2.3 W single transverse mode thulium-doped ZBLAN fiber laser at 1480 nm.

A 2.3 W single transverse mode thulium-doped fluoride fiber laser based on fiber Bragg gratings is presented. The laser has a conversion efficiency of 65% to be compared to the quantum limit of 72%. The performances of the laser are compared for two pump wavelengths of 1040 and 1064 nm and are analyzed based on a rate equation analysis.

Bragg gratings photoinduced in ZBLAN fibers by femtosecond pulses at 800 nm.

Fiber Bragg gratings were written in thulium-doped and undoped single-mode ZBLAN fibers by focusing femtosecond laser pulses on the fiber core through a phase mask. Maximum index modulation of the order of 1 x 10(-3) was induced in both types of fibers. Measurements of the transverse refractive index changes across the core and cladding regions indicate that the grating formation originates from a negative index change.

Facilitation of neonatal endotracheal intubation with mivacurium and fentanyl in the neonatal intensive care unit.

BACKGROUND: Endotracheal intubation in the neonate is painful and is associated with adverse physiological effects. Some premedication regimens have been shown to reduce these effects, but the optimal regimen is not yet determined. METHOD: Data on semi-elective intubations were prospectively collected in the neonatal intensive care unit over a six month period. Patients received 20 microg/kg atropine, 200 microg/kg mivacurium (a non-depolarising muscle relaxant) followed by 5 microg/kg fentanyl. RESULTS: Thirty three patients were electively intubated during this time period. The primary reason for intubation was surfactant administration (53%). Median (range) birth weight, gestational age, and age at intubation were 1360 g (675-4200), 29 weeks (25-38), and 33 hours (1-624) respectively. Twenty two of the infants were intubated on the first attempt. Median duration from initial insertion of the laryngoscope to successful intubation was 60 seconds (15 seconds to 20 minutes). In 18 cases, the first attempt was by a trainee with no previous successful intubation experience, 10 of whom intubated within two attempts. Muscle relaxation occurred at a mean (SD) of 94 (51) seconds, and mean (range) time to return of spontaneous movements was 937 seconds (480-1800). Intubation conditions were scored as excellent using a validated intubation scale. CONCLUSION: Effective analgesia can be administered and intubation performed with some brief desaturations, even when junior personnel are being taught their first intubation. In this first report of mivacurium for intubation in the newborn, effective bag and mask ventilation was easily achieved during muscle relaxation and was associated with excellent intubation conditions, permitting a high success rate for inexperienced personnel.

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.

[Laparoscopic posterior fundoplication in gastroesophageal reflux: mid-term results]. / Fundoplicature postérieure coelioscopique pour reflux gastro-oesophagien: résultats à moyen terme.

STUDY AIM: The aim of this study was to report the mid-term results of the surgical management of gastroesophageal reflux disease (GERD) by laparoscopic posterior partial fundoplication (Toupet technique) in 100 patients, and to evaluate their post-operative quality of life. PATIENTS AND METHOD: Between November 1993 and January 2000, 100 patients were surgically treated for a medically refractory GERD. Laparoscopic posterior partial fundoplication was performed by the Toupet technique. In the postoperative period, the patients were asked to answer a questionnaire by telephone. The aim of this survey was three-fold: to identify clinical symptoms indicative of recurrence; to evaluate postoperative functional impairment; to assess the postoperative quality of life. pH monitoring was also proposed in asymptomatic patients at a minimum follow-up of two years, and in all patients with clinical symptoms of GERD recurrence. RESULTS: Six laparotomy conversions were necessary. The mean duration of follow-up was 18 months (range: 6 to 57 months). The rate of clinically diagnosed recurrence was 7.6%. Intermittent dysphagia was observed in 2.3% of cases. Postoperative digestive functional disorders were noted in 53% of patients without clinical recurrence, and 95.3% of them were satisfied or very satisfied with the results of surgery. CONCLUSION: Laparoscopic posterior partial fundoplication by the Toupet technique can satisfactorily treat GERD without mid-term recurrence in about 94% of cases. Patient satisfaction seems mainly to depend on the disappearance of clinical symptoms of GERD. It was found that postoperative functional disorders frequently occurred, but were well tolerated. Their etiology has not yet been determined, and it is considered that factors other than the surgical procedure may also play a role.

Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger.

The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four-step procedure and p-nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246-fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 degrees C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.

Efficacy of sequential early systemic and inhaled corticosteroid therapy in the prevention of chronic lung disease of prematurity.

In order to assess the efficacy of a combination of systemic and nebulized corticosteroids in reducing the incidence and severity of chronic lung disease (CLD) in very low birthweight (VLBW) infants, 60 ventilator-dependent infants < or = 1500 g were randomly assigned to receive either steroids or placebo as of 7 d. The steroid group (n = 30, GA = 25.8 +/- 1.6 weeks, BW = 731 +/- 147 g) received systemic dexamethasone for 3 d, followed by nebulized budesonide for 18 d. Control infants (n = 30, GA = 25.9 +/- 1.8 weeks, BW = 796 +/- 199 g) received systemic and inhaled saline. Steroid-treated infants required less ventilatory support between 9 and 17 d (p < 0.01), and had greater lung compliance at 10 d (p = 0.01), but not subsequently. CLD incidence at 36 weeks was 45.5% vs 56.0% in controls, and fewer steroid-treated infants required dexamethasone rescue (23.3% vs 56.7%, p = 0.017). Survival to discharge was similar (73.3% vs 83.3%), as were the durations of mechanical ventilation, supplemental oxygen use, and hospitalization. Tracheal effluent elastase/albumin ratios and serum cortisol values did not differ between groups, and no adverse effects were noted. We conclude that early dexamethasone administration was associated with improved pulmonary function, which was not sustained with nebulized budesonide. However, the steroid regimen studied reduced the need for dexamethasone rescue in infants with CLD.

