Coexpression with beta(1)-subunit modifies the kinetics and fatty acid block of hH1(alpha) Na(+) channels

Voltage-gated cardiac Na(+) channels are composed of alpha- and beta(1)-subunits. In this study beta(1)-subunit was cotransfected with the alpha-subunit of the human cardiac Na(+) channel (hH1(alpha)) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na(+) currents were assessed. Current density was significantly greater in HEK293t cells coexpressing alpha- and beta(1)-subunits (I(Na,alpha beta)) than in HEK293t cells expressing alpha-subunit alone (I(Na,alpha)). Compared with I(Na,alpha), the voltage-dependent inactivation and activation of I(Na,alpha beta) were significantly shifted in the depolarizing direction. In addition, coexpression with beta(1)-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n-3)] significantly reduced I(Na,alpha beta) in a concentration-dependent manner and at 5 microM shifted the midpoint voltage of the steady-state inactivation by -22 +/- 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n-3)], alpha-linolenic acid [C18:3(n-3)], and conjugated linoleic acid [C18:2(n-6)] at 5 microM significantly inhibited both I(Na,alpha beta) and I(Na,alpha.) In contrast, saturated and monounsaturated fatty acids had no effects on I(Na,alpha beta). This finding differs from the results for I(Na,alpha), which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of beta(1)-subunit with hH1(alpha) modifies the kinetics and fatty acid block of the Na(+) channel.     

The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.     

Gravity independence of seed-to-seed cycling in Brassica rapa

 Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity. Received: 3 August 1999 / Accepted: 27 August 1999     

Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.     

Requirement of both tyrosine residues 1330 and 1337 in the C-terminal tail of the RON receptor tyrosine kinase for epithelial cell scattering and migration

RON is a receptor tyrosine kinase that mediates cell scattering, migration, and tubular formation. This study focused on the function of two tyrosines, Y1330 and Y1337, in the C-terminus of RON in regulating epithelial cell scattering and migration. Substitution of both tyrosine residues with phenylalanine causes complete loss of cell scattering and migration in kidney 293 cells. In contrast, single mutation of either tyrosine residue has no effect. We found that mutation at Y1330 or Y1337 alone does not significantly affect the association of RON with PI-3 kinase, whereas a double mutation abolishes the recruitment of substrates. RON-mediated cell migration was inhibited by PI-3 kinase inhibitor wortmannin. This effect was also achieved by a dominant inhibitory p85 of PI-3 kinase. We conclude that Y1330 and Y1337 are required for RON-mediated cell motility. By associating with PI-3 kinase, the Y1330-Y1337 docking site plays a critical role in transducing motile signals of RON.     

Molecular characterization of a Cryptosporidium isolate from a black bear

To further validate the observation of the existence of host-adapted strains of Cryptosporidium parvum, we genetically characterized an isolate of Cryptosporidium parasite from a black bear. Sequence analysis of the ribosomal RNA small subunit and the 70-kDa heat shock protein (HSP70) showed that this parasite represents a new genotype of C. parvum and is related to the C. parvum dog genotype. This finding is helpful for clarifying Cryptosporidium taxonomy.     

Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

An extremely thermostable restriction endonuclease, PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5′ ^CCWGG 3′ (W is A or T). PspGI digestion can be carried out at 65 to 85°C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR and pspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of α-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI from Micrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity to EcoRII and to regions of SsoII and StyD4I which have a closely related recognition sequence (5′ ^CCNGG 3′). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95°C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.     

Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe²⁺-pyoverdine than for Fe³⁺-pyoverdine. At pH 7.4, about 90% of Fe³⁺ was bound by pyoverdine Pa-C after 24 h whereas Fe²⁺ was bound by the pyoverdine completely in only 5 min. The possibility that Fe²⁺ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe²⁺ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe²⁺ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe³⁺-specific chelating agent, resulted in the formation of a Fe³⁺-hydroxyquinoline complex, suggesting that the iron in the Fe²⁺-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.     

A combinatorial synthesis of tyrphostins via the "directed sorting" method

Using solid-phase organic synthesis, we have prepared a 432-member (18 x 8 x 3) sample library based on the AG 490 "tyrphostin" template. By utilizing 432 reactors each equipped with a unique radiofrequency memory ID tag, the 432 products could be obtained as discrete entities (i.e., not as mixtures) via 18 + 8 + 3, or 29 reactions. Reading each ID tag after each reaction step permitted the "directed sorting" of reactors into appropriate reaction vessels containing multiple reactors. After synthesis, all products were cleaved from the solid-phase support and lyophilized to afford powders. Characterization of 5% of the library members by NMR and mass spectrometry provided verification of structure. In addition, TLC analysis of every library member provided evidence that most (or all) are composed of a single major organic compound. Some 88% of these samples were obtained in amounts of between 5 and 19 mg. Using this reaction sequence and the "directed sorting" approach, the synthesis of much larger AG 490-based libraries can be envisioned.