Complete genome sequences of two Pseudomonas aeruginosa temperate phages, MP29 and MP42, which lack the phage-host CRISPR interaction

We report the complete genome sequence of two Pseudomonas aeruginosa phages MP29 and MP42. Their genomes are similar to those of P. aeruginosa temperate phages DMS3 and MP22, whose lysogens are impaired in swarming motilities, involving the host CRISPR loci. Both MP29 and MP42 lysogens, however, were proficient in swarming, suggesting the absence of the phage-host CRISPR interaction.     

Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.     

Complex evolution of S5, a major reproductive barrier regulator, in the cultivated rice Oryza sativa and its wild relatives

? The hybrid sterility gene S5 comprises three types of alleles in cultivated rice. Such tri-allelic system provided a unique opportunity to study the molecular bases of evolutionary changes underlying reproductive isolation in plants. ? We analysed the sequence diversity and evolutionary history of S5 in 138 Oryza accessions. We also examined the effect of the two functional variations (C819A and C1412T) in determining hybrid sterility by transformation. ? Nineteen haplotypes were identified, which were classified into the indica-like, the japonica-like and the wide-compatibility gene (WCG)-like group, according to the sequence features of the tri-allelic system. The origin and evolutionary course of the three allelic groups were investigated, thus confirming the independent origins of indica and japonica subspecies. There were perfect associations between C819A and C1412T in the rice germplasm assayed, and the combination of C819 and C1412 was required for hybrid sterility. Evidence of positive selection in the WCG-like alleles suggested that they might have been favored by selection for higher compatibility in hybrids. ? The complex evolution of S5 revealed the counteractive function of the three allelic groups at the species level. S5 might perform an important primary function in an evolutionary scale, and hybrid sterility acts as a 'byproduct' of this speciation gene.     

Single nucleotide polymorphism-mismatch primer development for rapid molecular identification of selected microalgal species

Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.

     

Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture

The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.     

"Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.     

An update on TGN1412

In the last issue of ATLA, we assessed whether the existing methods for assessing the safety and efficacy of new candidate medicines was adequate for the testing of humanised therapeutic agents. We made specific reference to the failed TGN1412 first-in-man study that took place earlier this year. This paper was circulated to experts and those involved in the development or testing of TGN1412. More recently, the Focus on Alternatives group has made a submission to the Expert Working Group that is currently considering how such incidences can be avoided in the future. Here, we provide an update of the events relating to the TGN1412 clinical trial.     

Irgasan-induced pigmentation in Serratia marcescens and Pseudomonas aeruginosa.

Two irgasan-resistant micro-organisms (P. aeruginosa and S. marcescens) were used to study the effects of various antibiotic and chemotherapeutic agents on pigment production. These agents included streptomycin, thallium acetate, polymyxin B, hexachlorophene, irgasan, prodigiosin and DMSO (dimethyl sulphoxide). Only irgasan, compared to other drugs and membrane-active agents showed the unique property of inducing pigmentation in both P. aeruginosa and S. marcescens, i.e. prodigiosin in S. marcescens and pyocyanin in P. aeruginosa.     

BOX-PCR is an adequate tool for typing of clinical Pseudomonas aeruginosa isolates

In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of clinical Pseudomonas aeruginosa isolates. All isolates were typeable and nearly half showed unique banding patterns. According to our results, BOX-PCR fingerprinting is applicable for typing of Pseudomonas aeruginosa isolates and can be considered a useful complementary tool for epidemiological studies of members of this genus.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins

As an extension of previous work from this laboratory using Clostridium pasteurianum flavodoxin [Tollin, G., Cheddar, G., Watkins, J. A., Meyer, T. E., & Cusanovich, M. A. (1984) Biochemistry 23, 6345-6349], we have measured the rate constants as a function of ionic strength for electron transfer from the semiquinones of Clostridium MP, Anacystis nidulans, and Azotobacter vinelandii flavodoxins to the following oxidants: cytochrome c from tuna and horse, Paracoccus denitrificans cytochrome c2, Pseudomonas aeruginosa cytochrome c-551, and ferricyanide. The rate constants extrapolated to infinite ionic strength (k infinity) for the C. MP flavodoxin are all slightly smaller than for the C. pasteurianum flavodoxin, as would be predicted on the basis of the higher redox potential of the C. MP protein. This indicates that there is a close similarity between the surface topographies of the two proteins in the vicinity of the coenzyme binding site. Moreover, the electrostatic interactions between the two flavodoxins and the various oxidants are also approximately the same. These studies justify our previous use of the crystallographic structure of the C. MP flavodoxin to interpret kinetic results obtained with the structurally uncharacterized C. pasteurianum flavodoxin. Despite their lower redox potentials, both Anacystis and Azotobacter flavodoxins are appreciably less reactive toward all of these oxidants (as much as 2 orders of magnitude in some cases) than are the Clostridium flavodoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elaboration of PCR MP diagnostic kit for genetic typing of bacterial strains

