Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries

Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops.     

2-Aminomethyl piperidines as novel urotensin-II receptor antagonists

A series of 2-aminomethyl piperidines has been discovered as novel urotensin-II receptor antagonists. The synthesis, initial structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent, cross-species active, and functional urotensin-II receptor antagonists such as 1a and 11a are described.     

Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development

Mutation of either arginase structural gene (ARGAH1 or ARGAH2 encoding arginine [Arg] amidohydrolase-1 and -2, respectively) resulted in increased formation of lateral and adventitious roots in Arabidopsis (Arabidopsis thaliana) seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate and diamino-rhodamine-4M, respectively. Upon seedling exposure to the synthetic auxin naphthaleneacetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared with the wild type. In all genotypes, much 3-amino,4-aminomethyl-2',7'-difluorofluorescein diacetate fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven beta-glucuronidase (GUS) was expressed throughout the seedling; the ARGAH2::GUS expression pattern was more localized. Naphthaleneacetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type, consistent with increased internal NO leading to enhanced auxin signaling in roots. In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter DR5::GUS in root tips, emerging lateral roots, and hypocotyls. We propose that Arg, or an Arg derivative, is a potential NO source and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin action in roots. This model is supported by supplemental Arg induction of adventitious roots and increased NO accumulation in argah1-1 and argah2-1 versus the wild type.     

Receptor activity modifying proteins interaction with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), two closely related peptides, initiate their biological responses through their interaction with calcitonin receptor-like receptor (CRLR). The CRLR receptor phenotype can be determined by coexpression of CRLR with one of the three-receptor activity modifying proteins (RAMPs). In this report, we characterized the pharmacological properties of the human or porcine CRLR with individual RAMPs transiently expressed in human embroynic kidney cell line (HEK-293). Characterization of RAMP1/human or porcine CRLR combination by radioligand binding ([¹²⁵I] hCGRP) and functional assay (activation of adenylyl cyclase) revealed the properties of CGRP receptor. Similarly characterization of RAMP2/human or porcine CRLR and RAMP3/human or porcine CRLR combination by radioligand binding ([¹²⁵I]rADM) and functional assay (activation of adenylyl cyclase) revealed the properties of ADM (22–52) sensitive-ADM receptor. In addition, porcine CRLR/RAMP2 or 3 combination displayed specific high affinity [¹²⁵I] hCGRP binding also. Also, co-transfection of porcine CRLR with RAMPs provided higher expression level of the receptor than the human counterpart. Thus the present study along with earlier studies strongly support the role of RAMPs in the functional expression of specific CRLRs.     

Microvascular reconstruction of the hypopharynx: defect classification,treatment algorithm,and functional outcome based on 165 consecutive cases

The objectives of this study were three-fold: to develop a scheme for classification of hypopharyngeal defects, to establish a reconstructive algorithm based on this system, and to assess the functional outcome of such reconstruction. This study is a retrospective review of a 14-year experience with 165 consecutive microvascular reconstructions of the hypopharynx in 160 patients. The average patient age was 59 years (95 percent CI, 37 to 81). Thirty-four patients were operated on for recurrent disease; 71 had preoperative radiotherapy. Partial defects were reconstructed with radial forearm flaps (n = 52); circumferential defects were reconstructed with jejunum (n = 90); and extensive, noncircumferential longitudinal defects were reconstructed with rectus abdominis flaps (n = 23). The overall free flap success rate was 98 percent. Six flaps required reexploration, two of which were salvaged. The incidence of fistula was 7 percent and stricture, 4 percent. Preoperative radiotherapy was significantly associated with risk of recipient site complications (OR, 2.3; 95 percent CI, 1.0 to 5.0). Follow-up data were available on 95 percent of patients: 53 percent were able to tolerate an unrestricted diet, 23 percent a soft diet, 12 percent liquids only, and 12 percent were limited to tube feedings. The treatment algorithm for microvascular hypopharyngeal reconstruction is based on the type of defect with partial defects with radial forearm flaps, circumferential defects reconstructed with free jejunal flaps, and extensive, multilevel defects reconstructed with rectus abdominis myocutaneous flaps. Microvascular reconstruction of pharyngeal defects is highly successful with few postoperative complications. With appropriate flap selection, functional outcome can be optimized.     

