Polymerase chain reaction-based analysis using deaminated DNA of dodecamer expansions in CSTB, associated with Unverricht-Lundborg myoclonus epilepsy

Progressive myoclonus epilepsy of the Unverricht-Lundborg type is an autosomal recessive disorder that is characterized clinically by myoclonic seizures and ataxia. The majority of affected individuals carry repeat expansions of a dodecamer in the promoter region of the cystatin B gene. The unusually high GC content of this tract is refractory to conventional polymerase chain reaction (PCR), and, as a result, a circumventive procedure involving the deamination of DNA with sodium bisulfite has been proposed. This study evaluates the effectiveness of this deamination modification for the detection of dodecamer repeat variants. An analysis of 258 healthy Japanese individuals revealed an allele with four copies of the dodecamer repeat with a frequency of 0.01, in addition to the more commonly observed two and three copy repeat alleles. Homozygous repeat expansions 600 and 680 base pairs in length were detected in the analyses of two affected individuals. For these cases, sequencing, along with an alternative PCR-stutter formation, revealed 41 and 48 copies, respectively, of the dodecamer repeat. The complete conversion of C to T was observed in the expanded tracts, indicating that no methylation occurred at the CpG sites. Based on these results, it was concluded that the use of deaminated DNA allows for a precise analysis of consecutive GC tracts.     

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 1: Benzimidazole-5-sulfonamides

A new class of benzimidazole-5-sulfonamides has been identified as nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Initial structure-activity relationships are presented resulting in compounds 19 and 28 with submicromolar dual functional activity on human and rat receptors.     

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.     

Characterization of human CD4 helper T cell responses against Aurora kinase A

Aurora kinase A (Aurora-A) is a cell cycle-associated serine–threonine kinase that is overexpressed by various types of cancer and is highly associated with poor prognosis. Since the expression of Aurora-A in normal tissues has been shown to be significantly lower as compared to tumor cells, this protein is being considered as a potential tumor-associated antigen for developing immunotherapies. The goal in the present study was to identify CD4 helper T lymphocyte (HTL) epitopes for Aurora-A for the design of T cell-based immunotherapies against Aurora-A-expressing tumors. Synthetic peptides corresponding to potential HTL epitopes were identified from Aurora-A and used to stimulate CD4 T lymphocytes in vitro to generate antigen-specific HTL clones that were evaluated for antigen specificity, MHC restriction and for their ability to interact with Aurora-A-expressing tumor cells. The results show that two peptides (Aurora-A_161–175 and Aurora-A_233–247) were effective in generating HTL responses that were restricted by more than one MHC class II allele (i.e., promiscuous responses). The CD4 HTL clones were able to directly recognize Aurora-A-expressing tumor cells in an antigen-specific and MHC class II-restricted manner and some of the clones displayed cytolytic activity toward Aurora-A + tumor cells. Both of these peptides were capable of stimulating in vitro T cell responses in patients with bladder cancer.     

The fern Adiantum capillus-veneris lacks stomatal responses to blue light

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.     

Successive analysis of antigen trapping and enzymatic digestion on membrane-immobilized avidin

Avidin from egg white was migrated toward a cathode of nondenaturing electrophoresis and then immobilized on a polyvinylidene difluoride membrane. Adrenocorticotropic hormone (ACTH) was specifically captured after the biotinylated anti-ACTH antibody was bound to the membrane-immobilized avidin, and the captured ACTH was digested by the biotinylated trypsin on the membrane after extraction. The digested polypeptides from the ACTH were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results indicate that target substances can be specifically trapped and digested on membrane-immobilized avidin.     

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.