MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Canine bile acid enterohepatic circulation

The enterohepatic circulation (EHC) of bile acids has been studied in fasting dogs with portacaval shunt maintained in the steady state. In such animals the rate of EHC is proportional to systemic blood bile acid concentration. Bile acid EHC was irregular (20 to 100% variation) when measured at 15 minute or hourly intervals. Studies showed that the variations persisted in cholecystectomized and sphincterectomized animals. The irregularities were enhanced by bethanechol chloride which increases intestinal peristalsis and suppressed by diphenoxylate HCl which slows peristalsis. The variations appear to arise from irregular patterns of intestinal peristalsis. This phenomenon may explain some variations in blood bile acid concentration observed in patients with liver disease.     

Acoustic and Morphological Differentiation in the Frog Allobates femoralis: Relationships with the Upper Madeira River and Other Potential Geological Barriers

We studied patterns of call acoustics and external morphological differentiation in populations of the dart-poison frog Allobates femoralis occurring in forested areas along a 250-km stretch of the upper Madeira River, Brazil. Multivariate analyses of variance using principal components representing shared acoustic and morphological parameters distinguished three groups in relation to call structure and external morphology: (1) populations belonging to a two-note call morphotype; (2) populations with four-note calls inhabiting the left riverbank; and (3) populations with four-note calls inhabiting the right riverbank. Our results report a case of Amazonian anuran diversity hidden by current taxonomy and provide evidence for the upper Madeira River being a boundary between distinct populations of A. femoralis , and suggest a new taxonomic interpretation for these groups. Samples that did not fit into the general differentiation pattern and the existence of a well-defined contact zone between two morphotypes on the left riverbank indicate that mechanisms complementary to river-barrier hypotheses are necessary to explain the phenotypic differentiation between populations. Our study shows that at least one anuran species shows congruence between population differentiation and separation by a large Amazonian river, as documented for birds and mammals. Conservation efforts should not consider the taxon now known as A. femoralis as a homogeneous entity. There is much within-taxon variability, which can be probably explained partly by the existence of cryptic species, partly by geological barriers and part of which currently has no obvious explanation.     

Reproductive ecology and growth of a population of brown trout (Salmo trutta L.) in an aquifer-fed stream of Old Castile (Spain)

In the River Lobos-Ucero and its tributary the River Avión-Milanos (Duero basin, Old Castile, Central Spain), two limestone streams fed by aquifers, the population of brown trout, as compared with the populations of other European streams, shows a high growth rate, high condition coefficients, short life-span and early age at first maturity. Gonad cycle was also studied. Size distributions of unshed eggs exhibit a dynamic activity with a bi-modal distribution from June onwards, spawning occurred in the last days of November. Fecundity (F) can be predicted from trout length (L, mm) according to the equation: F= –646.47+5.6167 · L. Numbers and standing crop of trout range from 18 to 3903 ind. ha^–1 and 3.6 to 452.9 Kg ha^–1, reaching higher values in the sites close to the aquifers. Egg production had values of 22.4 and 18.0 eggs m^–2 in the Rivers Ucero and Avión-Milanos respectively. Some factors suggested as regulators of these demographical characteristics are discussed in the light of recent literature.     

Synthesis and structure-activity relationship of 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives: a novel series of 5-HT(2A/2C) receptor antagonists. Part 2.

Following the programme started at Janssen Research Foundation searching for 5-HT(2A/2C) antagonists, we now report on the synthesis of a series of substituted 2-(Dimethylaminomethyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives. The 5-HT(2A), 5-HT(2C) and H(1) receptor affinities as well as the mCPP antagonistic activity of the compounds synthesised is described.     

Sampling in aerobiology. Differences between traverses along the length of the slide in Hirst sporetraps

Two years of data from four longitudinal traverses along each day's slide prepared from a continuously running Burkard sporetrap have been analyzed statistically. Using the Friedman test, a statistically significant difference was found between the four traverses, with a greater than 7% loss of pollen grains in the two outer traverses in relation to the inner. Four slides were then selected for more detailed analysis, using 18 longitudinal traverses with a 1-mm separation from the upper to the lower edge of the Melinex tape. There was found to be a progressive decline from the centre to the outside, and more than 4% of pollen grains were found outside the typical 14 mm width of the impaction orifice. There was no correlation between pollen grain size and the decline in counts from the centre to the outside. For the complete data set, there was a general rise in the diversity of pollen types with increasing sample counts, but above about 1000 pollen grains per sample there were no more than 27 pollen types found, often even fewer. A discussion is presented of whether four traverses really should be a fixing sample size, or whether it might be better to fix the total pollen count beginning with a traverse in the middle of the slide and ending with a variable number of traverses when that count is reached.     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.     

Microbial Synthesis of Antiviral Nucleosides Using Escherichia coli BL21 as Biocatalyst

Experimental conditions such as shaking (aeration) rate, concentration of reagents and extent of culture growth for the optimal synthesis of adenosine using Escherichia coli BL21 as biocatalyst were assessed, achieving 95% yield in 30 min of reaction using microorganisms harvested from late exponential phase. The ability of E. coli BL21 to synthesise purine nucleosides containing sugar residues such as 2'-deoxyribose, 2',3'-dideoxyribose and arabinose was also verified. 2'-Deoxyribo- and arabinonucleosides could be prepared in high yield, while the results obtained with 2',3'-dideoxyribonucleosides were not satisfactory. In the case of 2'-deoxyadenosine, using thymidine as a starting material, a yield of 94% was achieved at 45°C.     

Stream Noise,Hybridization, and Uncoupled Evolution of Call Traits in Two Lineages of Poison Frogs: Oophaga histrionica and Oophaga lehmanni

According to the acoustic adaptation hypothesis, communication signals are evolutionary shaped in a way that minimizes its degradation and maximizes its contrast against the background noise. To compare the importance for call divergence of acoustic adaptation and hybridization, an evolutionary force allegedly promoting phenotypic variation, we compared the mate recognition signal of two species of poison frogs (Oophaga histrionica and O. lehmanni) at five localities: two (one per species) alongside noisy streams, two away from streams, and one interspecific hybrid. We recorded the calls of 47 males and characterized the microgeographic variation in their spectral and temporal features, measuring ambient noise level, body size, and body temperature as covariates. As predicted, frogs living in noisy habitats uttered high frequency calls and, in one species, were much smaller in size. These results support a previously unconsidered role of noise on streams as a selective force promoting an increase in call frequency and pleiotropic effects in body size. Regarding hybrid frogs, their calls overlapped in the signal space with the calls of one of the parental lineages. Our data support acoustic adaptation following two evolutionary routes but do not support the presumed role of hybridization in promoting phenotypic diversity.