AMOTL2 mono-ubiquitination by WWP1 promotes contact inhibition by facilitating LATS activation.

Contact inhibition is a key cellular phenomenon that prevents cells from hyper-proliferating upon reaching confluence. Although not fully characterized, a critical driver of this process is the Hippo signaling pathway, whose downstream effector yes-associated protein plays pivotal roles in cell growth and differentiation. Here, we provide evidence that the E3 ligase WWP1 (WW-domain containing protein 1) mono-ubiquitinates AMOTL2 (angiomotin-like 2) at K347 and K408. Mono-ubiquitinated AMOTL2, in turn, interacts with the kinase LATS2, which facilitates recruitment of the upstream Hippo pathway component SAV1 and ultimately promotes yes-associated protein phosphorylation and subsequent cytoplasmic sequestration and/or degradation. Furthermore, contact inhibition induced by high cell density promoted the localization and stabilization of WWP1 at cell junctions, where it interacted with Crumbs polarity proteins. Notably, the Crumbs complex was functionally important for AMOTL2 mono-ubiquitination and LATS activation under high cell density conditions. These findings delineate a functionally important molecular mechanism in which AMOTL2 mono-ubiquitination by WWP1 at cell junctions and LATS activation are tightly coupled to upstream cell density cues.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Isolation and characterization of a novel endo-ß-1,4-glucanase from a metagenomic library of the black-goat rumen.

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-ß-1,4-glucanase. The recombinant KG35 endo-ß-1,4-glucanase showed optimal activity within the range of 30-50°C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50°C at a pH of 5-7.

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-ß-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.

Antiadhesive effect and safety of oxidized regenerated cellulose after thyroidectomy: a prospective, randomized controlled study.

PURPOSE: To evaluate the antiadhesive effects and safety of an oxidized regenerated cellulose (Interceed) after thyroidectomy. METHODS: Seventy-six thyroidectomized patients were prospectively randomized into two groups with regard to the use of Interceed. We evaluated each group for their adhesive symptoms using four subjective and four objective items at the 2nd week, 3rd and 6th month after thyroidectomy. All patients were examined for vocal cord motility by indirect laryngoscope at each period. RESULTS: Total adhesion scores at each postoperative follow-up period decreased with time, but were not significantly different in each group. The median score for swallowing discomfort for liquid was significantly lower in the Interceed group than in the control group 2 weeks after surgery. In addition, the severity of skin adhesion to the trachea was reduced in the Interceed group compared with the control group 6 months after surgery. During the study, there were no adverse effects or significant differences in postoperative complications between the groups. CONCLUSION: Interceed appeared to be safe and effective in improving neck discomfort at early postoperative periods and preventing skin adhesion to the trachea 6 months after thyroidectomy.

Recovery of sensation in the anterior chest area after bilateral axillo-breast approach endoscopic/robotic thyroidectomy.

BACKGROUND: There is concern that endoscopic/robotic thyroidectomy could impair anterior chest skin sensation. Here, this possibility was assessed. PATIENTS AND METHODS: Fifty-one patients who underwent bilateral axillo-breast approach endoscopic/robotic thyroidectomy were asked 8 to 115 days (median: 29 d) later to undergo Semmes-Weinstein pressure threshold testing and to complete a questionnaire. Patient clinicopathological details were extracted. RESULTS: Twenty-one patients (41.2%) showed sensory impairment (sum of sensory changes ≥ 1). The average sum of sensory change (scale of 0 to 95) was 1.4 (range, 0 to 8). Impaired patients did not differ from unimpaired patients in any clinicopathological parameter except for time from operation. Compared with 1 month postsurgery, there was significantly less sensory impairment 3 months postsurgery (P=0.0083). The questionnaires yielded similar observations. CONCLUSIONS: The temporary and mild nature of the sensory change in the anterior chest area will relieve surgeons and patients from concerns that bilateral axillo-breast approach endoscopic/robotic thyroidectomy could permanently impair chest sensation.

Postauricular and axillary approach endoscopic neck surgery: a new technique.

BACKGROUND: While the bilateral axillo-breast approach (BABA) to endoscopic neck surgery resolves various benign and malignant thyroid and parathyroid diseases with minimal adverse effects and excellent cosmetic outcomes, it involves circumareolar incisions. Many patients, especially young female patients, are reluctant to have their breast involved. Consequently, we developed the postauricular and axillary approach (PAA) that uses postauricular incisions. METHODS: From June 2006 to December 2007, we treated 10 patients with PAA endoscopic neck surgery. After subcutaneous infiltration with diluted epinephrine solution, the subplatysmal and subcutaneous spaces were dissected. Two axillary ports and two postauricular ports were used and low-pressure CO2 insufflation generated operative space. After dividing the midline between the strap muscles, the isthmus was divided and the thyroid lobe was dissected with ultrasonic shears and excised after careful identification of the recurrent laryngeal nerve and parathyroid glands. Malignant lesions were treated with contralateral thyroid lobectomy. For parathyroid adenomas, we performed parathyroidectomy after dividing the strap muscles in the midline. RESULTS: Two thyroid lobectomies, one parathyroidectomy, one subtotal thyroidectomy, and six total thyroidectomies were performed by PAA endoscopic neck surgery. The mean operation time was 210.0+/-43.7 min. There were no cases of conversion to open surgery, permanent vocal cord palsy, or facial nerve palsy. None of the thyroidectomy patients exhibited hypocalcemia. The cosmetic outcomes were excellent and all patients were satisfied. CONCLUSIONS: PAA endoscopic neck surgery is a feasible method for thyroid and parathyroid surgery that permits good operative visualization and has minimal adverse effects and excellent cosmetic outcomes.

Cloning of genes differentially expressed during the initial stage of fruit development in melon (Cucumis melo cv. Reticulatus).

We have cloned genes involved in the initial stage of fruit development in the melon by suppression subtractive hybridization. A cDNA library of unfertilized ovules was subtracted from that of fruit 9 days after pollination (DAP); 10 of the 40 selected cDNA clones were identified by reverse Northern analysis as genes differentially expressed in fruit at 9 DAP. Seven of the ten genes were homologous to genes of known function; two were related to genes with unknown functions, and one was novel. With the exception of cucumisin, none of the cDNAs had been previously identified in melon. According to Northern analyses, six of the genes were expressed at high levels early in fruit development. Expression of cucumisin, Cmf-25, Cmf-30, and Cmf-124 was highest at 9 DAP, implying that these genes are involved in the initial stage of fruit development. Cmf-30, a seed nucellus-specific gene, was also expressed early in seed development. The other genes were expressed at a moderate level throughout fruit development, with the highest expression occurring in fruit at 9 and 18 DAP. In conclusion, nine new genes involved in early fruit development in melon were cloned, and their temporal and spatial expression patterns indicate that they are preferentially expressed during the active growing stage of fruit.