Severe hyperhomocysteinemia and peripheral neuropathy as side effects of nitrous oxide in two patients with sickle cell disease.

Nitrous oxide (N2O) is a widely used anesthetic agent. We report two patients with sickle cell disease (SCD) who presented with complications following the use of N2O. Patient 1, a 15-year-old girl, presented severe hyperhomocysteinemia, pancytopenia, vitamin B12 deficiency, and peripheral polyneuropathy after massive use of N2O for pain management. At the 1-year follow-up, hyperhomocysteinemia and B12 deficiency had resolved, but she had persisting mild symptoms of polyneuropathy. Patient 2, a 17-year-old boy, presented only severe hyperhomocysteinemia, only partially corrected by initial B12 supplementation. Careful monitoring of N2O use, especially in patients with SCD, is mandatory to prevent complications.

Infant botulism: Two case reports and electroneuromyogram findings.

Botulism is an uncommon severe neuromuscular disorder. We report two recent cases of confirmed infant botulism diagnosed in an 11-week and a 5-month-old infant along with electroneuromyogram (ENMG) findings. Then, we discuss the EMG features of infant botulism. In severe forms of infant botulism, presence of these features might help decide to use botulinum immune globulin. To our knowledge, case 1 is the first case reported in France based on confirmed dust contamination.

Clinical interest of bone texture analysis in osteoporosis: a case control multicenter study.

UNLABELLED: We demonstrate the clinical interest of bone texture analysis with a new high resolution X-ray device. We have found that the combination of BMD and texture parameter values provided a better assessment of the fracture risk than that obtainable solely by BMD measurement. INTRODUCTION: Osteoporosis is characterized by BMD and trabecular bone microarchitecture. We have developed a new high-resolution X-ray device with direct digitization. The aim of this study was to demonstrate in a multicenter case control study the clinical interest of bone texture analysis with this new device. METHODS: In this cross-sectional multicenter case-control population study in post-menopausal women, 159 osteoporotic fractures were compared with 219 control cases. Images were obtained on calcaneus with a direct digital X-ray device (BMA, D3A Medical Systems). Co-occurrence, run-length matrices and the fractal parameter Hmean were evaluated. BMD was measured at the lumbar spine (LS), femoral neck (FN) and total hip (TH) by DXA. RESULTS: The three texture parameters were significantly lower in osteoporotic fracture cases than in control cases. These differences persisted after adjustment for TH BMD. Receiver operating characteristic curves were used to compare the discriminant capacity of texture parameters and BMD measurements for fracture. The highest areas under curve (AUC) were 0.721 for TH BMD and 0.706 for Hmean (AUC THBMD vs. AUC Hmean, p = NS). We determined the threshold between high and low Hmean parameter values and then the odds ratios (OR) of fracture for low Hmean, for BMD < or =2.5 SD in the T-score and for combinations of both parameters. The OR of fracture for low H was 2.72 (95% CI, 1.36-5.4). For a FN BMD < or = -2.5 SD, the OR of 4.78 (2.19-10.43) shifted to 14.06 (4.41-44.85) adding H. CONCLUSIONS: These data confirmed the clinical interest of the combination of BMD and texture parameters to improve the assessment of the risk of fracture other that obtainable by the sole BMD measurement.

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.

In vitro pyrogenicity of Gram-positive bacteria--validation of the kit using fresh human whole blood.

The two conventional tests to detect pyrogen contaminants in injectable pharmaceutical drugs are the Rabbit Model and the Limulus amoebocyte lysate (LAL) test. To replace these models, a new system on human whole blood is developed, using the release of Interleukin 1 beta (IL1beta) after cell stimulation with gram-positive and gram negative pyrogens. The purpose of this study was to validate the ENDOSAFE-IPT kit using the quantitative ELISA enzyme immunoassay. The assay is divided into two parts: blood cell stimulation with Lipopolysaccharides (LPS) and Lipoteichoic acid (LTA) and quantitation of IL1beta using the ELISA method. In each assay, blood from a particular donor were stimulated with the Endotoxin Standard, and with a sample of a commercial antibiotic preparation (Clavulanic acid/Ticarcillin) spiked with the Endotoxin Standard. LTA from Bacillus subtilis and a sample of diphtheria toxoid were also used. At least, six assays were tested. A polynomial regression of the Endotoxin Standard series showed a correlation coefficient greater than 0.99. The spiked antibiotic sample recoveries were 50-121%. The LTA quantitation limit was 0.1 microg/ml and the range of detection of pyrogens from Gram positive diphtheria toxoid was 0.77 to 2.5 EEU/ml. The IL1beta production varied markedly between donors. However the coefficient of variation was less than 20 % intra-assay. In conclusion, the ENDOSAFE-IPT kit can be used for the quantitative and qualitative detection of pyrogens from Gram negative and Gram positive bacteria.

A naturally processed mitochondrial self-peptide in complex with thymic MHC molecules functions as a selecting ligand for a viral-specific T cell receptor.

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4(+)CD8(+) double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K(b) molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K(b)-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K(b). The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68-75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO(68-75)). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2(-/)- transporter associated with antigen processing 1(-/)- fetal thymic organ culture, NUbO(68-75) induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO(68-75) to support differentiation of a second K(b)-restricted TCR indicates that its inductive effects are not general.

Cloning and preliminary characterization of a 121 kDa protein with multiple predicted C2 domains.

In the course of characterizing proteins present in a preparation of vesicles from rat adipocytes containing glucose transporters, we examined a protein that migrated at 115 kDa upon SDS gel electrophoresis (designated vp115). Sequences of tryptic peptides were obtained, and from this information the cDNA for rat vp115 was cloned. The cDNA encodes an open reading frame for a protein of 121 kDa. Computer-aided sequence analysis predicted that vp115 has a potential membrane-inserted or membrane-spanning domain toward its amino terminus, followed by five C2 domains. Immunoblotting revealed that vp115 was not actually a component of the glucose transporter-containing vesicles, was most abundant in the plasma membranes and high density microsome fractions of rat adipocytes, and was expressed in all the major rat tissues.

