Mutation analysis of the RET proto-oncogene in Dutch families with MEN 2A, MEN 2B and FMTC: two novel mutations and one de novo mutation for MEN 2A

Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.     

Behavioural Changes in Free-ranging Red Foxes (Vulpes vulpes) due to Sarcoptic Mange

Sarcoptic mange (Sarcoptes scabiei var. vulpes) reached Scandinavia in the mid-1970’s and is mainly prevalent among red foxes (Vulpes vulpes) (Borg 1987). In the laboratory, foxes succumb 2–4 months after being infested, and it is commonly thought that carriers in the wild exhibit an abnormal behaviour and quickly die (Mörner & Christensson 1984, Holt & Berg 1990). However, there is a lack of comparative data from free-ranging animals to contribute to the ecology of the species in general and ta supplement the content of the above selected references in particular (e.g. Plowright 1988). Using telemetry studies, I have compared the behaviour in winter of 2 mange infested and 2 healthy red foxes. The work took place in 1987 and 1990 in a boreal area adjacent to farmland in central Norway (63° 20′N 10° 45′E).     

Spatio-temporal profiling and degradation of alpha-amylase isozymes during barley seed germination

Ten genes from two multigene families encode barley alpha-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the alpha-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 alpha-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding alpha-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both alpha-amylase 2 entries co-migrated in five full-length and one fragment spot. The alpha-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that alpha-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (betaalpha)(8)-barrel between beta-strand 3 and alpha-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other alpha-amylase 2 (gi|166985). The 2D gels of dissected tissues showed alpha-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length alpha-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.     

Engineering high-speed allosteric hammerhead ribozymes

Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg(2+). The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k (obs)) as high as 8 min(-1) in the presence of theophylline and maximal k (obs) increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.     

Tissue distribution of cytochrome P450 3A (CYP3A) in brushtail possums (Trichosurus vulpecula) exposed to Eucalyptus terpenes

We evaluated the distribution pattern of a specific xenobiotic metabolizing enzyme, cytochrome P450 3A (CYP3A) in the common brushtail possum (Trichosurus vulpecula). Western blot studies using CYP3A antibodies were used to compare CYP3A levels in the intestine, liver, kidney, brain, testes and adrenal gland in possums fed diets with and without a mixture of terpenes. Possums appear to produce at least 3 different CYP3A-like isoforms that are differentially expressed in various tissues. The liver and duodenum produce all three isoforms (CYP3A P1, P2, P3), the jejunum only produces CYP3A P1, the ileum, kidney, testes and adrenal only produce CYP3A P2 and the brain only produces CYP3A P3. Terpene treatment did not alter relative levels of isoforms present in any tissue type. This study is the first to identify the presence and differential expression of several CYP3A-like isoforms in a variety of tissues of a wild mammalian herbivore. Data suggest that CYP3A-like enzymes are not induced by terpenes. However, the wide distribution of CYP3A-like isoforms in a variety of tissues, suggests that these enzymes are an important mechanism for metabolism in possums and may contribute to the high tolerance possums have to a wide range of xenobiotics.     

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons

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Castor patatin-like phospholipase A IIIβ facilitates removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds

Plant Molecular Biology - Castor patatin-like phospholipase A IIIβ facilitates the exclusion of hydroxy fatty acids from phosphatidylcholine in developing transgenic Arabidopsis seeds. Hydroxy...     

Dense sub-ice bloom of dinoflagellates in the Baltic Sea, potentially limited by high pH

The phytoplankton community, carbon assimilation, chlorophylla (Chl a), pH, light and attenuation and inorganic nutrientswere monitored under the ice in the coastal Gulf of Finland,Baltic Sea. Maximum ice and snow thickness was 40 and 15 cm,respectively. Freshwater influence had created a halocline 1–2m below the ice–water interface, and above this halocline,a dense bloom of dinoflagellates developed (max: >300 µgChl a L^–1). The photosynthetic uptake of carbon dioxideby this "red tide" increased the pH to a maximum of 9.0. Thesub-ice phytoplankton community was dominated by the dinoflagellateWoloszynskia halophila (max: 3.6 x 10⁷ cells L^–1). ThepH tolerance of this species was studied in a monoculture andthe results indicate that pH >8.5 limits growth of this speciesat ambient irradiance. This study shows that primary productivitymay raise the pH to growth limiting levels, even in marine,low-light environments where pH normally is not considered important.