Physiological function as regulation of large transcriptional programs: the cellular response to genotoxic stress

The responses to ionizing radiation and other genotoxic environmental stresses are complex and are regulated by a number of overlapping molecular pathways. One such stress signaling pathway involves p53, which regulates the expression of over 100 genes already identified. It is also becoming increasingly apparent that the pattern of stress gene expression has some cell type specificity. It may be possible to exploit these differences in stress gene responsiveness as molecular markers through the use of a combined informatics and functional genomics approach. The techniques of microarray analysis potentially offer the opportunity to monitor changes in gene expression across the entire set of expressed genes in a cell or organism. As an initial step in the development of a functional genomics approach to stress gene analysis, we have recently demonstrated the utility of cDNA microarray hybridization to measure radiation-stress gene responses and identified a number of previously unknown radiation-regulated genes. The responses of some of these genes to DNA-damaging agents vary widely in cell lines from different tissues of origin and different genetic backgrounds. While this again highlights the importance of a cellular context to genotoxic stress responses, it also raises the prospect of expression-profiling of cell lines, tissues, and tumors. Such profiles may have a predictive value if they can define regions of ‘expression space’ that correlate with important endpoints, such as response to cancer therapy regimens, or identification of exposures to environmental toxins.     

Biochemical and physiological characterization of a new Na+-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12 Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na⁺-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Nucleotide sequence and characterization of a maize cytoplasmic ribosomal protein S11 cDNA

We isolated a Zea mays cDNA encoding the 40S subunit cytoplasmic ribosomal protein S11. The nucleotide sequence was determined and the derived amino acid sequence compared to the corresponding Arabidopsis thaliana protein showing an homology of 90%. This ribosomal protein is encoded by a small multigene family of at least two members. The mRNA steady-state level is about one order of magnitude higher in rapidly growing parts of the plant such as the roots and shoots of seedlings compared to fully expanded leaf tissue.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

A Novel Rhamnose-binding Lectin Family from Eggs of Steelhead Trout (Oncorhynchus mykiss) with Different Structures and Tissue Distribution

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities).

The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.     

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.