Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Sulphate induces very fast cell rounding and detachment

Within a few minutes of incubation with SO4(2-), cultured monolayer cells retract into round shapes with drastically reduced surface area. Concomitant elevation of phosphoinositide second messenger levels, viz, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), is observed. A causal relationship with sulphation seems to be suggested by finding (a) sulphation of an added acceptor, 4-methylumbelliferone, (b) sulphation of endogenous glycosaminoglycan (GAG) polymers, (c) inhibition by phenol sulphotransferase inhibitor, DCNP (2,6-dichloro-4-nitrophenol). DCNP also inhibits the second messenger production and cell rounding. Reduced surface area appears to be caused by massive plasma membrane internalization in a distinctive endocytosis which is also seen in cell rounding from directly imposed ionic gradients. Reducing the surface area would reduce the adhesive or attachment sites. Besides demonstrating a highly efficient cell detachment potential, huge macromolecules appear to be readily internalized. The association of sulphation, signal transduction and cell detachment is novel.     

The RootChip: an integrated microfluidic chip for plant science

Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.     

Prediction of a novel internal rearrangement of the insulin receptor

The insulin receptor (IR) plays critical roles in metabolism and growth, directed by the binding of insulin. Decades of research to understand the mechanism of insulin binding and activation of the IR have identified a region of the receptor, the C-terminal (CT) peptide, to be crucial for insulin binding. In particular, a truncated IR consisting of the first three domains fused to the CT peptide was found to bind insulin with nanomolar affinity, with undetectable binding in the absence of fused or soluble CT peptide. Problematically, all current crystal structures of the IR indicate the fusion point of the CT peptide to the three domains is located far from the position of the CT peptide as resolved in such structures. We have attempted to address this problem using molecular modelling and dynamics simulations. The results led to the identification of a potential inter-domain interaction between the L2 domain and the CT peptide that is not observed in any of the crystal structures of the IR. Investigations into this new interaction found a conformational change that could potentially be in response to insulin binding. Additionally, further simulation work with the new conformation demonstrated its compatibility with the position and orientation of insulin from the latest insulin-bound IR crystal structure.     

The design,synthesis and structure-activity relationships associated with C28 amine-based betulinic acid derivatives as inhibitors of HIV-1 maturation

The design and synthesis of a series of C28 amine-based betulinic acid derivatives as HIV-1 maturation inhibitors is described. This series represents a continuation of efforts following on from previous studies of C-3 benzoic acid-substituted betulinic acid derivatives as HIV-1 maturation inhibitors (MIs) that were explored in the context of C-28 amide substituents. Compared to the C-28 amide series, the C-28 amine derivatives exhibited further improvements in HIV-1 inhibitory activity toward polymorphisms in the Gag polyprotein as well as improved activity in the presence of human serum. However, plasma exposure of basic amines following oral administration to rats was generally low, leading to a focus on moderating the basicity of the amine moiety distal from the triterpene core. The thiomorpholine dioxide (TMD) 20 emerged from this study as a compound with the optimal antiviral activity and an acceptable pharmacokinetic profile in the C-28 amine series. Compared to the C-28 amide 3, 20 offers a 2- to 4-fold improvement in potency towards the screening viruses, exhibits low shifts in the EC₅₀ values toward the V370A and ΔV370 viruses in the presence of human serum or human serum albumin, and demonstrates improved potency towards the polymorphic T371A and V362I virus variants.     

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.     

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P?=?3.9?×?10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P?=?1.9?×?10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.     

Rheumatic fever susceptibility in four ascertainments: regressive segregation on a geometric ascertainment pattern

On resolving the ascertainment biases of the observed data in the geometric continuum vaffected-1 x P(sibship), where 0 less than v----infinity, four published ascertainments of rheumatic fever show excellent conformation with Mendelian recessive segregation, even in multiplex sibships. In two surveys in which ascertainment bias is near or a little above random sampling (v = 1), this conclusion is further corroborated by classical segregation analysis. The other two surveys have bias trends declining (v less than 1) very much below random sampling. Such levels of ascertainment bias, if defined through the ascertainment probability parameter pi, would be out of range because the range is from single ascertainment, where pi----0 to random sampling where pi = 1 and probability cannot exceed unity. Highly successful antimicrobial measures that would reduce the number of diseased sibs independent of the distribution of susceptible sibs could produce a dissociation of the gene-to-"rheumatic" relationship and thus explains the declining ascertainment bias.