Deep neuromuscular block improves the surgical conditions for laryngeal microsurgery.

BACKGROUND: Adequate neuromuscular block is required throughout laryngeal microsurgery. We hypothesized that the surgical conditions would improve under a deeper level of rocuronium-induced neuromuscular block. METHODS: Seventy-two patients undergoing laryngeal microsurgery were randomly allocated to either the 'post-tetanic counts 1-2' (PTC1-2) group or the 'train-of-four counts 1-2' (TOFcount1-2) group according to the level of neuromuscular block used. Two different doses of rocuronium (1.2 or 0.5 mg kg(-1)) were used after anaesthetic induction, and two respective targets of neuromuscular block (post-tetanic counts ≤2 or train-of-four count of 1 or 2) were used. Surgical conditions were assessed by the surgeon using a five-point rating scale (extremely poor/poor/acceptable/good/optimal), and clinically acceptable surgical conditions were defined as those which were rated acceptable, good, or optimal. The occurrence of vocal cord movement and postoperative adverse events was assessed. RESULTS: The surgical conditions were significantly different between the PTC1-2 and TOFcount1-2 groups (extremely poor/poor/acceptable/good/optimal: 0/2/1/7/26 and 3/10/2/14/7, respectively, P<0.001). The incidence of clinically acceptable surgical conditions was significantly higher in the PTC1-2 group than in the TOFcount1-2 group (94 vs 64%, P=0.003). The percentage of patients who exhibited vocal cord movement was significantly lower in the PTC1-2 group than in the TOFcount1-2 group (3 vs 39%, P<0.001). The incidence of postoperative adverse events was not significantly different except for the less frequent occurrence of mouth dryness in the PTC1-2 group (P=0.035). CONCLUSIONS: Deep neuromuscular block (post-tetanic count of 1-2) surgical conditions in patients undergoing laryngeal microsurgery improves. CLINICAL TRIAL REGISTRATION: NCT01980069.

Identification of Kunitz trypsin inhibitor mutations using SNAP markers in soybean mutant lines.

The Kunitz trypsin inhibitor (KTi) in soybean has several polymorphic types that are controlled by multiple alleles, which behave in a co-dominant fashion. Of these, Tia and Tib, which differ by nine amino acids, are the predominant types. In order to develop a single nucleotide amplified polymorphism (SNAP) marker for the classification of the predominant KTi types, Tia and Tib, and evaluate KTi activities by differing KTi type total 451 soybean mutant lines (M(12)-M(16) generation) were incorporated in this study. Among 451 soybean mutants, 144 and 13 mutant lines showed decreased and increased trypsin inhibitor activity when compared with the original cultivars, respectively. To identify the KTi type, we designed a SNAP marker. Among 451 mutant lines from 12 soybean cultivars and landraces, 8 mutant lines derived from cvs. Baekwoon, Paldal and Suwon115 showed a change in KTi type when compared with the original cultivars using the SNAP marker. Five mutant lines in Suwon115 changed from Tib to Tia, while two mutant lines derived from cv. Baekwoon and one mutant line derived from cv. Paldal were changed from Tia to Tib. These changes of KTi types were confirmed by sequencing of the KTi genes and non-denaturing polyacrylamide gel electrophoresis of the KTi proteins. To identify the effect of KTi activity based on the change in KTi type, we measured the KTi activity using the three cultivars and eight mutant lines that showed changes in KTi type. Two mutant lines (BW-1 and 7-2) derived from cv. Baekwoon and one mutant line (PD-5-10) from cv. Paldal that changed from Tia to Tib showed lower activity than the original cultivar. In cv. Suwon115, five mutant lines that changed from Tib to Tia showed higher activity than the original cultivar. These results indicate that the designed SNAP marker was capable of identifying the KTi type and that Tia activity was higher than Tib activity in soybean.

KITENIN recruits Dishevelled/PKC delta to form a functional complex and controls the migration and invasiveness of colorectal cancer cells.

