Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.     

Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, N(omega)-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and Ca(2+)-activated K(+) (K(Ca)) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the K(Ca) channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked.     

Inhibition mechanism of human galectin-7 by a novel galactose-benzylphosphate inhibitor

Galectins are involved in many cellular processes due to their ability to bind carbohydrates. Understanding their functions has shown the necessity for potent and specific galectin inhibitors. Human galectin-7 (hGal-7), in particular, has been highlighted as an important marker in many types of cancer by either inhibiting or promoting tumour growth. Producing ligands able to selectively target hGal-7 will offer promising tools for deciphering cancer processes in which hGal-7 is involved as well as present potential solutions for future therapeutics. Here we report the high resolution crystal structure of hGal-7 in complex with a synthetic 2-O-benzylphosphate-galactoside inhibitor (which is > 60-fold more potent than its parent galactoside). The high resolution crystallographic analysis highlights the validity of using saccharide derivatives, conserving properties of the galactose binding, while enhanced affinity and specificity is provided by the added phosphate group. This structural information will allow the design of further inhibitors with improved potency and specificity.     

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.     

Mobster: accurate detection of mobile element insertions in next generation sequencing data

Mobile elements are major drivers in changing genomic architecture and can cause disease. The detection of mobile elements is hindered due to the low mappability of their highly repetitive sequences. We have developed an algorithm, called Mobster, to detect non-reference mobile element insertions in next generation sequencing data from both whole genome and whole exome studies. Mobster uses discordant read pairs and clipped reads in combination with consensus sequences of known active mobile elements. Mobster has a low false discovery rate and high recall rate for both L1 and Alu elements. Mobster is available at http://sourceforge.net/projects/mobster.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0488-x) contains supplementary material, which is available to authorized users.     

Carbon monoxide contributes to hypotension-induced cerebrovascular vasodilation in piglets

The gaseous compound carbon monoxide (CO) has been identified as an important endogenous biological messenger in brain and is a major component in regulation of cerebrovascular circulation in newborns. CO is produced endogenously by catabolism of heme to CO, free iron, and biliverdin during enzymatic degradation of heme by heme oxygenase (HO). The present study was designed to test the hypothesis that endogenously produced CO contributes to hypotension-induced vasodilation of cerebral arterioles. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of the HO substrate heme-l-lysinate caused dilation of pial arterioles that was blocked by a metal porphyrin inhibitor of HO, chromium mesoporphyrin (CrMP). In normotensive piglets (arterial pressure 64 +/- 4 mmHg), CrMP did not cause vasoconstriction of pial arterioles but rather a transient dilation. Hypotension (50% of basal blood pressure) increased cerebral CO production and dilated pial arterioles from 66 +/- 2 to 92 +/- 7 microm. In hypotensive piglets, topical CrMP or intravenous tin protoporphyrin decreased cerebral CO production and produced pial arteriolar constriction to normotensive diameters. In additional experiments, because prostacyclin and nitric oxide (NO) are also key dilators that can contribute to cerebrovascular dilation, we held their levels constant. NO/prostacyclin clamp was accomplished with continuous, simultaneous application of indomethacin, N(omega)-nitro-l-arginine, and minimal dilatory concentrations of iloprost and sodium nitroprusside. With constant NO and prostacyclin, the transient dilator and prolonged constrictor responses to CrMP of normotensive and hypotensive piglets, respectively, were the same as when NO and prostaglandins were not held constant. These data suggest that endogenously produced CO contributes to cerebrovascular dilation in response to reduced perfusion pressure.     

