Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity

Human Molecular Genetics (2006) 15, 921–931;     

Circular dichroism detection in high-performance liquid chromatography: Evaluation of the anisotropy factor

The application of a circular dichroism (c.d.) detection system in HPLC using a chiral stationary phase is presented. The simultaneous measurement of the absorbance and c.d. signal allows the evaluation of the anisotropy factor (g = Δ/) and thus the determination of the enantiomeric excess (e.e.) of the eluates. When this detection system is used in preparative chiral chromatography the collection of the enantiomeric fractions can be readily optimized.     

BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Arthroscopic Latissimus Dorsi Transfer

The patient is placed in lateral decubitus. A 6-cm incision made in the axilla allows access to the latissimus dorsi tendon and its neurovascular pedicle. Holding the arm in internal rotation, the surgeon detaches sharply the tendon off the humeral shaft and then reinforces it with wrapping sutures. Pulling the free limbs of the sutures exposes the under surface of the muscle and helps to identify the neurovascular pedicle. Special lighting retractors suited for a large diameter scope are helpful. Mobilization is completed when 2 cm of the tendon crosses the posterior edge of the acromion. The standard lateral portal is used for visualization. A silicon drain tube stiffened by a Wissinger rod is advanced from the posterior portal under direct visualization in the space between teres minor and deltoid, exiting in the auxiliary incision. A suture loop passed down the tube retrieves the tendon sutures out the posterior portal. These are then moved out the anterior portal, thus pulling the tendon over the tuberosity. The first anchor is inserted at the anterior aspect of the greater tuberosity, close to the articular cartilage and long head of the biceps tendon. Two to 3 anchors are inserted fixing the tendon to the tuberosity until it is stable.     

Endodontic surgery using 2 different magnification devices: preliminary results of a randomized controlled study.

PURPOSE: The introduction of microsurgical instruments and magnification devices has brought advantages in root-end management and the application of root-end filling materials. The main purpose of this prospective clinical study was to monitor the outcome of ultrasonic root-end preparation using magnification loupes or an endoscope. Tooth location and the presence of post restoration were also examined as potentially affecting the outcome. MATERIALS AND METHODS: Teeth treated surgically showed a periradicular lesion of strictly endodontic origin. A total of 59 patients were included in the study, according to specific selection criteria. Following the reflection of a full mucoperiosteal tissue flap, residual soft tissues were curetted, root ends were resected, and root-end cavities were prepared ultrasonically with a zirconium nitrate tip, and zinc oxide EBA-reinforced cement root-end fillings were placed. Thirty-two root-end management procedures were performed using magnification loupes and 39 using an endoscope. All cases followed for a period of 1 year were classified into 3 groups (success, uncertain healing, and failure) according to radiographic and clinical criteria. RESULTS: Of the 71 teeth evaluated at 1-year follow-up, 67 teeth (92.95%) successfully healed, 3 teeth had uncertain healing, and 2 failed. In the group using endoscopy, 94.9% of successful healing was achieved, while for the other group, 90.6% was recorded. We found no statistically significant differences in treatment results related to the arch (P = .20), post restoration (P = .21), or type of magnification device (P = .08). CONCLUSIONS: In the present study, adherence to a strict endodontic surgical protocol and the use of modern surgical endodontic procedures, together with visual magnifications, resulted in an overall high success rate.     

Hypophysiotrophic neurons are capable of altering the ratio of co-packaged neurohormones.

Neuronal dense-core vesicles provide a mechanism whereby peptide messengers are secreted in discrete quanta. Here we report on the capacity of rat hypophysiotrophic corticotropin releasing factor-41 neurons to alter the peptide content as well as the size of dense-core vesicles after removal of glucocorticoid negative feedback by adrenalectomy. We demonstrate, using quantitative immunoelectron microscopy, that long-term adrenalectomy induces a progressive increase in the ratio of vasopressin to corticotropin releasing factor-41-immunoreactive sites in the dense-core vesicle compartment. The intravesicular concentration of vasopressin appeared to be the variable parameter while that of the corticotropin releasing factor-41 remained stable at all survival times after adrenalectomy. Moreover, observations for up to 5 weeks indicate that adrenalectomy results in a progressive increase in the mean volume of dense-core vesicles to about three times normal. These results suggest that the quantal size and the composition of dense-core vesicles are subject to long-term modulation. The capacity of corticotropin releasing factor-41 neurons to alter dense-core vesicles could enhance or diminish the efficacy of the hypothalamohypophyseal communication underlying physiological adaptation to stress, as well as pathological changes.