Formaldehyde causes an increase in blood pressure by activating ACE/AT1R axis.

Previous studies suggest some link between formaldehyde exposure and harmful cardiovascular effects. But whether exposure to formaldehyde can cause blood pressure to rise, and if so, what the underlying mechanism is, remains unclear. In this study, C57BL/6 male mice were exposed to 0.1, 0.5, 2.5 mg/m3 of gaseous formaldehyde for 4 h daily over a three-week period. The systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR) of the mice were measured by tail-cuff plethysmography, and any histopathological changes in the target organs of hypertension were investigated. The results showed that exposure to formaldehyde did cause a significant increase in blood pressure and heart rate, and resulted in varying degrees of damage to the heart, aortic vessels and kidneys. To explore the underlying mechanism, a specific inhibitor of angiotensin converting enzyme (ACE) was used to block the ACE/AT1R axis. We observed the levels of ACE and angiotensin II type 1 receptor (AT1R), as well as the bradykinin (BK) in cardiac cytoplasm. The data suggest that exposure to formaldehyde induced an increase in the expression of ACE and AT1R, and decreased the levels of BK. Strikingly, treatment with 5 mg/kg/d ACE inhibitor can attenuate the increase in blood pressure and the pathological changes caused by formaldehyde exposure. This result has improved our understanding of whether, and how, formaldehyde exposure affects the development of hypertension.

Control of ribosome synthesis in bacteria: the important role of rRNA chain elongation rate.

Bacteria growth depends crucially on protein synthesis, which is limited by ribosome synthesis. Ribosomal RNA (rRNA) transcription is the rate-limiting step of ribosome synthesis. It is generally proposed that the transcriptional initiation rate of rRNA operon is the primary factor that controls the rRNA synthesis. In this study, we established a convenient GFP-based reporter approach for measuring the bacterial rRNA chain elongation rate. We showed that the rRNA chain elongation rate of Escherichia coli remains constant under nutrient limitation and chloramphenicol inhibition. In contrast, rRNA chain elongation rate decreases dramatically under low temperatures. Strikingly, we found that Vibrio natriegens, the fastest growing bacteria known, has a 50% higher rRNA chain elongation rate than E. coli, which contributes to its rapid ribosome synthesis. Our study demonstrates that rRNA chain elongation rate is another important factor that affects the bacterial ribosome synthesis capacity.

Genome-wide screening of lipoproteins in Actinobacillus pleuropneumoniae identifies three antigens that confer protection against virulent challenge.

Actinobacillus pleuropneumoniae is an important veterinary pathogen that causes porcine pleuropneumonia. Lipoproteins of bacterial pathogens play pleiotropic roles in the infection process. In addition, many bacterial lipoproteins are antigenic and immunoprotective. Therefore, characterization of lipoproteins is a promising strategy for identification of novel vaccine candidates or diagnostic markers. We cloned 58 lipoproteins from A. pleuropneumoniae JL03 (serovar 3) and expressed them in Escherichia coli. Five proteins with strong positive signals in western blotting analysis were used to immunize mice. These proteins elicited significant antibody responses, and three of them (APJL_0922, APJL_1380 and APJL_1976) generated efficient immunoprotection in mice against lethal heterologous challenge with A. pleuropneumoniae 4074 (serovar 1), both in the active and passive immunization assays. Then immunogenicity of these three lipoproteins (APJL_0922, APJL_1380 and APJL_1976) were further tested in pigs. Results showed that these proteins elicited considerable humoral immune responses and effective protective immunity against virulent A. pleuropneumoniae challenge. Our findings suggest that these three novel lipoproteins could be potential subunit vaccine candidates.

Functional single-walled carbon nanotubes 'CAR' for targeting dopamine delivery into the brain of parkinsonian mice.