BACKGROUND AND AIMS: KITENIN was previously reported to promote metastasis in mouse colon tumour models; however, the signalling mechanism of KITENIN at the cellular level was unknown. Here the functional role of KITENIN with respect to colorectal cancer (CRC) cell invasion and its expression in CRC tissues were investigated. METHODS: The effect of KITENIN on cell motility was analysed in a migration and invasion assay upon its overexpression and knockdown. Immunoprecipitation was used to elucidate binding partners, and immunohistochemistry was used to study expression levels. RESULTS: KITENIN overexpression enhanced the migration of rat intestinal epithelial cells, whereas a loss of invasiveness was observed in CRC cells after KITENIN knockdown. Mechanically, KITENIN served as a scaffolding molecule that simultaneously recruited both Dishevelled (Dvl) and protein kinase C delta (PKC delta) through the membrane-spanning C-terminal region to form a complex that stimulated extracellular signal-regulated kinase (ERK)/activating protein-1 (AP-1) via a PKC delta component but also organised the actin filament via a Dvl component. The KITENIN complex controlled the invasiveness of CRC cells aetiologically harbouring various mutations in APC, beta-catenin or K-ras, in which AP-1 activation is redundant but the organisation of the actin filament is indispensable for cell motility. Clinically, KITENIN expression was significantly higher in colon cancer tissues from advanced stage (III, IV) than that of stage I CRC and also in corresponding metastatic tissues. CONCLUSIONS: The functional KITENIN complex acts as an executor with regard to cell motility and thereby controls CRC cell invasion, which may contribute to promoting metastasis.

Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants.

In an earlier investigation, homologous mutant lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from a callus that had been irradiated with a 50-Gy gamma ray during embryo culture. In order to identify the 5MT-resistant mechanism, we have continued our investigations of these mutant lines and studied the anthranilate synthase activity of the M5) advanced lines by direct fluorometric detection of the anthranilate formed in both control plants and mutant lines grown on 500 microM 5MT. The anthranilate synthase activity of the mutant plants was 2.2- to 3-fold higher than that of the control. In a kinetic analysis with tryptophan, an anthranilate synthase of the mutant lines was insensitive to feedback inhibition. These lines showed an enhanced accumulation of storage proteins and amino acids. The increased rates of protein synthesis in the mutant lines, relative to that of the control seeds, were 17-28.5%. The amino acid contents were 2.4-fold (MRI-40-2) to 2.6-fold (MRI-110-6) higher in the MRI lines than in the control seeds, and 2.4-fold (MRII-12-5) to 3.5-fold (MRII-8-1) higher in the MRII lines than in the control seeds. Significant increases among the amino acids of the MR lines were observed for tryptophan, phenylalanine, and tyrosine, which had been biosynthesized through the shikimate pathway. The transcript levels of putative OASA2, which is one of the key-regulating enzyme subunits in the tryptophan biosynthesis pathway, were studied in the control and 5MT-resistant mutant lines subjected to inhibition by two tryptophan analogs (5MT and alphaMT) and to other abiotic stresses (ABA, NaCl, and cold). The putative OASA2 gene in the 5MT-resistant mutant lines was highly expressed in at a low 5MT concentration and at an early stage of the 5MT and alphaMT treatments. However, mRNA accumulation of the putative OASA2 gene in the mutant plants gradually decreased when the plants were subjected to abiotic stresses such as NaCl and cold. These results indicated that the 5MT resistance in the mutant lines is due to altered anthranilate synthase forms.

Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants.

To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryo-cultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M4 lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4% greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8%) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1-OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M2 plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M2 plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.

Expressed sequence tags from a wheat-rye translocation line (2BS/2RL) infested by larvae of Hessian fly [Mayetiola destructor (Say)].