The rules of diagnostic kit elaboration for genetic typing of microorganisms, designed for epidemiological studies, have been shown in this paper. PCR MP method has been used for diagnostic kit elaboration. Well defined epidemiologically Enterococcus faecium strains have been applied as a research model. The optimisation of the method has been carried out using different amount of reagents and time of the particular stages. Critical parameters, which have significant influence on the quality of obtained results, have been assigned. Optimalised procedure, named PCR MP unique, has been validated for genetic typing of different species of microorganisms and its potential application for routine epidemiological studies. The PCR MP method has been successfully used for elaboration of diagnostic PCR MP unique-KIT, which allows intra-species differentiation of bacterial strains. The PCR MP unique-KIT enables fast, easy and cheap analysis of strains, using elementary laboratory equipment--gradient thermocycler.     

Pseudomonas aeruginosa septic arthritis of knee after intra-articular ozone injection

We describe a case of septic arthritis caused by Pseudomonas aeruginosa in an immunocompetent patient following intra-articular ozone injection into the knee. To the best of our knowledge, and after considering the current literature,we believe this case is unique as no other reports of septic arthritis caused by P. aeruginosa following intra-articular ozone injection has been made.     

Immunocytochemical localization of cell wall polysaccharides in the root tip ofAvena sativa

Summary Two polyclonal antisera, anti-xyloglucan (anti-XG) and anti-polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), which recognize, respectively, noncellulosic -(14)-D-glucan containing polysaccharides and the unesterified forms of the acidic pectic polysaccharide polygalacturonic acid/rhamnogalacturonan I, were used to localize epitopes recognized by the two antisera in the root tip of oat (Avena sativa). Immunoblot analysis shows that epitopes recognized by the anti-XG antibodies are present in both the mixed linkage -(13)-(14)-D-glucans (MG) and in xyloglucan (XG). Immunogold electron microscopy shows that the cell walls of meristematic, cortical, epidermal, columella, and peripheral cells contain significant amounts of such epitopes. In contrast, the molecules that carry these MG/XG epitopes appear to be sparse in the expanded middle lamella of meristematic cells, but dense in the expanded middle lamella of peripheral root cap cells. This finding suggests that the porosity of the middle lamella is altered in peripheral root cap cells to facilitate mucilage secretion. In contrast, few PGA/RG-I epitopes were detected in any cell walls of any of the cell types examined. Double immunogold labeling experiments revealed an intriguing localization pattern of MG/XG and of PGA/RG-I epitopes in the peripheral mucilage-secreting cells of the root cap. Whereas MG/XG epitopes were abundant in the cell wall, they were sparse in both the secreted mucilage and in intracellular secretory vesicles. In marked contrast, PGA/RG-I epitopes were detected at high density in intracellular secretory vesicles, but unexpectedly, were quite sparse in both the cell wall and in the mucilage. These immunolabeling patterns are consistent with the hypotheses that the synthesis and secretion of particular -D-glucans is subject to both activation and down-regulation during cell development and differentiation and that post-secretory alterations of pectic polysaccharides, such as enzymatic release of RG-I-type mucilage molecules from PGA/RG-I precursors, may occur in the peripheral cell walls of the oat root cap.Abbreviations MG mixed linkage -(13)-(14)-D-glucan - PGA/RG-I polygalacturonic acid/rhamnogalacturonan I - SEPS sycamore extracellular polysaccharides - TGN trans Golgi network - XG xyloglucan     

Quantitative proteome profiling of normal human circulating microparticles

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.     

Membrane interactions of mastoparan analogues related to their differential effects on protein kinase C, Na, K-ATPase and HL60 cells.

Membrane interactions of tetradecapeptide toxin mastoparan (MP) and analogues (MP-3, MP-X and polistes MP), as indicated by inhibition of various enzymatic and cellular activities, were investigated. MP-3 was found to be the least active in inhibiting protein kinase C (PKC; activated by phosphatidylserine vesicles, synaptosomal membranes or phorbol ester), synaptosomal membrane Na,K-ATPase and proliferation and viability of leukemia HL60 cells. MP-3, however, was as active as others in inhibiting PKC activated by arachidonate monomers and phorbol ester binding. The unique properties of MP-3, the [des-Ile1-Asn2]-analogue of MP, might be related to its low functional amphiphilicity compared to others and useful in further delineating biological activities associated with or regulated by membranes.