The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.     

The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.     

Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.     

Pefabloc – A sulfonyl fluoride serine protease inhibitor blocks induction of Diptericin in Drosophila l(2)mbn cells

Abstract Insects protect themselves against microbial infection by an efficient innate immune system that is activated by recognition of invariant microbial surface molecules. In the fruit fly Drosophila melanogaster the presence of bacteria is associated with expression of antimicrobial peptides in host immune‐competent tissues. Host receptors detect infection and relay the signal to mount the appropriate immune response. In Drosophila hemocyte‐like l(2)mbn cells pre‐infection treatment with Pefabloc, a commonly used serine protease inhibitor, induced two major effects: it blocked expression of the antibacterial peptide Diptericin in response to live Gram‐negative bacteria and bacterial surface molecules (crude lipopolysaccharide contaminated by peptidoglycans) and it induced morphological changes.     

Anatomical defences against bark beetles relate to degree of historical exposure between species and are allocated independently of chemical defences within trees

Conifers possess chemical and anatomical defences against tree‐killing bark beetles that feed in their phloem. Resins accumulating at attack sites can delay and entomb beetles while toxins reach lethal levels. Trees with high concentrations of metabolites active against bark beetle‐microbial complexes, and more extensive resin ducts, achieve greater survival. It is unknown if and how conifers integrate chemical and anatomical components of defence or how these capabilities vary with historical exposure. We compared linkages between phloem chemistry and tree ring anatomy of two mountain pine beetle hosts. Lodgepole pine, a mid‐elevation species, has had extensive, continual contact with this herbivore, whereas high‐elevation whitebark pines have historically had intermittent exposure that is increasing with warming climate. Lodgepole pine had more and larger resin ducts. In both species, anatomical defences were positively related to tree growth and nutrients. Within‐tree constitutive and induced concentrations of compounds bioactive against bark beetles and symbionts were largely unrelated to resin duct abundance and size. Fewer anatomical defences in the semi‐naïve compared with the continually exposed host concurs with directional differences in chemical defences. Partially uncoupling chemical and morphological antiherbivore traits may enable trees to confront beetles with more diverse defence permutations that interact to resist attack.     

Structural and Functional Properties of Glycerol-3-Phosphate Dehydrogenase from a Mammalian Hibernator

Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K_m G3P and DHAP decreased) at low temperature whereas K_m NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K_m G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal.     

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.