Variation in carbon isotope composition among years in the riparian tree Populus fremontii

Water availability is an important factor limiting the productivity of desert plants but little is known about the impact of water-limiting conditions on the physiology of plants in mesic environments. Riparian ecosystems of the western US receive significantly more water than the surrounding desert environments but experience dramatic interannual fluctuations in water availability because both stream flow and precipitation are highly variable over time. This variability results in different growing conditions each year which may influence the physiology of riparian species such as Fremont cottonwood (Populus fremontii), the dominant, native canopy tree species in lowland southwestern US river systems. We wished to determine if the physiology of this species varies among years, what climatic parameters are related to any observed physiological variation and if individuals within a P. fremontii population differ in their physiological response to variation through time. We collected tree ring cores from a central New Mexico cottonwood population and analyzed carbon isotope composition (δ¹³C) in each year from 1981 to 1995. We used δ¹³C analysis in this study because it allowed us to obtain multi-year estimates of physiological activity. During these years, mean stream flow at our study site ranged over two orders of magnitude from 0.82 to 80.94 m³ s⁻¹, precipitation ranged fourfold from 49 to 215 ccmm and mean temperature ranged from 20.5 to 22.6°C during the growing season. δ¹³C varied from a low of −26.7‰ in 1984 to a high of −24.7‰ in 1981. Low δ¹³C values were associated with years in which stream flow and/or precipitation were high and temperature was low. The opposite was true of years with high δ¹³C values. We observed a strong linear relationship between δ¹³C and stream flow during years when stream flow was <25 m³ s⁻¹ but no significant relationship between these variables when stream flow was >25 m³ s⁻¹. Additionally, there was a linear relationship between δ¹³C and precipitation during years when stream flow was <25 m³ s⁻¹ but not in years when stream flow was >25 m³ s⁻¹. These data suggest that above a threshold of total stream flow, increased flow does not influence physiology. Below this threshold, precipitation can be an important water source. The ten individuals within our study population varied significantly in mean δ¹³C values but responded to interannual variation in a similar manner (i.e., all individuals had low δ¹³C values when water was abundant). These results suggest that precipitation as well as stream flow are important factors influencing the physiology of this riparian tree. Received: 25 November 1998 / Accepted: 9 February 1999     

Multiple soluble vertebrate galactoside-binding lectins

All vertebrates synthesize soluble galactoside-binding lectins. Many are expressed at high levels in the embryo and at lower levels in the adult, whereas others show an inverse pattern of expression. Most lectins tend to be concentrated in one or a number of specific cell types. In the past few years, the multiplicity of these lectins has become more apparent. For example, in Xenopus laevis 3 galactoside-binding lectins, 2 with a preference for alpha-galactosides, have been purified and partially characterized. They have subunit molecular weights ranging from 16,000 to 69,000. More detailed studies have been done in mammals. For example, rat lung contains 3 soluble beta-galactoside-binding lectins, RL-14.5, RL-18 and RL-29, with subunit molecular weights, respectively, of 14,500, 18,000 and 29,000. A notable feature of these lectins is that, although they all bind lactose about equally well, their carbohydrate-binding sites are actually quite different, as shown by competitive binding studies with a range of complex mammalian glycoconjugates. Human lung also contains several beta-galactoside-binding lectins, including HL-14, HL-22 and HL-29 with subunit molecular weights, respectively, of 14,000, 22,000 and 29,000. They too show significant differences in their carbohydrate-binding sites when analyzed with naturally occurring mammalian glycoconjugates. Sequencing of purified lectins and cDNA clones indicates that at least 4 distinct genes code for what appears to be a family of HL-14. Heterogeneity is also indicated from isoelectric focusing studies which resolve at least 6 acidic forms of HL-14 and 5 acidic forms of HL-29.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats during normoxia and hypoxia.

Effects of exogenous prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats were investigated during normoxia and hypoxia. Pulmonary arterial infusion of prostaglandins E1 and E2 decreased pulmonary vascular resistance 20% and 14%, respectively, without systemic effects. Prostaglandin E1 abolished the pulmonary pressor response to hypoxia. Prostaglandin E2 was less effective in counteracting this hypoxic response. The increased pulmonary vascular resistance and augmented response to hypoxia following indomethacin administration was reversed by prostaglandin E1. Infusion of prostaglandin E1 directly into the pulmonary circulation may be of benefit to the distressed newborn with elevated pulmonary vascular resistance.