Soft-tissue flap coverage maximizes limb salvage after allograft bone extremity reconstruction

Limb salvage after extremity tumor ablation may include the use of allograft bone. The primary complication of this method is infection of the allograft, which can lead to limb loss in up to 50 percent of cases. The purpose of this study is to evaluate the efficacy of primary muscle flap coverage in the setting of allograft bone limb salvage surgery. This study is a prospective review of all patients with flap coverage of extremity allografts over the 10-year period 1991 to 2001. There were 20 patients (11 male and nine female patients) with an average age of 28 years (range, 6 to 72 years). Flap coverage was primary in 16 patients and delayed in four. Delayed coverage was performed for failed wounds that did not have a primary soft-tissue flap. Pathologic findings included osteosarcoma in nine patients, Ewing sarcoma in five patients, malignant fibrohistiocytoma in two patients, chondrosarcoma in two patients, synovial sarcoma in one patient, and leiomyosarcoma in one patient. Allograft reconstruction was performed for the upper extremity in 12 patients and for the lower extremity in eight patients. Flap reconstruction was accomplished with 20 pedicle flaps in 17 patients (latissimus dorsi, 12; gastrocnemius, four; soleus, three; and fasciocutaneous flap, one) and four free flaps (rectus abdominis, three; latissimus dorsi, one) in four patients. All pedicled flaps survived. There was one flap failure in the entire series, which was a free rectus abdominis flap. This case resulted in the only limb loss noted. The follow-up period ranged from 1 to 50 months (average, 12.35 months). At the time of final follow-up, three patients were dead of disease and 17 were alive with intact extremities. The overall limb salvage rate in the setting of bone allograft and soft-tissue flap coverage was 95 percent (19 of 20). Reoperation for bone-related complications was required in 50 percent (two of four) of cases receiving delayed flap coverage compared with 19 percent (three of 16) of patients with primary flap coverage (statistically not significant). The results of this study support the use of soft-tissue flap coverage for allograft limb reconstruction. In this series, no limb was lost in the setting of a viable flap. Reoperation was markedly reduced in the setting of primary flap coverage. Pedicled or microvascular transfer of well-vascularized muscle can be used to wrap the allograft and minimize devastating wound complications potentially leading to loss of allograft and limb.     

A classification system and reconstructive algorithm for acquired vaginal defects

Although multiple flaps have been used for vaginal reconstruction, a logical approach to reconstruction of these often complex defects has not been described. The objective of this study was to establish a classification system for acquired vaginal defects and to develop a reconstructive algorithm derived from this system. This study is a retrospective review of a 7-year experience with 51 flaps in 37 consecutive vaginal reconstructions. Twenty-two partial defects and 15 circumferential defects were reconstructed in 35 patients. Average patient age was 48 years (range, 19 to 69 years). Of the 22 patients with partial vaginal defects, six involved primarily the anterior and lateral wall and 16 the posterior vaginal wall. Among the 15 patients with circumferential defects, four included only the upper two-thirds of the vagina and 11 encompassed the entire vagina. On the basis of these defects, a classification system was developed. Partial defects involving the anterior or lateral vaginal wall were classified as type IA defects and were reconstructed primarily with pedicled Singapore fasciocutaneous flaps. Partial defects involving the posterior wall were classified as type IB and were reconstructed with pedicled rectus abdominis myocutaneous flaps. Circumferential defects involving the upper two-thirds of the vagina were classified as type IIA defects and were reconstructed with a rolled rectus flap or, less commonly, sigmoid colon (one patient). Total circumferential defects, type IIB, were reconstructed largely with bilateral gracilis flaps. Six patients had major complications, including one perioperative death, one complete flap loss, one partial flap loss, and three pelvic abscesses. Three patients had minor complications that included delayed wound healing and donor-site infection. Vaginal defects can be categorized into one of four types on the basis of the location and extent of resection. Flap selection is determined on the basis of the type of defect. Using this algorithm, immediate vaginal reconstruction with pedicled regional flaps can be performed with minimal patient morbidity and few surgical complications.