The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line.

The human galanin receptor has been characterized pharmacologically from the Bowes melanoma cell line. Using porcine [125I]galanin as the radioligand, a single population of non-interacting high-affinity binding sites (KD = 0.05 +/- 0.01 nM; Bmax = 135 +/- 7 fmol/mg protein) was demonstrated. Human galanin peptide competitively inhibited the specific binding of [125I]galanin (IC50 = 0.35 +/- 0.13 nM) and decreased the forskolin-stimulated cAMP production (EC50 = 0.46 +/- 0.05 nM) with a maximal inhibition of 63 +/- 2% at 10(-7) M. Rat and porcine galanin peptides and the chimeric peptides M15, M35, M32, M40 and C7 also dose-dependently inhibited the forskolin-stimulated cAMP production, while the fragment porcine galanin-(3-29) and [D-Trp2]galanin were found to be inactive. The specific binding of [125I]galanin was decreased in a dose-dependent manner by GTP and the cAMP response was inhibited by the pertussis toxin, suggesting the activation of a G-protein dependent process. The Bowes cell line thus appears to be a relevant tool for the study of human galanin receptor.

Polypeptide composition of an adenovirus type 5 used in cancer gene therapy.

For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25-0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.

[Simple tic disease in children. A report on 93 cases (author's transl)]. / Maladie des tics de l'enfant. A propos de 93 cas.

Based upon a study of 93 cases, clinical data and evolution of tic disease in childhood are described. A correlation within familes is ascertained; a resemblance with Gilles de la Tourette disease is suggested. Pimozide or N. dipropylacetate are recommended as a therapy.

[Long-term results of proximal osteocartilaginous autografts in extensive cartilagenous destruction of the knee. Apropos of 5 cases]. / Résultats a long terme de l'autogreffe ostéo-cartilagineuse de voisinage dans les destructions cartilagineuses etendues du genou. A propos de 5 cas.

Five patients with a large defect in the articular cartilage at the knee joint were treated by transplantation of an autogenic osteochondral fragment. The graft was harvested from the posterior portion of the ipsilateral femoral condyle in 4 cases of osteochondritis dissecans, and from the lateral third of the patella pedicled on the patellar ligament in one case of posttraumatic necrotic collapse of the lateral tibial plateau. One patient underwent concomitant high tibial osteotomy. Two months postoperatively bony union was achieved in all cases but in one case, the grafted articular cartilage did not survive after weight bearing because of an overlooked varus deformity. At the follow-up examination (8 to 20 years) all 5 patients were asymptomatic ; the range of flexion was somewhat restricted (120 degrees) ; roentgenogram revealed slight narrowing of the articular space or at least flattening of the grafted zone and subchondral osteosclerosis. At arthroscopic exploration, the grafted zones were recognizable from the surrounding cartilage, and histologic examination of their border revealed fibrocartilage and proliferating vessels; late gonarthrosis might ensue over time. Therefore the procedure should be performed only in large osteochondral defects where neither reattachment of a loose body, nor hemiarthroplasty, nor isolated osteotomy are suitable and before degenerative changes have developed. Morever any associated varus deformity requires concomitant correction by high tibial osteotomy to relieve stress from the graft.

[Clinical and radiologic outcome of humeral implants in shoulder arthroplasty]. / Devenir clinique et radiologique des implants huméraux des arthroplasties d'épaule.

The clinical and radiological evolution of the glenoid socket in total shoulder arthroplasty is well documented, whereas evaluation of the humeral component has received much less attention. The outcome of 40 humeral replacements performed in 39 patients was studied with a minimum follow-up of one year. There were 26 Neer prostheses, 9 Modular Shoulder prostheses and 5 Aequalis prostheses. Twenty-nine prostheses were implanted with cement and 11 were cementless. The clinical and radiological results were assessed according to the scoring system used for the 1994 symposium of SO.F.C.O.T. X rays in the coronal plane were used to assess the position of the stem, radiolucencies around the humeral component and the percentage of the cross-section of the diaphysis filled by the humeral stem. The average follow-up period was respectively 55.6 months for the Neer prostheses, 17.9 months for the Modular Shoulder prostheses and 12.4 months for the Aequalis prostheses. Radiolucent lines were noted in 20 cases, all with uncemented humeral components. Two prostheses were loose; however, none required revision. The underlying pathology, surgical approach, position of the humeral component, implant design or association with glenoid resurfacing were not correlated with the presence of radiolucent lines or with a loose implant. Cementless fixation was the only parameter statistically related with the presence of radiolucent lines. This report confirms the good results obtained with cemented humeral prostheses. Radiological evaluation of the smooth cementless humeral implant showed many progressive radiolucent lines, without clinical expression. The authors analyze the various studies in the literature and discuss the interest of cementless implants with ingrowth surface.

