Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIbeta,a type IA enzyme

Drosophila topoisomerase (topo) IIIbeta is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5'-end of DNA. We show here that a purified preparation of topo IIIbeta is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 degrees C, maximal cleavage occurs between 23 and 30 degrees C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo IIIbeta possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo IIIbeta cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo IIIbeta may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.     

The slippage of the Ca2+ pump and its control by anions and curcumin in skeletal and cardiac sarcoplasmic reticulum

Ca(2+) transport by sarcoplasmic reticulum (SR) ATPase occurs with an optimal coupling ratio of 2 Ca(2+) per ATP in pre-steady state. However, slippage of the pump and lower coupling ratios are observed in steady state. Slippage depends on the presence of high Ca(2+) in the lumen of SR vesicles and high nucleotide in the medium. Thereby, Ca(2+) and/or nucleotide-bound phosphoenzyme intermediates accumulate and undergo uncoupled cleavage, before vectorial translocation of bound Ca(2+) in the forward direction of the cycle or before productive reversal to ATP synthesis. Transport efficiency and coupling ratios are improved by reduction of nucleotide concentration in the presence of ATP regenerating systems and/or complexation of luminal Ca(2+) with phosphate or oxalate. Curcumin (1-5 microm) lowers the concentration of phosphate or oxalate required to reduce slippage of the Ca(2+) pump. Thereby, under appropriate conditions, curcumin favors kinetic flow, completion of productive cycles, and improvement of coupling ratios. The findings obtained with isolated SR vesicles suggest that slippage is an important phenomenon under prevailing conditions of muscle fibers in situ. Ca(2+) transport and its slippage can be improved by curcumin in cardiac as well as in skeletal SR, raising the possibility of pharmacological interventions to correct defective Ca(2+) homeostasis. Higher curcumin concentrations (5-30 microm), however, inhibit overall ATPase activity and Ca(2+) transport by interfering with phosphoenzyme formation with ATP or P(i).     

Apoptotic pathways: involvement of lysosomal proteases

Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.     

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.     

Spectroscopy on individual light-harvesting 1 complexes of Rhodopseudomonas acidophila

In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.     

The mitochondrial DNA polymerase beta from Crithidia fasciculata has 5'-deoxyribose phosphate (dRP) lyase activity but is deficient in the release of dRP

DNA polymerase beta (pol beta) has long been described as a nuclear enzyme involved in DNA repair. A pol beta from the trypanosomatid parasite Crithidia fasciculata, however, is the first example of a mitochondrial enzyme of this type. The mammalian nuclear enzyme functions not only as a nucleotidyl transferase but also has a dRP lyase activity that cleaves 5'-deoxyribose phosphate (dRP) groups from DNA, thus contributing to two consecutive steps of the base excision repair pathway. We find that the mitochondrial pol beta also has dRP lyase activity. Interestingly, the K(m) of this enzyme for a dRP-containing substrate is similar to that for the rat enzyme, but its k(cat) is very low. This difference is due to a deficiency of the mitochondrial enzyme in the release of dRP from the enzyme following its cleavage from the DNA.     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.