Exploring the open pore of the potassium channel from Streptomyces lividans

The tetrameric potassium channel from Streptomyces lividans (KcsA) embedded in planar bilayers exhibits the following electrophysiological characteristics: (i) K+ ions can cross the pore in a highly hydrated state (nH2O > or = 6), (ii) the selectivity for K+ exceeds that for Na+ ions by 11 times, and both Ca2+ and Mg2+ are permeant, (iii) the internal side is blocked by Ba2+ ions in a voltage-dependent manner, (iv) intrinsic rectification is due to gating, depending on the direction of the electric field, (v) the internal side is pH-sensitive, and (vi) the open pore has a diameter of approximately 5.8 A. In conclusion, our results show that ion conduction and selectivity of KcsA cannot easily be reconciled with the properties deduced from the rigid crystal structure [Doyle et al., Science 280 (1998) 69-77], which must be concluded to have the pore trapped in its closed state.     

5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons

The purpose of this study was to determine whether the 5-hydroxytryptamine7 (5-HT7) receptor is expressed by nociceptor-like neurons in the rat PNS and whether 5-HT activates these nociceptors via the 5-HT7 receptor subtype. Using a polyclonal antibody and the method of immunofluorescence staining, we demonstrated that the 5-HT7 receptor appears predominately on "nociceptor-like" neurons of the rat lumbar dorsal root ganglia. Using immunocytochemical methods, we showed that the immunoreactivity of the 5-HT7 receptor antibody complex is localized in the superficial layers of the spinal cord dorsal horn, which corresponds with laminae I, IIouter and IIinner. Furthermore, we demonstrated that noxious stimulation produced by knee injection of 5-HT or a 5-HT7 agonist dose-dependently increases c-Fos production of the rat spinal cord dorsal horn. This effect was significantly inhibited by the preinjection of a 5-HT7 antagonist. We conclude that the 5-HT7 receptor is expressed by rat primary afferent nociceptors which terminate in the superficial layers of the spinal cord dorsal horn and that the 5-HT7 receptor subtype is involved in nociceptor activation by 5-HT.     

Insights into [FeFe]-hydrogenase structure, mechanism, and maturation

Hydrogenases are metalloenzymes that are key to energy metabolism in a variety of microbial communities. Divided into three classes based on their metal content, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases are evolutionarily unrelated but share similar nonprotein ligand assemblies at their active site metal centers that are not observed elsewhere in biology. These nonprotein ligands are critical in tuning enzyme reactivity, and their synthesis and incorporation into the active site clusters require a number of specific maturation enzymes. The wealth of structural information on different classes and different states of hydrogenase enzymes, biosynthetic intermediates, and maturation enzymes has contributed significantly to understanding the biochemistry of hydrogen metabolism. This review highlights the unique structural features of hydrogenases and emphasizes the recent biochemical and structural work that has created a clearer picture of the [FeFe]-hydrogenase maturation pathway.     

Combined treatment of heterocyclic analogues and benznidazole upon Trypanosoma cruzi in vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via i.p. route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals.     

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H₂ production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.     

Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.     

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.     

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O₂ evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O₂ evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.Microalgae are able to efficiently convert sunlight, water, and CO₂ into a variety of products suitable for renewable energy applications, including H₂, carbohydrates, and lipids (11, 12, 16, 21, 38, 41, 44). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model organism for studying algal physiology, photosynthesis, metabolism, nutrient stress, and the synthesis of bioenergy carriers (12, 15, 19, 24, 32). During acclimation to nitrogen deprivation, C. reinhardtii cells accumulate significant quantities of starch and form lipid bodies (4, 5, 8, 26, 28, 30, 34, 43, 46, 48). Despite the significance of these products in algal physiology and in biofuels applications, the metabolic, enzymatic, and regulatory mechanisms controlling the partitioning of metabolites into these distinct carbon stores in algae are poorly understood. Several C. reinhardtii starch mutants with various phenotypic changes in starch content and structure have been isolated (2,–4). Two of these, the sta6 and sta7 mutants, contain single-gene disruptions that result in “starchless” phenotypes with severely attenuated levels of starch granule accumulation (2, 4, 34, 39, 40, 48).The disrupted loci in the two isolated starchless mutants are distinct and each mutant has a unique phenotype (7, 40). In the sta6 mutant, the small, catalytic subunit of ADP-glucose pyrophosphorylase (AGPase-SS) is disrupted (2, 4, 48), and this mutant accumulates less than 1% of the starch observed in wild-type (WT) cells under conditions of nitrogen deprivation. The sta7 mutant contains a disrupted isoamylase gene (7, 8, 10, 39, 40) and also has severely attenuated levels of starch, but it accumulates a soluble glycogen-like product (4, 9). In this study, we conducted an examination of the unique physiological acclimations that are utilized by these mutants to adapt to the loss of starch synthesis. As the genetic lesions in these two mutants are distinct and block starch synthesis via two very different mechanisms, we investigated the physiological consequences of starch inhibition in both of these mutants from a holistic bioenergy perspective, which included photosynthetic parameters and the overall yields of lipids and carbohydrates, the two primary bioenergy carriers in C. reinhardtii. Specifically, we examined whether the inability to synthesize starch would result in the accumulation of additional lipid, alter cellular growth or cell size, affect acetate utilization, and/or influence photosynthetic O₂ evolution. Our data indicate that both the sta6 (BAFJ5) and sta7 (sta7-10) mutants accumulate more lipid than the CC124 control during nitrogen deprivation. However, the additional lipid does not completely offset the loss of starch synthesis from a complete energetic perspective. Increased lipid accumulation during nitrogen stress has also been reported for a variety of starch mutants in recent papers (26, 27, 46). A significant feature in both of the starchless mutants studied here is that O₂ evolution and acetate utilization are diminished during nitrogen stress, which is undesirable from an overall bioenergy perspective. Remarkably, complementation of sta7-10 with genomic DNA encoding the wild-type isoamylase gene resulted in cells that were larger than those of the sta6, sta7-10, and CC124 strains, exhibited the highest total lipid levels during nitrogen deprivation, and overaccumulated starch even in nutrient-replete medium.