[3H](azidophenyl)ureido taxoid photolabels peptide amino acids 281-304 of alpha-tubulin.

The taxoid binding site on porcine brain tubulin was covalently labeled, in the presence or absence of Taxotere, with the photoaffinity reagent [3H]-p-(azidophenyl)ureido taxoid derivative [3H]TaxAPU [Combeau, C., Commercon, A., Mioskowski, C., Rousseau, B., Aubert, F., & Goeldner, M. (1994) Biochemistry 33, 6676-6683]. After disulfide reduction and carboxymethylation, the alkylated tubulin samples were treated with trypsin and the mixtures of peptides were first fractionated by gel filtration over Sephadex G50. Anion exchange chromatography of the radioactive areas showed, for one area, three major radioactive signals which were further analyzed by reversed phase C18 HPLC, leading to well-resolved radioactive peaks. Microsequencing of these different peaks gave a complete sequence of a tryptic fragment on alpha-tubulin (alpha-281-304) and two partial peptide sequences of a tryptic fragment on beta-tubulin (beta-217-229) in addition to sequences of mixture of peptides. The radioactive signals were lost while concentrating the samples for microsequencing, preventing the identification of the modified amino acids. These results identify the first peptide on alpha-tubulin which binds to the taxoids and confirm the involvement of both alpha- and beta-tubulin in the taxoid binding site.

A Ras-GTPase-activating protein SH3-domain-binding protein.

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

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.

Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis.

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Identification of the cytoskeletal protein alpha-actinin as a platelet thrombospondin-binding protein.

Binding of the alpha-granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified 125I-labeled TSP, we observed a strong interaction of [125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, alpha-actinin. We further demonstrated that [125I]TSP interacts with alpha-actinin in a specific manner and with a high affinity (Kd = 6.6 nM) in a solid-phase binding assay.

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

The major biotinyl protein from Pisum sativum seeds covalently binds biotin at a novel site.

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins.

Ontogeny of arginine vasopressin-immunoreactive neurons in the hypothalamus of fetal and newborn sheep.

Arginine vasopressin (AVP) is a peptide hormone which is found in neurons within the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus. In fetal sheep, this neuropeptide is involved in maturational processes and adaptive responses to 'stress'. This study examined the effect of age on the total number and distribution of AVP-containing neurons in the PVN and SON of fetal sheep and newborn lambs by quantitative light-microscopic immunocytochemistry. Serial coronal sections of hypothalami from three groups of animals were studied: fetuses at 104-109 days of gestation (n = 6) comprising the early group, fetuses at 130-139 days of gestation (n = 5) comprising the late group and newborn lambs at 12-20 postnatal days (n = 5) comprising the neonatal group. This period of development was chosen since adaptive mechanisms to stress are operative at or near the time of birth. Hypothalamic dimensions were measured to determine if maturation had an effect on the size of the AVP-containing subregions of the hypothalamus during this period of development. Dimensions included: ventricle height, optic chiasm width, distances from the dorsal margin of the ventricle to the lateral and medial margins of the optic tract, and distance between the medial margins of the optic tracts. As expected, with increase in maturational age, overall dimensions of the AVP-containing subregions increased significantly (p < 0.05). When early- and late-gestation fetuses were compared to newborn lambs, there was a significant increase in the total number of immunoreactive neurons in both the PVN (p < 0.01, Anova) and SON (p < 0.001, Anova) with age. With advancing age, we also observed an increase in the density of AVP neurons in the middle subregion of the PVN and in the midrostral subregion of the SON. These data suggest that, during the late gestational and early postnatal period, de novo synthesis of AVP genes occurs in these hypothalamic nuclei. This study provides a baseline for further investigation to study the effects of stress on these neurons in the developing ovine fetus and newborn lamb.

Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Topical anesthesia during circumcision in newborn infants.

OBJECTIVE: To determine the efficacy of topical anesthetic cream, eutectic mixture of local anesthetics (EMLA), in alleviating pain from neonatal circumcision. DESIGN: Randomized, controlled trial. SETTING: Normal newborn nursery in a university teaching hospital. PATIENTS: Twenty-seven newborn, full-term male neonates, aged 1 to 3 days. OUTCOME MEASURES: Heart rate, transcutaneous oxygen saturation, facial activity, and crying. RESULTS: Compared with baseline, all newborns experienced pain as evidenced by increased heart rate of an average of 40 beats per minute (F [8, 25] = 14.12; P < .0001), decreased oxygen saturation of 3% (F [8, 25] = 15.02; P < .0001), and more facial actions indicative of pain (F [8, 25] = 5.25; P < .002) during all phases of the procedure. Compared with placebo, EMLA significantly attenuated the pain response as shown by lower heart rate of an average of 25 beats per minute (F [1, 25] = 14.92; P < .001), higher oxygen saturation of 5%, particularly during the clamping and lysis phases (F [1, 25] = 19.83; P < .0001), 20% less facial activity (F [1, 25] = 12.01; P < .002), and 15% less crying during the clamping, Gomco clamp application, and incision of the foreskin. There were no differences between groups in the spectral crying parameters. CONCLUSIONS: Circumcision procedure produces pain responses that EMLA diminishes. Thus, EMLA may be a useful agent for pain management in neonatal circumcision.

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.