A previously unrecognized large fraction of cytotoxic lymphocyte precursors is present in CD4+ human peripheral blood T cells

We describe a limiting dilution (LD) culture system in which cell sorter-purified CD4+ (and CD8+) peripheral blood T cells are cocultured with irradiated, anti-CD3 mab-producing OKT3 hybridoma cells. Under these conditions, one out of 2-3 CD4+ (and CD8+) T cells is induced to clonal proliferation. In striking contrast to previously described LD culture systems, every growing CD4+ cell clone displayed cytotoxic activity when tested in a lectin-facilitated 51Cr release assay against P815 target cells. This contrasts with the development of cytotoxic CD4+ T cells in alloantigen-stimulated LD cultures, where only one out of 15-20 proliferating CD4+ cells killed P815 in the presence of PHA, and one out of 300-500 proliferating CD4+ cells displayed alloantigen-specific cytotoxic activity. Furthermore, we have established antigen-specific proliferating CD4+ T cell clones which do not exert antigen-specific cytotoxicity but can be cytotoxic when crosslinked to target cells via lectin or monoclonal antibody (anti-CD3, anti-TCR). Our results show that a previously unrecognized large fraction (at least 30-50%) of all peripheral blood CD4+ T cells can give rise to cytotoxic effector cells. The mode of CD4+ T cell activation (OKT3 hybridoma versus alloantigen) thus determines whether the intrinsic cytotoxic capacity of CD4+ T cells is functionally activated or not.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli

Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K _D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K _D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K _m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V _max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP alkaline phosphatase - Pi inorganic orthophosphate - Km kanamycin     

MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

Histidine is involved in coupling proton uptake to electron transfer in photosynthetic proteins

In photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake and release. Despite intense investigations of different photosynthetic protein complexes, including the photosystem II (PS II) in plants and the reaction center (RC) in bacteria, the molecular details of this fundamental process remain incompletely understood. In the RC of Rhodobacter (Rb.) sphaeroides, fast formation of the charge separated state, P(+)Q(A)(-), is followed by a slower electron transfer from the primary acceptor, Q(A), to the secondary acceptor, Q(B), and the uptake of a proton from the cytoplasm. The proton transfer to Q(B) takes place via a protonated water chain. Mutation of the amino acid AspL210 to Asn (L210DN mutant) near the entry of the proton pathway can disturb this water chain and consequently slow down proton uptake. Time-resolved step-scan Fourier transform infrared (FTIR) measurements revealed an intermediate X in the Q(A)(-)Q(B) to Q(A)Q(B)(-) transition. The nature of this transition remains a matter of debate. In this study, we investigated the role of the iron-histidine complex located between Q(A) and Q(B). We used time-resolved fast-scan FTIR spectroscopy to characterize the Rb. sphaeroides L210DN RC mutant marked with isotopically labeled histidine. FTIR marker bands of the intermediate X between 1120 cm(-1) and 1050 cm(-1) are assigned to histidine vibrations and indicate the protonation of a histidine, most likely HisL190, during the disappearance of the intermediate. Based on these results we propose a novel mechanism of the coupling of electron and proton transfer in photosynthesis.     

The roles of host evolutionary relationships (genus: Nasonia) and development in structuring microbial communities

The comparative structure of bacterial communities among closely related host species remains relatively unexplored. For instance, as speciation events progress from incipient to complete stages, does divergence in the composition of the species’ microbial communities parallel the divergence of host nuclear genes? To address this question, we used the recently diverged species of the parasitoid wasp genus Nasonia to test whether the evolutionary relationships of their bacterial microbiotas recapitulate the Nasonia phylogenetic history. We also assessed microbial diversity in Nasonia at different stages of development to determine the role that host age plays in microbiota structure. The results indicate that all three species of Nasonia share simple larval microbiotas dominated by the γ‐proteobacteria class; however, bacterial species diversity increases as Nasonia develop into pupae and adults. Finally, under identical environmental conditions, the relationships of the microbial communities reflect the phylogeny of the Nasonia host species at multiple developmental stages, which suggests that the structure of an animal's microbial community is closely allied with divergence of host genes. These findings highlight the importance of host evolutionary relationships on microbiota composition and have broad implications for future studies of microbial symbiosis and animal speciation.     

Disease defence through generations: leaf‐cutter ants and their symbiotic bacteria

Microbial ecology of animals is taking on significance in the modern dialogue for the biology of species. Similar to a nuclear genome, the entire bacterial assemblage maintains an ancestral signal of the host's evolution leading to cophylogeny between the host and the microbes they harbour (Brucker & Bordenstein 2012b). The stability of such associations is of great interest as they provide a means for species to acquire new traits and genetic diversity that their own genomes lack (McFall‐Ngai et al. 2013). The role of gut microbiota, for example, in host health and nutrition is widely recognized and a shared characteristic among animals. The role of bacteria colonizing the outside surfaces of animals is less well understood, but rather than random colonization, these microbes on skin, cuticles, scales and feathers in many cases provide benefits to the host. The symbiosis of leaf‐cutter ants, their fungus gardens and their microbiota is a fascinating and complex system. Whether culture‐independent bacterial diversity on the cuticle of leaf‐cutter ants is high or highly constrained by subcuticular gland secretions is one prominent question. In this issue of Molecular Ecology, Andersen et al. (2013) show that leaf‐cutting ants, Acromyrmex echinatior, maintain a dominant and colony‐specific bacterium called Pseudonocardia on their cuticles (the laterocervical plates in particular). This bacterium is involved in protecting the ants and their fungal gardens from disease. Other fungus‐gardening attine species as well as soil and vegetation can harbour Pseudonocardia. However, it was previously unknown how stable the bacterial strain–ant colony association was through the lifetime of the colony.     

Disruption of the termite gut microbiota and its prolonged consequences for fitness

The disruption of host-symbiont interactions through the use of antibiotics can help elucidate microbial functions that go beyond short-term nutritional value. Termite gut symbionts have been studied extensively, but little is known about their impact on the termite's reproductive output. Here we describe the effect that the antibiotic rifampin has not only on the gut microbial diversity but also on the longevity, fecundity, and weight of two termite species, Zootermopsis angusticollis and Reticulitermes flavipes. We report three key findings: (i) the antibiotic rifampin, when fed to primary reproductives during the incipient stages of colony foundation, causes a permanent reduction in the diversity of gut bacteria and a transitory effect on the density of the protozoan community; (ii) rifampin treatment reduces oviposition rates of queens, translating into delayed colony growth and ultimately reduced colony fitness; and (iii) the initial dosages of rifampin had severe long-term fitness effects on Z. angusticollis. Taken together, our findings demonstrate that the antibiotic-induced perturbation of the microbial community is associated with prolonged reductions in longevity and fecundity. A causal relationship between these changes in the gut microbial population structures and fitness is suggested by the acquisition of opportunistic pathogens and incompetence of the termites to restore a pretreatment, native microbiota. Our results indicate that antibiotic treatment significantly alters the termite's microbiota, reproduction, colony establishment, and ultimately colony growth and development. We discuss the implications for antimicrobials as a new application to the control of termite pest species.