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.     

Amino acid analysis of bone from a possible case of prehistoric iron deficiency anemia from the American southwest

It is possible that dietary conditions can result in the production of abnormal bone protein. For example, a heavily maize-dependent diet could be deficient in one or more essential amino acids necessary to normal human biochemistry and consequently necessary for normal bone protein synthesis. Amino acid analysis of bone tissues, thus, could provide a useful diagnostic tool in paleopathology. To test this potential we have compared the amino acid analyses of bone samples from a prehistoric Southwest Indian child exhibiting porotic hyperostosis with samples taken from (1) two children's skeletons lacking bone lesions but from the same area and time, (2) a modern child who died from accidental causes, and (3) adult human compact bone. Analytical results of the nonpathological prehistoric specimens were virtually identical to that of the modern infant, indicating remarkable preservation of bone protein. The pathological bone sample differed from the three control specimens by having as much as 25% less of those amino acids containing hydroxyl group and acidic side chains. We interpret the amino acid profile for the diseased child as indicating the presence of a greater proportion of helical protein (or less noncollagenous protein) as well as a lowered degree of hydroxylation of proline and lysine. One explanation for our data is that protein biosynthesis is altered in the child exhibiting porotic hyperostosis, and either some proteins important in the early phases of mineralization are not produced in sufficient quantity, or some necessary enzyme cofactors (e.g., dietary ferrous ions) are missing. We conclude that our data are compatible with, but do not prove, the hypothesis that the porotic hyperostosis exhibited by the Southwest Indian child is the result of iron deficiency anemia.     

Elevated CO2 Levels do not Affect the Shell Structure of the Bivalve Arctica islandica from the Western Baltic

Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO₂ and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO₂ levels (from 380 to 1120 µatm) indicate no affect of elevated pCO₂ on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO₂ levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO₂ related bias.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

Antagonism of kainic acid lesions in the mouse hippocampus by U-54494A and U-50488H.

A morphometric study of kainic acid- (KA) induced lesions was designed for the study of the interaction of the diamines U-5449A and U-50488H with excitatory amino acids, and the dose-response relationship thereof. IC50S determined for binding at the kappa receptor and other opioid receptors demonstrated the lack of kappa activity of U-54494A, a structurally related analog of U-50488H. Both opiate kappa receptor related anticonvulsant diamines were tested for their ability to protect the mouse hippocampus from the cytopathological changes induced by KA in neurons and glia. The damage observed with i.c.v. KA in mouse was restricted to neurons of the CA3 pyramidal region and glia of the hippocampus. It involved massive cell loss and shrunken neurons with dark cytoplasm and nuclei. Groups treated with combinations of KA and U-54494A or U-50488H showed scarce damage, but patches of necrotic changes were still observed. Control animals treated with saline (i.c.v.) and U-54494A (s.c.) or U-50488H (s.c.) did not suffer any noticeable alterations of the polymorphic layers of the hippocampal formation. Image analysis of the CA3 area of the hippocampus was used to quantitate the vacuolization induced by KA lesions in the control and treated groups. By this method, both U-54494A and U-50488H were shown to protect this area in a dose-related fashion as evidenced by reduced vacuolization. The anticonvulsant properties of these compounds may result in the antagonism of the excitotoxic lesions. More specifically, the ability of these diamines to block depolarization-induced influxes of Ca++ may protect the CA3 cells from the cytotoxic effects of persistent depolarization.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

Non-Selective Calcium Channel Blocker Bepridil Decreases Secondary Pathology in Mice after Photothrombotic Cortical Lesion

Experimental studies have identified a complex link between neurodegeneration, β-amyloid (Aβ) and calcium homeostasis. Here we asked whether early phase β-amyloid pathology in transgenic hAPP_SL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPP_SL (n = 33) and non-transgenic (n = 30) male mice (4–5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aβ and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aβ and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aβ accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPP_SL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia.