Reconstruction of the synthetic gene for human interleukin-2

Chemical-enzymatic synthesis and cloning of the DNA fragment coding for the human interleukin-2 signal sequence was accomplished. A hybrid plasmid pSIL-2 containing the gene of the human interleukin-2 with this signal sequence was constructed for effective expression of the gene in eukaryotic systems. A variant permitting the removal of the interleukin-2 stop-codons was obtained, which is suitable for the construction of chimeric genes containing the interleukin-2 gene sequence at the 5'-end.     

Low-molecular-weight ligands in plants: role in metal homeostasis and hyperaccumulation

Photosynthesis Research - Mineral nutrition is one of the key factors determining plant productivity. In plants, metal homeostasis is achieved through the functioning of a complex system governing...     

Comparative Impacts of Heavy Metals on Root Growth as Related to Their Specificity and Selectivity

Two-day-old maize (Zea mays L.) seedlings were incubated on the solutions of Ag, Cd, Pb, Zn, Cu, Tl, Co, and Hg salts (0.001 to 3 g/l). Toxicity of heavy metals was assessed as the inhibition of root growth on the first, second, and third days, the change in the length of the lateral root zone, and the duration of lateral root development from the first division in pericycle to emergence. For all salts under study, the ratio of the lethal concentration to the lowest concentration slowing down root growth was about ten, and growth inhibition was not almost enhanced in the course of three days. With concentrations calculated as g/l, metal toxicity declined in the following order: Cu Tl > Ag > Cd > Hg > Co > Zn > Pb; for molar concentrations, the order was the following: Tl³⁺ > Cu²⁺ > > Ag⁺ > Hg²⁺ Cd²⁺ > Zn²⁺ Pb²⁺ Co²⁺. Duration of lateral root development was least affected by heavy metals. Metal affinity of biological compounds for SH-groups was closely correlated (r = 0.9) with the molar concentration that inhibited primary root growth by 50%. Because of the narrow range of effective concentrations, only slightly increasing inhibition over the exposure time, tolerant root branching, and close relationship between the toxicity and the constant of binding to SH-groups, we conclude that the salts under study exert nonselective inhibition and root growth is slowed down due to the general toxicity of heavy metals rather than selective inhibition of any particular process or processes.     

Nickel Toxicity and Distribution in Maize Roots

A new histochemical method for Ni determination has been developed and employed to study the pattern of Ni distribution in plant tissues. Two-day-old seedlings of maize (Zea mays L.) were transferred onto 15, 20, 25, and 35 M Ni(NO₃)₂ solutions in the presence of 3 mM Ca(NO₃)₂, and Ni localization in shoot and root tissues was investigated at days 2 and 7 of the incubation. Following two days of incubation, Ni was found in all root tissues, and its content increased with the period of exposure and from the tip to the root base. Independent of root region and tissue, Ni content in the protoplasts exceeded that in the cell walls. Ni penetrated the endodermal barrier and accumulated in the endodermis and pericycle to the highest concentration. Ni accumulation in the pericycle restricted root branching. Ni did not affect the final cell length, and the inhibition of root growth resulted from suppressed cell division. In the shoots, Ni content was below the level discerned by the dimethylglyoximine method; we therefore conclude that maize belongs to excluder plants, with their root systems functioning as a barrier limiting heavy metal intake by aboveground organs. The pattern of Ni transport differs from that of Cd and Pb; this difference stands for specific toxic effects of Ni, including an arrest of root branching.     

Orthopoxvirus Genes for Kelch-like Proteins: II. Construction of Cowpox Virus Variants with Targeted Gene Deletions

Integrative plasmids pC, pD, and pG were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chick embryos.     

Histochemical methods for detection of heavy metals and strontium in the tissues of higher plants

Elaboration and application of histochemical methods for detection of heavy metals (Cd, Pb, Ni, Zn) and strontium for the purpose of investigating their distribution, accumulation, and translocation within the tissues of higher plants are discussed. Detailed protocols of metal detection with metallochrome indicators dithizone (Cd, Pb), dimethylglyoxime (Ni), sodium rhodizonate (Sr), zincon (Zn), and fluorescent indicator Zinpyr-1 (Zn) by light and fluorescence microscopy are described. Special attention is given to interpretation of the obtained results, advantages and drawbacks of these methods, as well as potential problems associated with histochemical analysis of distribution of heavy metals and strontium.     

A common single nucleotide polymorphism in endoplasmic reticulum aminopeptidase 2 induces a specificity switch that leads to altered antigen processing

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (k(cat)) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.     

Junín virus infection activates the type I interferon pathway in a RIG-I-dependent manner

Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.