Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Performance of a simplified HEART score and HEART-GP score for evaluating chest pain in urgent primary care

BackgroundChest pain is a common symptom in urgent primary care. The distinction between urgent and non-urgent causes can be challenging. A modified version of the HEART score, in which troponin is omitted (‘simplified HEART’) or replaced by the so-called ‘sense of alarm’ (HEART-GP), may aid in risk stratification.MethodThis study involved a retrospective, observational cohort of consecutive patients evaluated for chest pain at a large-scale, out-of-hours, regional primary care facility in the Netherlands, with 6‑week follow-up for major adverse cardiac events (MACEs). The outcome of interest is diagnostic accuracy, including positive predictive value (PPV) and negative predictive value (NPV).ResultsWe included 664 patients; MACEs occurred in 4.8% (n = 32). For  simplified HEART and HEART-GP, we found C‑statistics of 0.86 (95% confidence interval (CI) 0.80–0.91) and 0.90 (95% CI 0.85–0.95), respectively. Optimal diagnostic accuracy was found for a simplified HEART score ≥2 (PPV 9%, NPV 99.7%), HEART-GP score ≥3 (PPV 11%, NPV 99.7%) and HEART-GP score ≥4 (PPV 16%, NPV 99.4%). Physicians referred 157 patients (23.6%) and missed 6 MACEs. A simplified HEART score ≥2 would have picked up 5 cases, at the expense of 332 referrals (50.0%, p < 0.001). A HEART-GP score of ≥3 and ≥4 would have detected 5 and 3 MACEs and led to 293 (44.1%, p < 0.001) and 186 (28.0%, p = 0.18) referrals, respectively.ConclusionHEART-score modifications including the physicians’ ‘sense of alarm’ may be used as a risk stratification tool for chest pain in primary care in the absence of routine access to troponin assays. Further validation is warranted.Supplementary InformationThe online version of this article (10.1007/s12471-020-01529-4) contains supplementary material, which is available to authorized users.     

Unique Gene Expression and MR T2 Relaxometry Patterns Define Chronic Murine Dextran Sodium Sulphate Colitis as a Model for Connective Tissue Changes in Human Crohn’s Disease

Introduction

Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease.

Materials and Methods

C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T ₂ relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis.

Results

Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn’s colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T₂ relaxometry of the colon showed a clear shift towards higher T₂ values in the acute stage and a gradual regression of T₂ values with increasing cycles of DSS.

Conclusions

Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn’s disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T₂ relaxometry is a promising non-invasive assessment of inflammation and fibrosis.     

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

Cas3p belongs to a seven-member family of capsule structure designer proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.     

Heart design: free ADP scales with absolute mitochondrial and myofibrillar volumes from mouse to human

Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005+/-50 (n=16), 460+/-32 (n=10) and 330+/-22 (n=5) mM(-1) and a free cytosolic [ADP] concentration of 13, 18 and 22 microM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (-0.28, r=0.99 and -0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=-0.26, r=0.99) and myofibrillar volumes (slope=-0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.     

Impact of clock gene Bmal1 deficiency on nutritionally induced obesity in mice

To evaluate the hypothesis that the clock gene Bmal1 (brain and muscle arnt like protein-1) plays a role in the development of obesity, 5-week-old male Bmal1-deficient (Bmal1(-/-)) mice and wild-type littermates (Bmal1(+/+)) were kept on a high-fat diet (HFD) for 15 weeks. Despite an initial accelerated weight gain of Bmal1(-/-) mice, body weight and subcutaneous (SC) and gonadal (GON) adipose tissue mass were comparable to Bmal1(+/+) mice at the end of the diet period. Noninvasive magnetic resonance imaging scanning revealed a modest increase in fat content in Bmal1(-/-) mice after 10 weeks of HFD, whereas at the start and the end of the HFD feeding no differences were observed between both genotypes. After 15 weeks of HFD, adipocyte and blood vessel size and density were similar for Bmal1(+/+) and Bmal1(-/-) mice. However, the weight of major organs was significantly reduced in Bmal1(-/-) mice, confirming the premature ageing phenotype. Thus, we hypothesize that an initial accelerated increase in body weight and fat mass of Bmal1(-/-) mice on HFD may have been offset by the effect of premature ageing on organ weight, resulting in comparable weights after 15 weeks of HFD.