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.     

The mitochondrial and nuclear genetic structure of Myotis capaccinii (Chiroptera: Vespertilionidae) in the Eurasian transition, and its taxonomic implications

Allopatric isolation in glacial refugia has caused differentiation and speciation in many taxa globally. In this study, we investigated the nuclear and mitochondrial genetic differentiation of the long fingered bat, Myotis capaccinii during the ice ages in south-eastern Europe and Anatolia. The mitochondrial DNA (mtDNA) analyses indicated a suture zone similar to those recorded in other animal species, including bats, suggesting the association of more than one refugium with the region. Contrary to most of the other species where a suture zone was seen in Anatolia, for M. capaccinii the geographical location of the genetic break was in south-eastern Europe. This mitochondrial differentiation was not reflected in the nuclear microsatellites, however, suggesting that the lack of contact during the ice ages did not result in reproductive isolation. Hence taxonomically, the two mitochondrial clades cannot be treated as separate species.     

Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin-protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.     

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.     

Electrophysiological characteristics of cardiac pacemaker cells of the frog Caudiverbera caudiverbera

1. The cardiac pacemaker cells of the frog Caudiverbera caudiverbera are centrally located in the sinus venosus. These cells are rounded, smaller than contractile fibres and have large nuclei. 2. Intracellular recording confirmed the existence of primary and transitional pacemaker cells. 3. Action potentials from primary cells were resistant to blockade by tetrodotoxin (TTX), but were abolished by verapamil suggesting that their bioelectric activity is dependent on a slow inward current. 4. Transitional cells appeared to have two different inward currents contributing to the upstroke: a fast TTX-sensitive and a slow verapamil-sensitive current.     

Natural antibodies to interferon-gamma

Natural antibodies to interferon (IFN)-γ were detected in the serum of virus-infected patients and also, at a low titre, in the serum of healthy subjects. The increased titre of antibodies to IFN-γ in the sera of virus-infected patients, and its decrease with clinical resolution, indicate that these antibodies are related to viral infection and probably reflect IFN-γ production as a result of antigenic stimulationin vivo. Natural antibodies to IFN-γ were affinity purified and studied for their capability to interferein vitro with the multiple activities of the lymphokine. Data obtained show that these human anti-IFN-γ antibodies have no inhibitory effect on the antiviral and antiproliferative activity of IFN-γ and do not interfere with the binding of the lymphokine to its specific cell receptor. Instead, they can inhibit the expression of HLA-DR antigens induced by IFN-γ on U937 cells and interfere, in mixed lymphocyte culture, with the proliferation of lymphocytes and the generation of cytotoxic lymphocytes. Experiments in animal models suggest that natural antibodies to IFN-γ may have a role in the immunoregulatory process limiting the intensity and/or duration of immune response. As they can interfere only with the immunomodulating activities of IFN-γ, these antibodies might open up new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

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.