The effects of mispair and nonpair correction in hybrid DNA on base ratios (G + C content) and total amounts of DNA

Base ratios and total DNA amounts can vary substantially between and within higher taxa and genera, and even within species. Gene conversion is one of several mechanisms that could cause such changes. For base substitutions, disparity in conversion direction is accompanied by an equivalent disparity in base ratio at the heterozygous site. Disparity in the direction of gene conversion at meiosis is common and can be extreme. For transitions (which give purine [R]/pyrimidine [Y] mispairs) and for transversions giving unlike R/R and Y/Y mispairs in hybrid DNA, this disparity could give slow but systematic changes in G + C percentage. For transversions giving like R/R and Y/Y mispairs, it could change AT/TA and CG/GC ratios. From the extent of correction direction disparity, one can deduce properties of repair enzymes, such as the ability (1) to excise preferentially the purine from one mispair and the pyrimidine from the other for two different R/Y mispairs from a single heterozygous site and (2) to excise one base preferentially from unlike R/R or Y/Y mispairs. Frame-shifts usually show strong disparity in conversion direction, with preferential cutting of the nonlooped or the looped-out strand of the nonpair in heterozygous h-DNA. The opposite directions of disparity for frame-shifts and their intragenic suppressors as Ascobolus suggest that repair enzymes have a strong, systematic bias as to which strand is cut. The conversion spectra of mutations induced with different mutagens suggest that the nonlooped strand is preferentially cut, so that base additions generally convert to mutant and deletions generally convert to wild-type forms. Especially in nonfunctional or noncoding DNA, this could cause a general increase in DNA amounts. Conversion disparity, selection, mutation, and other processes interact, affecting rates of change in base ratios and total DNA.      

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.     

Methods of modelling viral disease dynamics across the within- and between-host scales: the impact of virus dose on host population immunity

We study the epidemiology of a viral disease with dose-dependent replication and transmission by nesting a differential-equation model of the within-host viral dynamics inside a between-host epidemiological model. We use two complementary approaches for nesting the models: an agent-based (AB) simulation and a mean-field approximation called the growth-matrix (GM) model. We find that although infection rates and predicted case loads are somewhat different between the AB and GM models, several epidemiological parameters, e.g. mean immunity in the population and mean dose received, behave similarly across the methods. Further, through a comparison of our dose-dependent replication model against two control models that uncouple dose-dependent replication from transmission, we find that host immunity in a population after an epidemic is qualitatively different than when transmission depends on time-varying viral abundances within hosts. These results show that within-host dynamics and viral dose should not be neglected in epidemiological models, and that the simpler GM approach to model nesting provides a reasonable tradeoff between model complexity and accuracy of results.     

Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.     

Critical role of helix 12 of the vitamin D(3) receptor for the partial agonism of carboxylic ester antagonists.

The carboxy-terminal alpha-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Delta424-27) and augmented even more, when in addition helix 12 of VDR's dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1alpha,25(OH)(2)D(3). The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR's ability to complex with coactivator proteins and inversely correlated with the strength of the compound's antagonistic action on 1alpha,25(OH)(2)D(3) signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.     

Involvement of Golgin-160 in cell surface transport of renal ROMK channel: co-expression of Golgin-160 increases ROMK currents.

The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins.     

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

Background

At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences.

Results

Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences.

Conclusion

High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.     

Desflurane consumption during automated closed-circuit delivery is higher than when a conventional anesthesia machine is used with a simple vaporizer-O2-N2O fresh gas flow sequence

Background

Current analgesics have drawbacks such as delays in acquisition, lag-times for effect, and side effects. We recently presented a preliminary report of a new analgesic method involving a two-minute sciatic nerve press, which resulted in immediate short-term relief of pain associated with dental and renal diseases. The present study investigated whether this technique was effective for pain associated with other disease types, and whether the relief was effective for up to one hour.

Methods

This randomized, placebo-controlled, parallel-group trial was conducted in four hospitals in Anhui Province, China. Patients with pain were sequentially recruited by participating physicians during clinic visits, and 135 patients aged 15 – 80 years were enrolled. Dental disease patients included those with acute pulpitis and periapical abscesses. Renal disease patients included those with kidney infections and/or stones. Tumor patients included those with nose, breast, stomach and liver cancers, while Emergency Room patients had various pathologies. Patients were randomly assigned to receive a "sciatic nerve press" in which pressure was applied simultaneously to the sciatic nerves at the back of both thighs, or a "placebo press" in which pressure was applied to a parallel region on the front of the thighs. Each fist applied a pressure of 11 – 20 kg for 2 minutes. Patients rated their level of pain before and after the procedure.

Results

The "sciatic nerve press" produced immediate relief of pain in all patient groups. Emergency patients reported a 43.5% reduction in pain (p < 0.001). Significant pain relief for dental, renal and tumor patients lasted for 60 minutes (p < 0.001). The peak pain relief occurred at the 10 – 20^th minutes, and the relief decreased 47% by the 60^th minutes.

Conclusion

Two minutes of pressure on both sciatic nerves produced immediate significant short-term conduction analgesia. This technique is a convenient, safe and powerful method for the short-term treatment of clinical pain associated with a diverse range of pathologies.

Trial registration

Current Controlled Trials ACTRN012606000439549     

Detecting cathepsin activity in human osteoarthritis via activity-based probes

IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0586-5) contains supplementary material, which is available to authorized users.