Isolation and characterization of two genes, waaC (rfaC) and waaF (rfaF), involved in Pseudomonas aeruginosa serotype O5 inner-core biosynthesis.

Recent studies have provided evidence to implicate involvement of the core oligosaccharide region of Pseudomonas aeruginosa lipopolysaccharide (LPS) in adherence to host tissues. To better understand the role played by LPS in the virulence of this organism, the aim of the present study was to clone and characterize genes involved in core biosynthesis. The inner-core regions of P. aeruginosa and Salmonella enterica serovar Typhimurium are structurally very similar; both contain two main chain residues of heptose linked to lipid A-Kdo2 (Kdo is 3-deoxy-D-manno-octulosonic acid). By electrotransforming a P. aeruginosa PAO1 library into Salmonella waaC and waaF (formerly known as rfaC and rfaF, respectively) mutants, we were able to isolate the homologous heptosyltransferase I and II genes of P. aeruginosa. Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. Similarly, plasmid pCOREf1 was able to complement the Salmonella waaF mutant. Sequence analysis of the DNA insert of pCOREc2 revealed one open reading frame (ORF) which could code for a protein of 39.8 kDa. The amino acid sequence of the deduced protein exhibited 53% identity with the sequence of the WaaC protein of S. enterica serovar Typhimurium. pCOREf1 contained one ORF capable of encoding a 38.4-kDa protein. The sequence of the predicted protein was 49% identical to the sequence of the Salmonella WaaF protein. Protein expression by the Maxicell system confirmed that a 40-kDa protein was encoded by pCOREc2 and a 38-kDa protein was encoded by pCOREf1. Pulsed-field gel electrophoresis was used to determine the map locations of the cloned waaC and waaF genes, which were found to lie between 0.9 and 6.6 min on the PAO1 chromosome. Using a gene-replacement strategy, we attempted to generate P. aeruginosa waaC and waaF null mutants. Despite multiple attempts to isolate true knockout mutants, all transconjugants were identified as merodiploids.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

Elucidation of the complete Azorhizobium nicotinate catabolism pathway.

A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.     

Coronary arterial calcification in rheumatoid arthritis: comparison with the Multi-Ethnic Study of Atherosclerosis

Introduction

Although cardiovascular morbidity and mortality are increased in rheumatoid arthritis, little is known about the burden of subclinical coronary atherosclerosis in these patients.

Methods

Using computed tomography, coronary artery calcification was measured in 195 men and women with rheumatoid arthritis aged 45 to 84 years without clinical cardiovascular disease and compared with 1,073 controls without rheumatoid arthritis enrolled in the Baltimore cohort of the Multi-Ethnic Study of Atherosclerosis.

Results

The prevalence of coronary calcification (Agatston score > 0) was significantly higher in men, but not women, with rheumatoid arthritis after adjusting for sociodemographic and cardiovascular risk factors (prevalence ratio = 1.19; P = 0.012). Among participants with prevalent calcification, those with rheumatoid arthritis had adjusted mean Agatston scores 53 units higher than controls (P = 0.002); a difference greater for men than women (P for interaction = 0.017). In all analyses, serum IL-6 attenuated the association between rheumatoid arthritis and coronary calcification, suggesting its role as a potential mediator of enhanced atherosclerosis. Notably, increasing severity of rheumatoid arthritis was associated with a higher prevalence and extent of coronary calcification among both men and women with rheumatoid arthritis, and for all age categories. The largest percentage difference in coronary arterial calcification between rheumatoid arthritis patients and their nonrheumatoid arthritis counterparts was observed in the youngest age category.

Conclusions

Increasing rheumatoid arthritis disease severity was associated with a higher prevalence and greater extent of coronary artery calcification, potentially mediated through an atherogenic effect of chronic systemic inflammation. Gender and age differences in association with coronary calcification suggest that preventive measures should be emphasized in men with rheumatoid arthritis, and considered even in younger rheumatoid arthritis patients with low levels of traditional cardiovascular risk factors.     

Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express

Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult-to-express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult-to-express antibody (mAb-A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb-A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb-A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point-mutant versions of difficult-to-express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.     

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O₂ evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.     

Crop residue incorporation negates the positive effect of elevated atmospheric carbon dioxide concentration on wheat productivity and fertilizer nitrogen recovery

Background and purpose

Rapid increases in atmospheric carbon dioxide concentration ([CO₂]) may increase crop residue production and carbon: nitrogen (C:N) ratio. Whether the incorporation of residues produced under elevated [CO₂] will limit soil N availability and fertilizer N recovery in the plant is unknown. This study investigated the interaction between crop residue incorporation and elevated [CO₂] on the growth, grain yield and the recovery of ¹⁵N-labeled fertilizer by wheat (Triticum aestivum L. cv. Yitpi) under controlled environmental conditions.

Methods

Residue for ambient and elevated [CO₂] treatments, obtained from wheat grown previously under ambient and elevated [CO₂], respectively, was incorporated into two soils (from a cereal-legume rotation and a cereal-fallow rotation) 1 month before the sowing of wheat. At the early vegetative stage ¹⁵N-labeled granular urea (10.22 atom%) was applied at 50 kg?N ha^?1 and the wheat grown to maturity.

Results

When residue was not incorporated into the soil, elevated [CO₂] increased wheat shoot (16 %) and root biomass (41 %), grain yield (19 %), total N uptake (4 %) and grain N removal (8 %). However, the positive [CO₂] fertilization effect on these parameters was absent in the soil amended with residue. In the absence of residue, elevated [CO₂] increased fertilizer N recovery in the plant (7 %), but when residue was incorporated elevated [CO₂] decreased fertilizer N recovery.

Conclusions

A higher fertilizer application rate will be required under future elevated [CO₂] atmospheres to replenish the extra N removed in grains from cropping systems if no residue is incorporated, or to facilitate the [CO₂] fertilization effect on grain yield by overcoming N immobilization resulting from residue amendment.