Telerehabilitation services using web-based telecommunication.

A World Wide Wide-based telerehabilitation platform has been demonstrated in a laboratory environment. This platform allows a rehabilitation provider to thoroughly evaluate the progress of a patient remotely with the same care and measurement precision that would be possible if the provider and the patient were in the same room. The platform was designed to be Web-based so that the service could be offered at the same price without regard to long distance telecommunication facility charges. The Web-based implementation allows enough bandwidth for a simultaneous video teleconference and a precision data acquisition mode even when the Web connection is a low cost analog modem computer interface at both ends of the connection.

[Is calcaneal osteotomy justified in Haglund's disease?]. / L'Ostéotomie calcanéenne est-elle justifiée dans la maladie de Haglund?

PURPOSE OF THE STUDY: The authors report their experience of Zadek's wedge osteotomy in the treatment of posterior talalgia in adults and evaluate the various radiographical measurements for calcaneus deformity measurement.... MATERIAL: This procedure was performed in 27 patients with a total of 36 calcanea treated between 1985 and 1996. The sex ratio was 20 women for 7 men with an average age of 27 years. Prior to this osteotomy nine feet were operated by resection of the postero-superior tuberosity with recurrence of pain and disability, and two patients presented an inflammatory arthritis. METHOD: For the surgical procedure, the patient was placed in lateral position. Skin incision was five to six centimeters long, curvilinear along the lateral side of the tendo achillis. Two dorsal pins were inserted, creating a triangle so that the apex pointed plantarly and the base dorsally. The sagittal saw was then used to create a wedge osteotomy with a dorsal base of seven to ten millimeters. A screw was then driven across the osteotomy site for ultimate fixation. RESULTS: Eighty per cent (29 osteotomies) of patients had been examined clinically and radiologically with a mean follow-up of 29 months. Considering pain, daily activity and discomfort with footwear, results were excellent in 12 cases, good in 10, fair in 5 and poor in two cases. No radiological criteria used for the diagnosis of Haglund's disease were absolutely reliable. The "total angle" of Ruch, the "c.l angle" of Chauveaux and the "parallel pitch lines" of Heneghan reflect the amount of deformation most accurately, but especially, the calcaneal inclination angle was always increased (16 cases between 18 degrees and 30 degrees, 6 cases > 30 degrees). All patients had a weight-bearing calcaneal talus, either alone or in the context of posterior pes cavus. CONCLUSION: This study confirms the responsibility of rearfoot morphostatic disorders in posterior talalgia and the goods results obtained by Zadek osteotomy.

[Temporary antibiotic-loaded cemented prosthesis for two-stage septic hip arthroplasty]. / Prothèse temporaire en ciment imprégné d'antibiotiques au cours des arthroplasties en deux temps sur hanche infectée.

PURPOSE OF THE STUDY: During the excision period of a two-stage revision arthroplasty, the hip has a low function and an unacceptable leg length discrepancy. The goal of this study was to expose technical details in order to perform a simple articulated cement spacer which could be implanted during this period to improve hip function, to authorize partial weight bearing and to avoid leg length discrepancy. MATERIAL: This method was applied in three two-stage procedures justified because of particular immunodeficiency conditions: a 43 years old man who had bone marrow allograft and immunosuppressive therapy because of leukemia suffering of subacute septic hip arthritis; a 58 years old man suffering of diabetes and active C-hepatitis who had a septic loosening of a total hip arthroplasty (THA); a 76 years old woman suffering of diabetes who had a third septic loosening of THA. METHOD: The prosthesis was made of antibiotic-impregnated cement according to organisms antibiotic resistance. The prosthetic junction between head and diaphysis was reinforced with a tibial plate. Prosthetic shape was identical to the one of femoral broaches inserted in the femur after prosthetic and cement removal. The broach size was chosen when mechanical stability in the femur was obtained, and avoided leg length discrepancy after trials with cups. The tibial plate was bent in order to reinforce the junction with regard to the shape of the determined broach. Two doses of antibiotic-impregnated cement were mixed and molded with hands, then the plate was incorporated at the appropriate location, finally the broach was applied on this composite and cement in excess was removed before polymerisation. For prosthetic head, two options were available: to mold the cephalic zone of the cement at the patient acetabulum diameter with a soft aluminium cup previously molded in the acetabulum; to mold the cement cephalic zone with a trial cup in order to obtain a 22 or 28 ball. For this last option, a third dose of antibiotic-impregnated cement was prepared and placed in the acetabulum, a trial femoral head was applied in it to mold the location for the 22 or 28 prosthetic head. Before insertion, a collar was applied on the stem to prevent migration. Active mobilization was encouraged, and partial weight-bearing authorized. RESULTS: The mean range of hip flexion during period was 60 degrees. The patients were discharged approximatively 12 days after the first stage. Two patients had effective painless partial weight-bearing. The second stage was performed six weeks later on the average. The second procedure was easier than the second stage of a conventional two-stage procedure because of: easy and low hemorrhagic dissection authorized by the prosthesis; low difficulties with soft tissue tension as the prosthesis prevents leg length discrepancy; preservation of the articular space which prevents soft tissue sacrifice during the second stage. CONCLUSION: This simple technique is effective to prevent complications related to the excision period of a two-stage hip revision arthroplasty. Likewise, the economical aspect (short delay of hospitalisation, quick functional recovery) should be considered when compared with the excision period of a conventional two-stage procedure.