Current treatments for Parkinson's disease (PD) are limited, partly due to the difficulties posed by the blood brain barrier (BBB) when delivering drugs to the brain. Herein, we explore the feasibility and efficacy of functional single-walled carbon nanotubes 'CAR' (SWCNT-PEGs-Lf) which carry and target-deliver dopamine (DA) to the brain in PD mice for treatment. SWCNTs can penetrate the cell-membrane remarkably, with the characteristics including high drug-loading and pH-dependent therapeutic unloading capacities. It has been reported that polyethylene glycol (PEG)-coated SWCNTs could increase the circulation time and thus prolong the concentration gradient of SWCNTs to the brain. Besides, an obvious lactoferrin-nanoparticle (Lf-NP) accumulation in the striatum, wherein the pharmacological target site of PD has been reported, a dual modification of PEG and Lf onto SWCNTs was applied and thus a specific 'CAR' to carry DA. The results from in vitro studies demonstrate that with 20 mol L-1 DA loaded onto SWCNT-polyethylene glycol (PEGs) in addition to 100 µmol L-1 6-hydroxydopamine (6-OHDA), the activity of PC12 cells increases significantly (p < 0.05), and that the lactate dehydrogenase (LDH) levels and reactive oxygen species (ROS) content also significantly decrease (p < 0.01). Furthermore, the levels of oxidative stress, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß are all reduced significantly in PD mice and the CAR-25 mg kg-1 DA group in comparison with that in 6-OHDA-lesioned mice with saline and 6-OHDA-lesioned mice, as well as the Tyrosine hydroxylase-immunoreactive (TH-ir) density increased (p < 0.01). The toxicity of CAR was in vitro and in vivo investigated, showing that the safe dose of SWCNT-PEG exposure to PC12 cells was 6.25 µg µl-1 or lower with a higher metabolic activity in comparison with that in the control group and the safe dose of CAR in the mice experiments was 3.25 mg kg-1 or less, given by intraperitoneal injection with a lower level of oxidative stress and inflammatory responses in comparison with that in the control group. This study suggests that 25 mg kg-1 DA loaded onto 3.25 mg kg-1 CAR can alleviate the oxidative stress and inflammatory responses in parkinsonian mice and increase the TH-ir density in the striatum.

Structure and Function of CW Domain Containing Proteins.

The CW domain is a zinc binding domain, composed of approximately 50- 60 amino acid residues with four conserved cysteine (C) and two to four conserved tryptophan (W) residues. The members of the superfamily of CW domain containing proteins, comprised of 12 different eukaryotic nuclear protein families, are extensively expressed in vertebrates, vertebrate-infecting parasites and higher plants, where they are often involved in chromatin remodeling, methylation recognition, epigenetic regulation and early embryonic development. Since the first CW domain structure was determined 5 years ago, structures of five CW domains have been solved so far. In this review, we will discuss these recent advances in understanding the identification, definition, structure, and functions of the CW domain containing proteins.

The design, synthesis and biological evaluation of novel thiamin diphosphate analog inhibitors against the pyruvate dehydrogenase multienzyme complex E1 from Escherichia coli.

Pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme when looking for inhibitors to combat microbial disease. In this study, we designed and synthesized a series of novel thiamin diphosphate (ThDP) analogs with triazole ring and oxime ether moieties as potential inhibitors of PDHc E1. Their inhibitory activities against PDHc E1 were examined both in vitro and in vivo. Most of the tested compounds exhibited moderate inhibitory activities against PDHc E1 (IC50 = 6.1-75.5 µM). The potent inhibitors 4g, 4h and 4j, had strong inhibitory activities with IC50 values of 6.7, 6.9 and 6.1 µM against PDHc E1 in vitro and with inhibition rates of 35%, 50% and 33% at 100 µg mL(-1) against Gibberella zeae in vivo, respectively. The binding mode of 4j to PDHc E1 was analyzed by a molecular docking method. Furthermore, the possible interactions of the important residues of PDHc E1 with compound 4j were examined by site-directed mutagenesis, enzymatic assays and spectral fluorescence studies. The theoretical and experimental results are in good agreement and suggest that compound 4j could be used as a lead compound for further optimization, and may have potential as a new microbicide.

