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Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin
No full textEvolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche
On the phytoplankton bloom in coastal waters of southern King George Island (Antarctica) in January 2010: An exceptional feature?
No full textSince the early 1990s, phytoplankton has been studied and monitored in Potter Cove (PC) and Admiralty Bay
(AB), King George/25 de Mayo Island (KGI), South Shetlands. Phytoplankton biomass is typically low
compared to other Antarctic shelf environments, with average spring–summer values below 1 mg chlorophyll a
(Chl a) m23. The physical conditions in the area (reduced irradiance induced by particles originated from the land,
intense winds) limit the coastal productivity at KGI, as a result of shallow Sverdrup’s critical depths (Zc) and large
turbulent mixing depths (Zt). In January 2010 a large phytoplankton bloom with a maximum of around 20 mg
Chl a m23, and monthly averages of 4 (PC) and 6 (AB) mg Chl a m23, was observed in the area, making it by far
the largest recorded bloom over the last 20 yr. Dominant phytoplankton species were the typical bloom-forming
diatoms that are usually found in the western Antarctic Peninsula area. Anomalously cold air temperature and
dominant winds from the eastern sector seem to explain adequate light : mixing environment. Local physical
conditions were analyzed by means of the relationship between Zc and Zt, and conditions were found adequate
for allowing phytoplankton development. However, a multiyear analysis indicates that these conditions may be
necessary but not sufficient to guarantee phytoplankton accumulation. The relation between maximum Chl a
values and air temperature suggests that bottom-up control would render such large blooms even less frequent in
KGI under the warmer climate expected in the area during the second half of the present century
The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid
Get PDFMost bacterial genomes contain different types of toxin–antitoxin (TA) systems. The ω-ε-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ε/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ε-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His6-Epsilon by the His6-LonBs, ClpPBs and ClpXBs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation
Cationic derivative of polyprenol, a potential component of liposomal drug carriers, does not alter renal function in rats
No full textPotential toxicity of a cationic derivative of polyprenol (APren‐7) with lipofecting activity was examined,especially its influence on renal haemodynamics and function, in order to conduct further investigation of its suitability as a component of liposomal drug carriers. Sprague–Dawley rats received daily injections (0.5 mL, s.c.) of liposomes comprised of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE) and APren‐7 (LP); classic liposomes composed of DOPE only (L); water solvent (W). After 4 weeks rats were
anaesthetised and a wide spectrum of functional parameters were measured. These included mean arterial blood pressure (MBP), total renal blood flow (RBF), renal cortical, outer and inner medullary blood flow (CBF, OMBF, IMBF, respectively), diuresis, and sodium and solute excretion. Most parameters did not significantly differ between treated and control groups. A lower OMBF/IMBF ratio in
liposome‐treated groups (L: 1.0±0.1; LP: 1.1±0.1 vs. W: 1.9±0.3, p<0.05) might be an early sign of renal function impairment, however, since the lowering occurred in both L and LP groups, there was no reason to suspect that APren‐7 had any untoward influence on renal haemodynamics or excretion. The present results did not reveal toxic effects of APren‐7 in experimental rats, indicating that it could be
suitable as a component of liposomal drug carriers.
Practical applications: Many currently used drugs have severe side effects, which present a serious problem in the treatment of numerous diseases. Searching for compounds augmenting efficacy of drugs to reduce their dosage seems to be a promising strategy. This can be accomplished by facilitating the bioavailability of the drug, e.g. by improving penetration of biological membranes or delivery to
the areas distant from the lumen of blood vessels and in particular instances by improving drug stability. The cationic derivative of polyprenol APren‐7 has lipofecting properties. In this study we found APren‐7 to be harmless to the experimental animals. Newly designed liposomal carriers could find application not only in the pharmaceutical and cosmetics industry but also in biological laboratories and in molecular medicine
cis-Urocanic acid as a potential nickel(ii) binding molecule in the human skin
Get PDFcis-Urocanic acid, a derivative of histidine, is one of the essential components of human skin. We found that it can bind nickel(II) ions in a pH-dependent manner, with the dissociation constant in the low millimolar range, as revealed by potentiometry, and confirmed by isothermal titration calorimetry and UV-vis spectroscopy. The binding occurs within the physiological skin pH range. Considering the fact that cis-urocanic acid is present in the human skin in concentrations as high as millimolar, this molecule may be a physiologically important player in nickel trafficking in the human organism
Highly immunogenic prime–boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus
Get PDFHighly pathogenic avian influenza viruses (HPAIVs) cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA), are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments) indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated) transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8 weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus
Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.
Get PDFThe A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response
NudC Nudix hydrolase from Pseudomonas syringae, but not its counterpart from Pseudomonas aeruginosa, is a novel regulator of intracellular redox balance required for growth, motility and biofilm formation.
No full textNudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria
Feto-maternal haemorrhage assessment in a woman with a large population of red blood cells containing fetal haemoglobin
Get PDFBackground: FMH quantification is necessary to calculate an individual dose of prophylactic anti-RhD immunoglobulin
and to diagnose fetal anaemia causes. We encountered a healthy woman with a numerous RBCs containing
fetal haemoglobin (HbF).
Aims: To investigate the cause of this sign and the correct evaluation of fetal RBCs in maternal circulation.
Materials and Methods: Patient’s samples and artificial mixtures were tested by microscopic Kleihaur-Betke (KB)
and flow cytometric (FC) tests with anti-HbF + anti-CA (carbonic anhydrase), and with anti-D. The patient’s blood
count with reticulocyte parameters, and concentration of bilirubin, haptoglobin, iron, transferrin, ferritin, hepcidin,
sTR, HbF, HbA2 were measured. Genes coding the �- and �-globin were sequenced.
Results: It was impossible to distinguish the population of fetal and maternal HbF positive cells using KBT and FC
with anti-HbF. Application of anti-CA and anti-D allowed to separate them.
Maternal blood haematological and biochemical parameters were normal but HbF was 3.3% of total Hb concentration
(normal <1%). There were no mutations in the �- and �-globin genes, but Xmn I polymorphism at –158 position
in �-globin gene was detected in the homozygous state.
Conclusion: A very large population of HbF positive cells sometimes can be detect in a healthy woman. Implementation
of the various procedures for FMH assessment is necessary in the such case, otherwise, the detection
of fetal erythrocytes may not be possible or can give false results
Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease
No full textMGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA