VH1/BRL2 receptor-like kinase interacts with vascular-specific adaptor proteins VIT and VIK to influence leaf venation

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.     

Identification of Diploid <Emphasis Type="Italic">Stylosanthes seabrana</Emphasis> Accessions from Existing Germplasm of <Emphasis Type="Italic">S. scabra</Emphasis> Utilizing Genome-Specific STS Markers and Flow Cytometry,and Their Molecular Characterization

Stylosanthes seabrana (Maass and ‘t Mannetje) (2n = 2x = 20), commonly known as Caatinga stylo, is an important tropical perennial forage legume. In nature, it largely co-exist with S. scabra, an allotetraploid (2n = 4x = 40) species, sharing a very high similarity for morphological traits like growth habit, perenniality, fruit shape and presence of small appendage at the base of the pod or loment. This makes the two species difficult to distinguish morphologically, leading to chances of contamination in respective germplasm collections. In present study, 10 S. seabrana accessions were discovered from the existing global germplasm stock of S. scabra represented by 48 diverse collections, utilizing sequence-tagged-sites (STS) genome-specific markers. All the newly identified S. seabrana accessions displayed STS phenotypes of typical diploid species. Earlier reports have conclusively indicated S. seabrana and S. viscosa as two diploid progenitors of allotetraploid S. scabra. With primer pairs SHST3F3/R3, all putative S. seabrana yielded single band of ~550 bp and S. viscosa of ~870 bp whereas both of these bands were observed in allotetraploid S. scabra. Since SHST3F3/R3 primer pairs are known to amplify single or no band with diploid and two bands with tetraploid species, the amplification patterns corroborated that all newly identified S. seabrana lines were diploid in nature. Flow cytometric measurement of DNA content of the species, along with distinguishing morphological traits such as flowering time and seedling vigour, which significantly differ from S. scabra, confirmed all identified lines as S. seabrana. These newly identified lines exhibited high level of similarity among themselves as revealed by RAPD and STS markers (>92% and 80% respectively). Along with the enrichment in genetic resources of Stylosanthes, these newly identified and characterized accessions of S. seabrana can be better exploited in breeding programs targeted to quality.     

Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin α are capable of generating biologically active IL-1β from its precursor pro-IL-1β. Amino-acid sequencing analysis reveals that meprin A and meprin α cleave pro-IL-1β at the His¹¹⁵-Asp¹¹⁶ bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin β site. The biological activity of the pro-IL-1β cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1β product produced by meprin β or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1β, meprin inhibitor actinonin significantly reduces levels of serum IL-1β. Meprin A and meprin α may therefore play a critical role in the production of active IL-1β during inflammation and tissue injury.     

Nitrooxyethylation reverses the healing-suppressant effect of Ibuprofen

Nonsteroidal antiinflammatory drugs like ibuprofen impede tissue repair by virtue of retarding inflammation. The present study was undertaken to explore if linking of nitrooxyethyl ester to ibuprofen reverses its healing-depressant propensity. Nitrooxyethyl ester of ibuprofen (NOE-Ibu) was synthesized in our laboratory through a well-established synthetic pathway. NOE-Ibu was screened for its influence on collagenation, wound contraction and epithelialization phases of healing, and scar size of healed wound in three wound models, namely, incision, dead space, and excision wounds. Besides, its influence on the oxidative stress (levels of GSH and TBARS) was also determined in 10-day-old granulation tissue. NOE-Ibu was further screened for its antiinflammatory activity in rat paw edema model. NOE-Ibu promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction and epithelialization phases of healing. NOE-Ibu also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to ibuprofen. Results vindicate that the esterification of ibuprofen with nitrooxyethyl group reverses the healing-suppressant effect of ibuprofen. The compound also showed equipotent antiinflammatory activity as ibuprofen.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

Microdissection approaches in tuberculosis research

Mycobacterium tuberculosis, a globally significant pathogen, results in active or latent tuberculosis. The granuloma is the characteristic lesion that offers insight into host–pathogen interactions in these distinct states. Microdissection provides a way to isolate and consequently investigate specific tissue sections. We review various techniques available and in use.     

The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

Longitudinal patterns in carbon and nitrogen fluxes and stream metabolism along an urban watershed continuum

An urban watershed continuum framework hypothesizes that there are coupled changes in (1) carbon and nitrogen cycling, (2) groundwater-surface water interactions, and (3) ecosystem metabolism along broader hydrologic flowpaths. It expands our understanding of urban streams beyond a reach scale. We evaluated this framework by analyzing longitudinal patterns in: C and N concentrations and mass balances, groundwater-surface interactions, and stream metabolism and carbon quality from headwaters to larger order streams. 52 monitoring sites were sampled seasonally and monthly along the Gwynns Falls watershed, which drains 170 km² of the Baltimore Long-Term Ecological Research site. Regarding our first hypothesis of coupled C and N cycles, there were significant inverse linear relationships between nitrate and dissolved organic carbon (DOC) and nitrogen longitudinally (P < 0.05). Regarding our second hypothesis of coupled groundwater-surface water interactions, groundwater seepage and leaky piped infrastructure contributed significant inputs of water and N to stream reaches based on mass balance and chloride/fluoride tracer data. Regarding our third hypothesis of coupled ecosystem metabolism and carbon quality, stream metabolism increased downstream and showed potential to enhance DOC lability (e.g., ~4 times higher mean monthly primary production in urban streams than forest streams). DOC lability also increased with distance downstream and watershed urbanization based on protein and humic-like fractions, with major implications for ecosystem metabolism, biological oxygen demand, and CO₂ production and alkalinity. Overall, our results showed significant in-stream retention and release (0–100 %) of watershed C and N loads over the scale of kilometers, seldom considered when evaluating monitoring, management, and restoration effectiveness. Given dynamic transport and retention across evolving spatial scales, there is a strong need to longitudinally and synoptically expand studies of hydrologic and biogeochemical processes beyond a stream reach scale along the urban watershed continuum.