percentages in the light, with respect to those incubated in the darkness, and showed rapid germination (time in days to reach 50% of germination: 0.5 days) at the warmer tested temperature (25°C). High germination (>80%) was also detected for seeds of all the investigated populations, except for those from the Apulian region (South Italy, ca. 60%). Our results highlight that L. avei has a high variability in seed morphology, probably habitat induced, and a fast germination response for all populations. Rapid germination may be an adaptive strategy that allows L. avei seeds to take advantage of transient favorable conditions during the germination stage, to ensure seedling establishment under the unpredictable rainfall pattern in the Mediterranean climate.     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

The IFN-alpha-inducing capacity of Sendai Virus in human leukocytes is independent from the hemagglutinating and hemolytic activities and from the viral proteins of the Sendai Virus preparations

About 30 Sendai Virus (SV) preparations, examined for their capacity to induce natural human interferon alpha from fresh human leukocytes (Le-IFN-alpha) of healthy donors, were characterized for hemagglutinating (HA) and hemolytic (HemA) activities and for SDS-PAGE proteic pattern. The SV preparations were produced by a single passage or by serial propagations through eggs in different conditions of multiplicity of infection (m.o.i.).The produced SV subpopulations showed variable IFN-inducing capacity, the values of which are distributed over a 6-fold range resembling a Gaussian distribution. No detectable difference was observed comparing the SV preparation obtained by serial propagations with those obtained by a single passage. The variability of the measured HA activity and HemA activity and as well as the SDS-PAGE proteic pattern of the SV preparations did not correlate with the induced amount of IFN per cell. In the same experimental conditions to produce Le-IFN-alpha, u.v.-treated SV preparations were unable to induce interferon depending on the u.v.-treatment. So it can be concluded that the distinct nHu-IFN-alpha-inducing capacity of SV subpopulation could be mainly associated with divergent compositions of the viral RNAs rather than with a different contents of viral proteins, among those detectable in SDS-PAGE and those responsible for HA activity and for HemA activity.     

Acanthamoeba castellanii promotion of in vitro survival and transmission of coxsackie b3 viruses

This work was undertaken to determine whether Acanthamoeba could play a role in the survival and transmission of coxsackieviruses and focused on in vitro interactions between Acanthamoeba castellanii and coxsackie B3 viruses (CVB-3). Residual virus titer evaluations and immunofluorescence experiments revealed a remarkable CVB-3 adsorption on amoeba surfaces and accumulation inside cells. The survival of viruses was independent of the dynamics of amoeba replication and encystment. In addition, our results indicated that virus-infected amoebas can release infectious viruses during interaction with human macrophages. On the basis of these data, Acanthamoeba appears to be a potential promoter of the survival of coxsackieviruses and their transmission to human hosts.     

Preliminary assessment of the genetic diversity in Lamyropsis microcephala (Asteraceae)

Endangered species with small and isolated populations has been a key topic of conservation biology studies in the last decade. Lamyropsis microcephala is among the most significant narrow endemic plants in the Mediterranean region, lying on the Gennargentu massif of the Sardinia island (Italy). Due to heavy threat factors, this species has rapidly become threatened with extinction. The inter-simple sequence repeat technique was used to assess the genetic variation and structure of the individuals growing in the four remnant localities known to date, with the aim to implement further conservation strategies. Results indicated a degree of differentiation among the four subpopulations, in particular for the Fonni one. The estimates of Nei's genetic diversity (H) ranged from 0.0563 (Fonni) and 0.1104 (Bau ‘e Laccos). Analysis of molecular variance values showed that 53% of the total variation may be attributed to the individuals within subpopulations, while 47% is due to differences among subpopulations (P < 0.001). Results also highlighted a scarce gene flow (N_m = 0.503).