Michal Shapira

Expression of heat shock protein 83 in Leishmania is regulated post-transcriptionally

Mechanisms for regulation of heat shock protein (hsp) 83 expression were examined in Leishmania amazonesis. Transcripts of hsp83 accumulated upon temperature elevation; however, in contrast to non-protozoan eukaryotes (i.e. Drosophila, yeast, avian or human cells), no transcriptional activation was observed. The increase in the hsp83 mRNA level evolved from temperature induced variations in mRNA turn-over: the hsp83 transcript was rapidly degraded at normal temperatures, whereas heat shock led to its stabilization. The quick decay of the mRNA at lower temperatures was dependent on active protein synthesis. A similar pattern of regulation was observed for the transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked by sequences from the hsp83 intergenic region (IR), and cloned into the pX transfection vector (pX-ICI). CAT mRNA was abundant at normal temperatures and further accumulated upon temperature elevation. The altered turn-over rates of CAT mRNA at the different temperatures were observed only in the presence of flanking hsp83 IR sequences. The increase in temperature also affected translational regulation of hsps, and synthesis of hsp83 was more efficient at 35 degrees C than at 26 degrees C. However, the effect of translation was transient, and the steady state level of the protein was hardly altered.

A synthetic vaccine against influenza with built-in adjuvanticity

In a previous publication we demonstrated that an anti-viral response against influenza can be achieved by immunization with a conjugate of the synthetic peptide corresponding to the sequence 91-108 of the hemagglutinin, when administered in complete Freunds adjuvant. In the present study we compare the adjuvant activity of the synthetic MDP with that of CFA and alum, in the above mentioned immunological system. The level of anti-peptide antibodies raised by the three adjuvants was similar, with only slight variations, yet, only CFA led to significant cross reaction with the virus. Nevertheless, MDP, when linked covalently to the conjugate (91-108)-TT was an efficient substitute for CFA in inducing anti viral protection against in vivo challenge infection. The administration of free MDP in a mixture with the peptide-toxoid conjugate did not lead to a significant protection.

Sequence analysis and transcriptional activation of heat shock protein 83 of Leishmania mexicana amazonensis

Changes in environmental temperature regulate the differential expression of genes during Leishmania stage differentiation. Therefore, molecular analysis of the heat shock proteins (HSPs) in these parasites is of interest as a model for thermoregulation of gene expression. Sequences of the HSP83 repetitive unit in the genome of Leishmania mexicana amazonensis, including both the coding and intergenic regions, are described. The 5 boundary of the message was mapped by S1 analysis, to potential AG splice sites located 293, 295 and 321 nucleotides upstream of the first ATG. A high degree of conservation (84%) is present between the coding sequence of HSP83 from L. mexicana amazonensis and similar sequences from Trypanosoma cruzi. The intergenic leishmanial sequences, however, were not homologous to similar sequences from HSP83 of trypanosomes, or from HSP70 of Leishmania major. A search for sequences that resemble eukaryote thermoregulated promoters was made and several regions with dyad symmetry were detected. However, only one of these regions was partially homologous with the consensus heat shock element present upstream of all eukaryotic HSPs studied to date.

Specificity and cross-reactivity of synthetic peptides derived from a major antigenic site of influenza hemagglutinin.

A synthetic peptide corresponding to sequence 138-164 of influenza hemagglutinin elicited in rabbits antibodies that recognized different parts of the peptide, namely the loop region (139-146) and the rest of the peptide, region 147-164. This was shown both by direct binding and by competitive-inhibition experiments. Individual antisera differed in their specificity. The contribution of the Asp residue at position 144 to the antigenic specificity was shown in various inhibition assays. These data are in accordance with the reported effect of the exchange Gly----Asp on the serological specificity of influenza virus.

Intergenic sequences from the heat-shock protein 83-encoding gene cluster in Leishmania mexicana amazonensis promote and regulate reporter gene expression in transfected parasites

Regulation of expression from hsp83 gene cluster encoding heat-shock protein (HSP) 83 of the protozoan parasite Leishmania mexicana amazonensis (L.m.a) was examined. The first gene from this cluster, along with 8 kb of flanking sequences, was cloned, and intergenic region (IR) sequences were found upstream from the cluster. L.m.a. parasites were electroporated with a plasmid (pICI) in which the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) was cloned between two IRs derived from an internal repeat unit of the hsp83 cluster, resulting in CAT activity at 26 degrees C. Exposure of cells transfected with this plasmid to a 35 degrees C heat shock led to an increase in CAT activity, within a range similar to that observed for the accumulation of hsp83 steady-state mRNA at 35 degrees C. S1 analysis of the hsp83 mRNA showed that the major part of the IR was transcribed and mostly present as 3 non-translated extensions. Deletion analysis of the flanking regions indicated that the presence of IR sequences, both upstream and downstream from cat, was critical to its expression. Partial deletions that removed the original AG splice acceptor site (leaving 289 bp upstream) and downstream IR sequences (leaving 200 bp) did not eliminate CAT activity. However, this combined deletion altered the effect of temperature on cat expression in transfected cells, as compared with the activity measured in cells transfected with the original plasmid.

In Vitro and in Vivo Differentiation of L.Mexicana-Hsp70 Gene Expression

Expression of heat shock proteins (Hsp’s) is a universal response to exposure of cells to stress of various kinds. It is believed to play an important role in protecting the organism against unphysiological growth conditions, and in acquisition of thermotolerance, through mechanisms which are still not uniquely agreed upon. For review see references1–3. Most abundant are proteins of Mr 70 and 83–90 Kd, which are both highly conserved throughout evolution. Proteins of the Hsp70 family all share the characteristic of binding to ATP4–6, and are believed to be involved in prevention or disruption of hydrophobic aggregates through an ATP-dependent mechanism7. Recently it has also been shown that RNA splicing is interrupted by heat shock and rescued by Hsp70 expression8. Most organisms also produce one or more small heat shock proteins, with Mr of 18–30 Kd. These small Hsp’s show a lower degree of structural conservation among species. In some organisms, heat shock genes are expressed independently of environmental stress at specific stages of development9.

Anti-influenza Response Induced with Synthetic Antigen

ABSTRACT The peptide corresponding to sequency 91–108 of the hemagglutinin of type A H3N2 influenza virus has been chemically synthesized. Its conjugate with tetanus toxoid elicited in rabbits antipeptide antibodies that showed marked crossreactivity with the intact virus of several subtype A strains. The antibodies inhibited the hemagglutination assay as well as in vitro plaque formation by the virus. Furthermore, mice immunized with the peptide toxoid conjugate were partially protected against further challenge infection. The results are thus indicative of the efficacy of the synthetic material in eliciting anti-influenza immune response.