Identification of Sarcocystis cymruensis in wild Rattus flavipectus and Rattus norvegicus from Peoples Republic of China and its transmission to rats and cats

Sarcocystis cymruensis was initially identified in skeletal muscles of 22 (11.6%) of 189 wild rats (Rattus spp.) captured in 2008 in Anning and Kunming, Peoples Republic of China. Sarcocyst walls were thin (<1 μm) and smooth. Ultrastructurally, the parasitophorous vacuolar membrane had small, osmiophilic knob-like invaginations covered with numerous vesicle-like invaginations toward the interior of the cyst. Domestic cats (Felis catus) fed sarcocysts shed sporocysts measuring 10.3 (9.8-11.0) × 7.6 (7.2-9.5) μm with a prepatent period of 6 to 8 days. Sarcocysts were infective orally to Norway rats, and oocysts and sporocysts developed in the lamina propria of the small intestine of rats fed sarcocysts. Thus, rats were both intermediate and definitive hosts for S. cymruensis.     

Structure and immunological characterization of the capsular polysaccharide of a pyrogenic liver abscess caused by Klebsiella pneumoniae: activation of macrophages through Toll-like receptor 4

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-β-D-Glc-(1→4)-[2,3-(S)-pyruvate]-β-D-GlcA-(1→4)-α-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C(2)-OH or C(3)-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.     

Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.     

Characterization of NocL involved in thiopeptide nocathiacin I biosynthesis: a [4Fe-4S] cluster and the catalysis of a radical S-adenosylmethionine enzyme

The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis. EPR and UV-visible absorbance spectroscopic analyses demonstrated the interaction between L-Trp and the [4Fe-4S] cluster of NocL, leading to the assumption of nonspecific interaction of [4Fe-4S] cluster with other nucleophiles via the unique Fe site. This notion is supported by the finding of the heterogeneity in the [4Fe-4S] cluster of NocL in the absence of AdoMet, which was revealed by the EPR study at very low temperature. Furthermore, a free radical was observed by EPR during the catalysis, which is in good agreement with the hypothesis of a glycyl radical intermediate. Combined with the mutational analysis, these studies provide new insights into the function of the [4Fe-4S] cluster of radical AdoMet enzymes as well as the mechanism of the radical-mediated complex carbon chain rearrangement catalyzed by MIA synthase.     

Mechanical transduction mechanisms of RecA-like molecular motors

A majority of ATP-dependent molecular motors are RecA-like proteins, performing diverse functions in biology. These RecA-like molecular motors consist of a highly conserved core containing the ATP-binding site. Here I examined how ATP binding within this core is coupled to the conformational changes of different RecA-like molecular motors. Conserved hydrogen bond networks and conformational changes revealed two major mechanical transduction mechanisms: (1) intra-domain conformational changes and (2) inter-domain conformational changes. The intra-domain mechanism has a significant hydrogen bond rearrangement within the domain containing the P-loop, causing relative motion between two parts of the protein. The inter-domain mechanism exhibits little conformational change in the P-loop domain. Instead, the major conformational change is observed between the P-loop domain and an adjacent domain or subunit containing the arginine finger. These differences in the mechanical transduction mechanisms may link to the underlying energy surface governing a Brownian ratchet or a power stroke.     

Binding site of Fe3+ at purine of ATP as studied by NMR

The binding site of Fe3+ in the purine base of adenosine 5'-triphosphate (ATP) was studied by nuclear magnetic resonance (NMR). The NMR relaxation rates (R1) of 1H and 31P in ATP solutions free of and containing ferric ions were measured in the pH range of 3-10. It was found that Fe3+ selectively enhanced the relaxation rate of protons. In the presence of Fe3+, the R1 of H2 was much bigger than that of H8 at a lower pH (3-4.5), while at a higher pH (5.5-7.5) the R1 of H8 was more enhanced than H2. At a pH of around 5, both H2 and H8, as well as all three phosphorous, showed a sudden jump in R1. When pH>8, Fe3+ failed to show appreciable enhancement of R1 to all protons and phosphorous. The quantitative data of relaxation rate enhancements suggest that the binding site of Fe3+ in ATP is strongly dependent on pH. At lower pH values, Fe3+ binds N1 but at higher pH it binds to N7. When pH is around 5, the whole purine base donates the aromatic pi-electrons to the ferric ion, forming a ferrocene-like complex, while when pH>8, ATP could not form complexes with Fe3+.     

Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

BACKGROUND: 3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg(2+) for activity. RESULTS: The first three-dimensional structure of the enzyme was determined at 1.4 A resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an alpha + beta fold having a complex linkage of beta strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. CONCLUSIONS: A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg(2+) cofactor within the active site.     

Floral vasculature and ontogeny in Canna indica

The identity of the labellum is a hot point in Zingiberales, which has long been discussed by many authors. In this study, floral vasculature and ontogeny of Canna indica (Cannaceae) was observed by LM and SEM in order to ascertain the identity of the labellum and the functional stamen of this species and provide evidence for the homologies of the floral organs in Zingiberales. The results indicate that the labellum of C. indica have incorporated two androecial members from both outer and inner whorls, rather than three, one or half member, as previously suggested by morphologists of Cannaceae flowers. The two labellum traces are here interpreted as: one from the outer androecial whorl (diverging from the carpellary dorsal bundle), while the other from the inner androecial whorl (diverging from the parietal bundle). The functional stamen also incorporates two androecial bundles, the same as the labellum: one trace from the carpellary dorsal bundle, and the other (the petaloid appendage) from the parietal bundle. In addition, the origin of the vascular system in the androecium of Zingiberales and its systematic significance are discussed.     

Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942

Glycogen synthesis initiated by glucose-1-phosphate adenylyltransferase (glgC) represents a major carbon storage route in cyanobacteria which could divert a significant portion of assimilated carbon. Significant growth retardation in cyanobacteria with glgC knocked out (ΔglgC) has been reported in high light conditions. Here, we knocked out the glgC gene and analyzed its effects on carbon distribution in an isobutanol-producing strain of Synechococcus elongatus PCC7942 and its parental wild-type strain. We showed that isobutanol production was able to partially rescue the growth of ΔglgC mutant where the growth rescue effect positively correlated with the rate of isobutanol production. Using NaH¹⁴CO₃ incorporation analysis, we observed a 28 % loss of total carbon fixation rate in the ΔglgC mutant compared to the wild-type. Upon expression of the isobutanol production pathway in ΔglgC mutant, the total carbon fixation rate was restored to the wild-type level. Furthermore, we showed that 52 % of the total carbon fixed was redirected into isobutanol biosynthesis in the ΔglgC mutant expressing enzymes for isobutanol production, which is 2.5 times higher than that of the wild-type expressing the same enzymes. These results suggest that biosynthesis of non-native product such as isobutanol can serve as a metabolic sink for replacing glycogen to rescue growth and restore carbon fixation rate. The rescue effect may further serve as a platform for cyanobacteria energy and carbon metabolism study.