Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

PdT-2: a novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.

Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides. (C) 2011 Published by Elsevier Inc.

Association between tortilla consumption and human urinary fumonisin B1 levels in a Mexican population

Fumonisins are mycotoxins produced by Fusarium spp. and commonly contaminate maize and maize products worldwide. Fumonisins are rodent carcinogens and have been associated with human esophageal cancer. However, the lack of a valid exposure biomarker has hindered both the assessment of human exposure and the evaluation of disease risk. A sensitive liquid chromatography-mass spectrometry method to measure urinary fumonisin B1 (FB1) following extraction on Oasis MAX cartridges was established and applied to urine samples from women in a cohort recruited in Morelos County, Mexico. Urinary FB1 was compared with dietary information on tortilla consumption. FB1 recovery in spiked samples averaged 94% as judged by deuterium-labeled FB1 internal standard. Urinary FB1 was determined in 75 samples from women selected based on low, medium, or high consumption of maize-based tortillas. The geometric mean (95% confidence interval) of urinary FB1 was 35.0 (18.8-65.2), 63.1 (36.8-108.2), and 147.4 (87.6-248.0) pg/mL and the frequency of samples above the detection limit (set at 20 pg FB1/mL urine) was 45%, 80%, and 96% for the low, medium, and high groups, respectively. Women with high intake had a 3-fold higher average FB1 levels compared with the "low intake" group (F = 7.3; P = 0.0015). Urinary FB1 was correlated with maize intake (P-trend = 0.001); the correlation remained significant after adjusting for age, education, and place of residence. This study suggests that measurement of urinary FB1 is sufficiently sensitive for fumonisin exposure assessment in human populations and could be a valuable tool in investigating the associated health effects of exposure.

When terror hits home: Identity Fused Americans who saw Boston bombing victims as “family” provided aid

When tragedy strikes a group, only some group members characteristically rush to the aid of the victims. What motivates the altruism of these exceptional individuals? Here, we provide one set of answers based on data collected before and shortly after the 15 April 2013, Boston Marathon bombings. The results of three studies indicated that Americans who were strongly “fused” with their country were especially inclined to provide various forms of support to the bombing victims. Moreover, the degree to which participants reported perceiving fellow Americans as psychological kin statistically mediated links between fusion and pro-group outcomes. Together, these findings shed new light on relationships between personal and group identity, cognitive representations of group members, and personally costly, pro-group actions.

Factors associated with spectacle-wear compliance in school-aged Mexican children.

PURPOSE: To study the prevalence and determinants of compliance with spectacle wear among school-age children in Oaxaca, Mexico, who were provided spectacles free of charge. METHODS: A cohort of 493 children aged 5 to 18 years chosen by random cluster sampling from primary and secondary schools in Oaxaca, Mexico, all of whom had received free spectacles through a local program, underwent unannounced, direct examination to determine compliance with spectacle wear within 18 months after initial provision of spectacles. Potential determinants of spectacle wear including age, gender, urban versus rural residence, presenting visual acuity, refractive error, and time since dispensing of the spectacles were examined in univariate and multivariate regression models. Children not currently wearing their spectacles were asked to select the reason from a list of possibilities, and reasons for noncompliance were analyzed within different demographic groups. RESULTS: Among this sample of children with a mean age of 10.4 +/- 2.6 years, the majority (74.5%) of whom were myopic (spherical equivalent [SE] < or = -0.50 D), 13.4% (66/493) were wearing their spectacles at the time of examination. An additional 34% (169/493) had the spectacles with them but were not wearing them. In regression models, the odds of spectacle wear were significantly higher among younger (OR = 1.19 per year of age; 95% CI, 1.05-1.33) rural (OR = 10.6; 95% CI, 5.3-21.0) children and those with myopia < or = -1.25 D (OR = 3.97; 95% CI, 1.98-7.94). The oldest children and children in urban-suburban areas were significantly more likely to list concerns about the appearance of the glasses or about being teased than were younger, rurally resident children. CONCLUSIONS: Compliance with spectacle wear may be very low, even when spectacles are provided free of charge, particularly among older, urban children, who have been shown in many populations to have the highest prevalence of myopia. As screening programs for refractive error become increasingly common throughout the world, new strategies are needed to improve compliance if program resources are to be maximized.

Correction of moderate myopia is associated with improvement in self-reported visual functioning among Mexican school-aged children.

PURPOSE: To quantify the impact on self-reported visual functioning of spectacle provision for school-aged children in Oaxaca, Mexico. METHODS: The Refractive Status Vision Profile (RSVP), a previously validated tool to measure the impact of refractive correction on visual functioning, was adapted for use in rural children and administered at baseline and 4 weeks (27.3 +/- 4.4 days) after the provision of free spectacles. Visual acuity with and without correction, age, sex, and spherical equivalent refraction were recorded at the time of follow-up. RESULTS: Among 88 children (mean age, 12 years; 55.7% girls), the median presenting acuity (uncorrected or with original spectacles), tested 4 weeks after the provision of free spectacles, was 6/9 (range, 6/6-6/120). Significant improvements in the following subscales of the RSVP were seen for the group as a whole after the provision of free spectacles: function, 11.2 points (P = 0.0001); symptoms, 14.3 points (P < 0.0001); total score, 10.3 points (P = 0.0001). After stratification by presenting vision in the better-seeing eye, children with 6/6 acuity (n = 22) did not have significant improvement in any subscale; those with acuity of 6/7.5 to 6/9 (n = 34) improved only on function (P = 0.02), symptoms (P = 0.005), and total score (P = 0.003); and those with acuity of 6/12 or worse improved on total score (P < 0.0001) and all subscales. Subjects (n = 31) with uncorrected myopia of -1.25 D or more had a mean improvement in total score of 15.9 points (P < 0.0001), whereas those with uncorrected myopia between -0.50 and -1.00 D inclusive (n = 53) had a mean improvement of 8 points (P = 0.01). CONCLUSIONS: Provision of spectacles to children in this setting had a significant impact on self-reported function, even at modest levels of baseline visual disability. The correlation between presenting vision/refraction and improvement and the failure of children 6/6 at baseline to improve offer evidence for a real effect.