Temperature dependent 2nd derivative absorbance spectroscopy of aromatic amino acids as a probe of protein dynamics

Proteins display a broad peak in 250–300 nm region of their UV spectrum containing multiple overlapping bands arising from the aromatic rings of phenylalanine, tyrosine, and tryptophan residues. Employing high resolution 2^nd derivative absorbance spectroscopy, these overlapping absorption bands can be highly resolved and therefore provide a very sensitive measure of changes in the local microenvironment of the aromatic side chains. This has traditionally been used to detect both subtle and dramatic (i.e., unfolding) conformational alterations of proteins. Herein, we show that plots of the temperature dependent 2^nd derivative peak positions of aromatic residues have measurable slopes before protein unfolding and that these slopes are sensitive to the dielectric properties of the surrounding microenvironment. We further demonstrate that these slopes correlate with hydration of the buried aromatic residues in protein cores and can therefore be used as qualitative probes of protein dynamics.     

Patterns of divergence of a large family of nodule cysteine‐rich peptides in accessions of Medicago truncatula

The nodule cysteine‐rich (NCR) groups of defensin‐like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant–microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non‐synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions.     

Time resolved secretion of chloride from a monolayer of mucin-secreting epithelial cells

Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (∼50 μm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875–2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl⁻ flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl⁻ may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kinase array design, back to front: biaryl amides

New kinase inhibitors can be found by synthesis of targeted arrays of compounds designed using system-based knowledge as well as through screening focused or diverse compounds. Most array strategies aim to add functionality to a fragment that binds in the purine subpocket of the ATP-site. Here, an alternative pharmacophore-guided array approach is described which set out to discover novel purine subpocket-binding groups. Results are shown for p38alpha and cFMS kinase, for which multiple distinct series with nanomolar potency were discovered. Some of the compounds showed potency in cell-based assays and good pharmacokinetic properties.     

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in beta-cells

Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in beta-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 +/- 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 +/- 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 +/- 311; WT, 425 +/- 231 pg x islet(-1) x 35 min(-1); P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 +/- 0.8 mM glucose, compared with 11.8 +/- 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 +/- 342; WT, 3,493 +/- 367 pg x islet(-1) x 35 min(-1); P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 +/- 0.4-fold in 20 mM glucose, compared with only 1.4 +/- 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.     

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.     

Designing a crop rotation strategy to manage Streptomyces scabies causing potato scab in north India

Common scab of potato caused by various species in the genus Streptomyces has assumed serious proportions in certain potato-growing regions of North India. Although it does not have significant effect on tuber yield, it downgrades quality and reduces marketability. Being soil-borne, this disease is often difficult to manage. Therefore, the present studies were conducted to evaluate different potato-based crop rotations prevalent in north India as a part of a strategy to manage Streptomyces scabies population in scab sick fields. Our results showed that S. scabies population in soil can be effectively reduced either by keeping the field fallow after potato or by including mungbean or sunhemp as green manure, in a single year cropping sequence over a period of four years. Three crop rotations, viz. fallow–rice–potato, mungbean–rice–potato and sunhemp–rice–potato, showed maximum decrease in the population of S. scabies in soil resulting in reduced scab severity. The mungbean- and sunhemp-based crop rotations also enhanced rhizospheric soil microflora especially fluorescent pseudomonads and Trichoderma spp.     

Genomic evidence for gene flow between monarchs with divergent migratory phenotypes and flight performance

Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 km to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analysing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to nonmuscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major‐effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression or by many alleles of small effect.