Targeted deletion of CASK-interacting nucleosome assembly protein causes higher locomotor and exploratory activities.

CASK-interacting nucleosome assembly protein (CINAP) has been shown to interact with the calcium/calmodulin-dependent serine kinase (CASK) and the T-box transcription factor T-brain-1 (Tbr1) thus modulating the expression of N-methyl-D-aspartic acid receptor subunit 2b (NMDAR2b) in cultured hippocampal neurons. To explore the physiological significance of CINAP in vivo, CINAP knockout mice were generated and subjected to biochemical, anatomical, and behavioral analyses. Unexpectedly, CINAP deletion did not impact NMDAR2b expression, and these CINAP knockout mice were consistently comparable to wild-type littermates in terms of immediate memory (assessed with the Y maze) and associative memory (evaluated by conditioned taste aversion and contextual and auditory fear conditioning). Although CINAP deletion did not obviously influence learning and memory behaviors, CINAP knockout mice exhibited higher locomotor and exploratory activities. Compared with wild-type littermates, the horizontal and vertical movements of the CINAP knockout mice were higher in a novel environment; in home cages, rearing, sniffing, and jumping also occurred more frequently in CINAP knockout mice. These observations suggest that although CINAP deletion in mice does not influence learning and memory behaviors, CINAP is required for restriction of locomotor and exploratory activities.

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway.

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N-methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.

Loss of telomerase activity may be a potential favorable prognostic marker in lung carcinomas.

Many cancer and immortal cells exhibit telomerase activity that stabilizes telomere lengths, possibly contributing to cell immortality and carcinogenesis. The aim of this study was to elucidate the clinicopathological relationship between telomerase activity and telomerase reverse transcriptase subunit (hTERT) status in non small cell lung cancer. hTERT status in non small cell lung cancer using telomeric repeat amplification protocol (TRAP) and RT-PCR assay, respectively. Telomerase activity and hTERT were detected in 85.7 and 80.3% of cancerous tissues, respectively. Telomerase activity does not correlate with clinicopathological variables. However, there was an association between p53-correlated expression and hTERT negative status. Lung cancer patients without telomerase activity survived for a significantly longer period than those with telomerase activity. In addition, hTERT was not associated with the prognosis. TERT expression did not correlate well with any clinical parameter. Reactivated telomerase activity may be a poor prognostic factor in NSCLCs.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

Specific detection of benzimidazole resistance in Colletotrichum gloeosporioides from fruit crops by PCR-RFLP.

Anthracnose diseases, caused by Colletotrichum gloeosporioides, are a worldwide problem and are especially important in Taiwan owing to the severe economic damage they cause to tropical fruits that are grown for local consumption and export. Benzimidazoles are systemic fungicides widely used for controlling these diseases in Taiwan. Thirty-one isolates of C. gloeosporioides from mango and strawberry grown in Taiwan were examined for their sensitivity to benzimidazole fungicides. The responses of the isolates grown on benzimidazole-amended culture media were characterized as sensitive, moderately resistant, resistant or highly resistant. Analysis of point mutations in the beta-tubulin gene by DNA sequencing of PCR-amplified fragments revealed a substitution of GCG for GAG at codon 198 in resistant and highly resistant isolates and a substitution of TAC for TTC at codon 200 in moderately resistant isolates. A set of specific primers, TubGF1 and TubGR, was designed to amplify a portion of the beta-tubulin gene for the detection of benzimidazole-resistant C. gloeosporioides. Bsh1236I restriction maps of the amplified beta-tubulin gene showed that the resistant isolate sequence, but not the sensitive isolate sequence, was cut. The PCR restriction fragment length polymorphism (PCR-RFLP) was validated to detect benzimidazole-resistant and benzimidazole-sensitive C. gloeosporioides isolates recovered from avocado, banana, carambola, dragon fruit, grape, guava, jujube, lychee, papaya, passion fruit and wax apple. This method has the potential to become a valuable tool for monitoring the occurrence of benzimidazole-resistant C. gloeosporioides and for assessment of the need for alternative management practices.

Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae.

Light is a major environmental factor that influences many biological processes. We characterized the roles of light in asexual development (including the formation of aerial hyphae and conidiophore) in Magnaporthe oryzae, which is the causal agent of rice blast disease. Our data revealed a complex nature of light regulation in the asexual developments of M. oryzae. Asexual development of M. oryzae is suppressed by blue light in a light/dark cycling environment and asexual spore release is controlled by both blue and red light. We demonstrated that even very dim light, about 10 micromol m(-2), is sufficient to suppress spore-release behavior in M. oryzae. We also generated knockout strains of a blue light receptor, mgwc-1, the M. oryzae homolog of white collar-1 in Neurospora crassa, and demonstrated blue-light-specific regulation in the asexual development and spore release in M. oryzae. Our findings in this agriculturally important pathogen, M. oryzae, broaden our understanding of the roles of light in fungal development.