Crystal structure of human Intersectin-2L C2 domain.

Intersectin-2L (ITSN-2L) is a long isoform of ITSN family, which is a multimodule scaffolding protein functioning in membrane-associated molecular trafficking and signal transduction pathways. ITSN-2L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain, suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. But the role of C2 domain is obscure in this process. Here we report the crystal structure of human ITSN-2L C2 domain at 1.56Å resolution. The sequence and structural alignment of ITSN-2L C2 domain with other members of C2 domain protein family indicate its vital cellular roles in membrane trafficking, the generation of lipid-second messengers and activation of GTPases. Moreover, our data show the possible roles of ITSN-2L C2 domain in regulating the activity of Cdc42.

Identification of Sare0718 as an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205.

BACKGROUND: Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated "amino acid adenylation domain". Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.

Crystal structure of inactive form of Rab3B.

Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

SigB plays a major role in Listeria monocytogenes tolerance to bile stress.

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ(B)) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD(ΔsigB), EGD(ΔprfA) and EGD(ΔprfAΔsigB) with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD(ΔsigB) and EGD(ΔprfAΔsigB) (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD(ΔsigB) and EGD(ΔprfAΔsigB)) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transcription were observed in EGD(ΔsigB) and EGD(ΔprfAΔsigB). Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ(B) might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.

[Analysis of gene structure, cloning and expression of cyp51 from Ustilago maydis].

The cyp51 primers and two pairs of mutant primers which removed different transmembrane region were designed based on Ustilago maydis cyp51 gene structure analysis. The full cyp51 DNA fragment as well as mutant cyp51 genes were amplified and cloned by using the total DNA from Ustilago maydis as template, then subcloned into different expression vectors. The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3), BL21 (DE3) pLysS and Rosetta (DE3) respectively. A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30 degrees C induced with 0.5 mmol/L IPTG The expressed protein (CYP51) showed biological activity by spectra analysis of the protein binding to 4 standard fungicides and to 14 XF-synthetic fungicide compounds, and only one XF-synthetic fungicide compound (XF-113) was similar to standard fungicides in binding constant. This compound is promising to be a new effective antifungal drug. These results will facilitate the further study on the mechanism of pathogenic fungi CYP51 and pesticide molecules, and will provide a new idea for efficient design and development of new anti-fungal drugs.

Some essential elements on the inlC promoter for prfA-dependent regulation in Listeria monocytogenes.

To study some essential elements of a PrfA-dependent promoter of Listeria monocytogenes, a series of promoter mutants incorporated into upstream of a promoterless lacZ gene were constructed from a known listerial PrfA-dependent promoter, inlC promoter, by PCR-mediated site-directed mutagenesis and recombinant PCR technique and then electroporated into L. monocytogenes wild-type strain P14, prfA * mutant Pl4a and prfA deletion mutant A42. The corresponding transcription activities of altered promoters were measured by the beta-galactosidase assay. The results showed that a PrfA-box-like sequence ("pseudo-PrfA-box"), TTAACAGCGTTTGTTAA, 22bp downstream of the transcriptional start site of PinlC had no ability to enhance or inhibit the PrfA-dependent transcription of inlC promoter, even it was modified to the "ideal PrfA-box" TTAACATTTGTTAA. However, there was almost no PrfA-dependent transcriptional activity from the mutants deletion of the inlC original PrfA-box. Moreover, altered spacing between 3'-end of the PrfA-box and 5'-end of the -10 box in the inlC promoter region affected transcription efficiency dramatically, which also happened in another promoter-dependent promoter, plcA promoter. Those above suggested that besides the "PrfA-box", additional unknown PrfA-dependent promoter structure(s) or sequence(s) might be required for the PrfA binding to the promoter and initiation of transcription. Furthermore, the distance between the PrfA-box and the -10 box should be fixed to 22 or 23bp for the PrfA-dependent transcription.