Improving international nurse training: an American-Italian case study.

BACKGROUND: Institutionalized international nurse training organized by national educational institutions is a relatively new phenomenon. This, descriptive case study examines an early example of an American-Italian initiative of such training, in order to stimulate future international education of nurses. AIM: To find out what factors have to be taken into account to improve training and what its potential effects are in exchange and also in the context of nurse migration. METHOD: A questionnaire was sent to the 85 nurses who all participated in this particular international programme (response rate: 30.6%). FINDINGS: The collected data indicate that personalized and well-aimed training, preparatory language courses, pre-departure exposure of nurses to the culture of the host country and well-prepared welcomes are among the most important ways to improve this programme. IMPLICATIONS FOR PRACTICE: While the specific circumstances and cultures involved in this particular case study should not be ignored, these factors might also be applied to maximize the positive effects of nurse-migration. Two-way learning is among the positive effects of such an international training experience. Motivational and team-building effects can result in enhanced quality of care and a more efficient allocation of resources. However, the mind-opening effect seems to be the most important learning experience. Therefore, regardless of whether one system is considered better or worse than another, experiencing a different way of nursing/education is considered the most important, enriching element of an international learning experience. The effects of this experience could include avoiding cultural imposition in the increased cultural diversity of nursing in the country of origin.

Labelling by axonal transport of myelin-associated proteins in the rabbit visual pathway.

After intraocular injections of [3H]leucine, six regions of the visual pathway of adult rabbit were used to study the spatio-temporal pattern of the slow anterograde axonal transport of radioactive proteins associated with the particulate fraction, the water-soluble fraction and the myelin fraction. Unlike other fractions, myelin-associated labelled proteins represented a time-constant (for a given region) percentage of total tissue radioactivity. This percentage increased from the first half to the second half of the optic nerve and remained high in the chiasma and tract. The peak specific radioactivity of myelin decreased in the same direction. Myelin proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the labelling patterns obtained in different regions and at different survival times were compared. At the peak of myelin radioactivity of a given region the label was typically associated with four protein bands, L1, L2, L3 and L4, of 40000, 44000, 62000, and 68000 mol.wts. respectively. The basic protein, the proteolipid protein and the W1 component (mol.wt. 51000-53000) of the Wolfgram proteins were not significantly labelled. The radioactivity associated with the W2 component (mol.wt 60000) of the Wolfgram proteins could be derived from the closely migrating L3 component. At shorter survival times no clear labelling pattern could be detected. At longer survival times radioactivity was almost totally localized around band L3. The results presented underline the importance of choosing appropriate experimental conditions to obtain a consistent labelling pattern of myelin-associated proteins and to investigate the possible mechanism responsible for this phenomenon.

Internal ophthalmoparesis: an uncommon complication of varicella, a common disease.

Two cases are represented with ocular complications after varicella. In one case the complication was diagnosed as internal ophthalmoparesis and in the second patient the complication could be the same; at any rate an iridoplegia was present. From the cases presented in the literature we found that there seems to be a predilection for males to contract this type of ocular complication. The association of this complication and meningo-encephalitis is rare. Recovery of the pupillary reactions and accommodative power is almost never complete. The interesting observation that decrease in cell-mediated immunity but not in humoral immunity decides whether the clinical disease will manifest itself as herpes zoster or varicella is brought forward.

An enzymic assay for uric acid in serum and urine compared with HPLC.

We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively.

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis.

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme.

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)

Purification, stabilization, and crystallization of a modular protein: Grb2.

We report here the purification and the crystallization of the modular protein Grb2. The protein was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione agarose. It was apparent from reverse phase chromatography that the purified protein was conformationally unstable. Instability was overcome by the addition of 100 mM arginine to the buffers. Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs. The presence of 8 to 14% glycerol was necessary to obtain monocrystals. These results are discussed in relation with the modular nature of Grb2.

Lipid-binding properties of synthetic peptide fragments of human apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.