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.     

Reducing the Occupational Risk of Infections for the Surgeon: Multicentric National Survey on More Than 15,000 Surgical Procedures

The objective of this study was to find the incidence of accidental exposures to blood and body fluids among surgeons during operations and to describe their dynamics. A probabilistic model was also used to predict the cumulative 30-year risk to the surgeon of contracting hepatitis B and C viruses (HBV, HCV) or human immunodeficiency virus (HIV) infection and estimate the effect of preventive strategies in reducing this risk. A multicentric prospective survey, based on self-administered questionnaires, was conducted during a period of 6 months in 39 Italian hospitals. An accidental exposure to blood or body fluids occurred in 9.2% of 15,375 operations. In about 2% of procedures a parenteral-type injury, such as actual skin puncture or eye contamination, was suffered by the operating surgeon. A needle-stick injury was the commonest accident, and its occurrence was found to vary with the phase of the procedure and its length. The current lifetime risk of acquiring HBV, HCV, and HIV infection in our regions was estimated to be as high as 42.7%, 34.8%, and 0.54%, respectively. The adoption of preventive strategies is expected to reduce this risk to 21% for HBV, 16.6% for HCV, and 0.23% for HIV infection. Active immunization of surgeons against HBV is strongly recommended. The case is also made for the use of a face-shield combined with a permanent change in our surgical practice capable of reducing the current high rate of parenteral injuries.     

Chronic peritoneal dialysis in paediatrics: Experience of a national registry

The results of the first 3 year' collaboration of the Italian Registry of Paediatric Chronic Peritoneal Dialysis (CPD) (1986–1988) are presented. This Registry acquired data on the majority of the paediatric patients treated with CPD in Italy, thus providing a national picture in a field where few nationwide surveys are available. Patients of less than 15 years of age at the start of dialysis were enrolled and clinical data collected until the age of 19 years. The number of nephrological paediatric centres participating in the Registry increased from 7 in 1986 to 11 in 1988. The total number of patients on CPD was 70 and the percentage of dialysed children treated with CPD ranged from 40.2% to 43.6%. Data on 89 peritoneal catheters were collected: during 1417 dialysis-months 70 catheter-related complications were observed (1:20.8 dialysis-months); actuarial catheter survival was 92.7% at 6 months, 84.8% at 1 year and 68.8% at 2 years. The incidence of peritonitis changed from 1 episode every 10.9 patient-months in 1986 to 1 every 19.8 in 1988. Abdominal hernias were the other main clinical complication observed. The survival of patients was 92.5% at 3 years, while the technique survival at the same time was 84%.     

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel α1 subunit

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis posses immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti–voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore–forming α₁ subunig of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel α₁ subunit–directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel–binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both α₁ and β subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.     

Relationship between maternal- and fetal-specific IgE

AIM: A positive correlation between maternal and cord-blood IgE levels is well documented for total IgEs, but not for specific IgEs. The difficulty in detecting specific cord-blood IgEs is due to their low concentrations, which hinder their dosage by low-sensitivity methods. The study aimed to correlate maternal and foetal specific IgEs against individual cow's milk proteins, detected by highly sensitive and specific techniques. METHODS: Cow's milk specific IgE detection was performed by chemiluminescence on 52 specimens of maternal and cord blood after cow's milk protein separation by 1D and 2D gel electrophoresis. Cow's milk protein (CMP) antigens were identified by mass spectrometry techniques. RESULTS: Specific IgEs for CMPs were found in 25/52 (48.1%) of maternal sera and in 19/52 (37%) of cord-blood sera. In order of decreasing frequency, the proteins found were BSA, IgG heavy chain, caseins and, in a single case, b-lactoglobulin. Positive cord-blood sera in all cases corresponded to a positive maternal result, and maternal and foetal immunoreactivity patterns were closely correlated. Moreover, in no case was there a positive cord-blood response with a negative maternal response. CONCLUSION: The study demonstrates a close relationship between maternal and cord-blood specific IgE patterns. The phenomenon observed could provide a model to elucidate the general production method of foetal IgEs, which might only be produced in the presence of both the corresponding maternal IgE and the related allergen.