Of the 16 known biotypes of the Hessian fly [ Mayetiola destructor (Say)], biotype L is recognized as being the most virulent. We have previously reported the development of near-isogenic lines (NILs) (BC(3)F(3:4)) by backcross introgression (Coker797*4/Hamlet) that differed by the presence or absence of the H21 gene on 2RL chromatin. Florescence in situ hybridization analysis revealed introgressed 2RLs in NILs possessing the H21 gene, but no signal was detected in NILs lacking 2RL. As part of an approach to elucidate molecular interactions between plants and the Hessian fly, a cDNA library from NILs with H21 infested by larvae of biotype L of the Hessian fly was constructed for expressed sequence tag (EST) analysis. Of 1,056 sequenced reactions attempted, 919 ESTs produced some lengths of readable sequences. Based on their putative identification, 730 ESTs that showed significant similarity with amino acid sequences registered in the gene bank were divided into 13 functional categories. Defense- and stress-related genes represented about 16.1%, including protease inhibition, oxidative burst, lignin synthesis, and phenylpropanoid metabolism. EST clones obtained from the cDNA library may provide a clue to the molecular interactions between plant and larva of the Hessian fly larval infestation.

Molecular characterization of a cDNA encoding putative calcium binding protein, HvCaBP1, induced during kernel development in barley (Hordeum vulgare L.).

Seed development is known to involve complex physiological and molecular events. In order to gain information on the molecular events that occur in the grains of barley during kernel development, we conducted suppression subtractive hybridization (SSH) using grains of barley cv. Karl at 14 days after fertilization (DAF) as the tester and grains at 5 DAF as the driver. We isolated an SSH clone that showed homology with a specific calcium binding protein in rice called EFA27. Screening the cDNA library, we identified two clones as a calcium binding protein. These clones, each carrying one calcium-binding EF-hand motif, were designated HvCaBP1 (Hordeum vulgare Calcium Binding Protein 1). HvCaBP1 possesses an N-terminal region with a conserved single Ca(2+)-binding EF-hand motif and one transmembrane helix. Northern hybridization showed that the highest expression occurred in grains and that expression increased in kernels at 8 DAF. As shown in situ hybridization, the HvCaBP1 gene was highly expressed in the embryo and tissues of the endosperm near the embryo and was detected in the vascular tissues of the glume in the kernel at 8 DAF. Accumulation of HvCaBP1 mRNAs subsequently increased in vegetative tissues for up to 48 h after abscisic acid (ABA) treatment. Transcripts of HvCaBP1 mRNAs may be regulated by endogenous ABA in the grains during kernel development.

Differential modulation of transcriptional activity of oestrogen receptors by direct protein-protein interactions with retinoid receptors.

Control of oestradiol-responsive gene regulation by oestrogen receptors (ERs) may involve complex cross-talk with retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently, we have shown that ERalpha directly interacts with RARalpha and RXRalpha through their ligand binding domains (LBDs). In the present work, we extend these results by showing that ERbeta binds similarly to RARalpha and RXRalpha but not to the glucocorticoid receptor, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull-down assays. These direct interactions were also demonstrated in gel-shift assays, in which the oestrogen response element (ERE) binding by ERalpha was enhanced by the RXRalpha LBD but was abolished by the RARalpha LBD. In addition, we showed that RARalpha and RXRalpha bound the ERE as efficiently as ERalpha, suggesting that competition for DNA binding may affect the transactivation function of the ER. In transient transfection experiments, co-expression of RARalpha or RXRalpha, along with ERalpha or ERbeta, revealed differential modulation of the ERE-dependent transactivation, which was distinct from the results when each receptor alone was co-transfected. Importantly, when the LBD of RARalpha was co-expressed with ERalpha, transactivation of ERalpha on the ERE was repressed as efficiently as when wild-type RARalpha was co-expressed. Furthermore, liganded RARalpha or unliganded RXRalpha enhanced the ERalpha transactivation, suggesting the formation of transcriptionally active heterodimer complexes between the ER and retinoid receptors. Taken together, these results suggest that direct protein-protein interactions may play major roles in the determination of the biological consequences of cross-talk between ERs and RARalpha or RXRalpha.