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.     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

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.     

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     

Emerging role of ferrous iron in bacterial growth and host–pathogen interaction: New tools for chemical (micro)biology and antibacterial therapy

Chemical tools capable of detecting ferrous iron with oxidation-state specificity have only recently become available. Coincident with this development in chemical biology has been increased study and appreciation for the importance of ferrous iron during infection and more generally in host–pathogen interaction. Some of the recent findings are surprising and challenge long-standing assumptions about bacterial iron homeostasis and the innate immune response to infection. Here, we review these recent developments and their implications for antibacterial therapy.     

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.