Cloning and characterization of a 22 kDa protein from rat adipocytes: a new member of the reticulon family.

In the course of our examination of proteins associated with the GLUT4-containing vesicles of rat adipocytes we have identified a new 22 kDa member of the family of endoplasmic reticulum (ER) proteins known as reticulons. The protein, which we refer to as vp20, was purified from a preparation of GLUT4-containing vesicles of rat adipocytes, and tryptic peptides were micro-sequenced. From this information a cDNA encoding a single open reading frame for a protein of 22 kDa was cloned. This protein is homologous to known members of the reticulon protein family. vp20 has two hydrophobic stretches of about 35 amino acids that could be membrane spanning domains and an ER retention motif at its carboxy-terminus. vp20 was most abundant in the high density microsome fraction of adipocytes, which is the fraction most enriched in ER. Only a small fraction of vp20 was present in the GLUT4 vesicle population, and that fraction appears to be due to ER vesicles that were non-specifically bound to the adsorbent. Analysis of tissue distribution of vp20 in rats revealed that it is concentrated in muscle, fat and the brain.

Binding motifs of copolymer 1 to multiple sclerosis- and rheumatoid arthritis-associated HLA-DR molecules.

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".

VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies.

Steady-state captopril renography: continuous monitoring of the captopril-induced increase in 99mTc-MAG3 mean parenchymal transit time in renovascular hypertension.

Steady-state captopril renography (SSCR) is an original technique for assessing the captopril-induced increase in technetium-99m mercaptoacetyltriglycine (99mTc-MAG3) mean parenchymal transit time (MPTT) in kidneys affected with functional renal artery stenosis (RAS). The steady-state parenchymal activity achieved by constant infusion of 99mTc-MAG3 is directly linked to the MPTT of the radiopharmaceutical. This steady-state parenchymal activity was continuously monitored from 15 min before to 60 min after a single dose of captopril in order to detect possible disruption of the steady state. SSCR was performed in 11 hypertensive patients with unilateral RAS and in two with RAS of a solitary kidney before renal revascularization (RR). In four of these patients, an additional SSCR was performed after RR. Of the ten patients whose hypertension was cured or improved by RR, one presented an uninterpretable SSCR and six presented a positive SSCR on the affected side. Control SSCR performed in four of these six patients was bilaterally negative. SSCR was also bilaterally negative in the three patients who showed no blood pressure response to RR. These preliminary results tend to indicate that, in spite of the stability of pre- and post-captopril hydration and data acquisition conditions allowed by this steady-state technique, the sensitivity is lower than expected. However, the reason for the false-negative results does not seem to be inherent to SSCR.

Purification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription.

The Tat protein is a transcriptional activator which is required for efficient human immunodeficiency virus 1 (HIV-1) gene expression Tat stimulates HIV-1 transcriptional elongation by increasing the processivity of RNA polymerase II. To address whether Tat-mediated effects on HIV-1 gene expression are due to modulation in the phosphorylation of the RNA polymerase II C-terminal domain (CTD), we developed a purification protocol to identify cellular kinases that are capable of binding to Tat and hyperphosphorylating the RNA polymerase II CTD. A 600 kDa protein complex with these properties was isolated, and specific components were identified using peptide microsequence analysis. This analysis indicated that proteins comprising the multi-subunit TFIIH complex, in addition to several novel factors, were associated with Tat using both in vitro and in vivo analysis. The Tat-associated kinase bound to the activation domain of Tat, and its ability to hyperphosphorylate RNA polymerase II was markedly stimulated by Tat. Furthermore, the addition of the Tat-associated kinase to in vitro transcription assays stimulated the ability of Tat to activate HIV-1 transcription. These results define a cellular kinase complex whose activity is modulated by Tat to result in activation of HIV-1 trancription.

The MHC class I homolog encoded by human cytomegalovirus binds endogenous peptides.

The ability of a human cytomegalovirus-encoded homolog of MHC class I molecules to serve as a peptide receptor was investigated. Sequencing of peptide material eluted from the purified viral protein revealed a mixture of endogenous peptides with characteristics similar to those eluted from conventional class I molecules, that is, anchor residues, and a predominance of short peptides derived from cytoplasmic proteins. The possible function(s) of this viral MHC homolog are discussed in light of the finding that it binds endogenous peptides.

[Direct pulmonary embolectomy without extracorporeal circulation. 5 cases]. / Embolectomie pulmonaire directe sans circulation extracorporelle. Cinq observations.

Five patients underwent pulmonary embolectomy without cardiopulmonary bypass. All had recent embolism completely obstructing the right branch of the artery but usually leaving the left branch open or almost totally free. In all patients fibrinolytic drugs were formally contra-indicated. Thoracotomy was performed through the right lateral approach. The embolus was completely removed; there were no post-operative complications and the results on follow-up are excellent.

[Two cases of traumatic rupture of the corpus cavernosum (author's transl)]. / A propos de deux cas de rupture traumatique d'un corps caverneux.

The authors report two cases of traumatic rupture of the corpus cavernosum and have insisted on the early repair of the tunica albuginea best guarantee of adequate cure without erection impairement.

[An unusual appearance of ureteric bilharzia (author's transl)]. / Sur un aspect particulier de bilharziose urétérale.

The authors report a case of bilharzia localised to one of the ureters in a case of double renal pelvis and ureter. The parasitic infection was identified only at the time of exploratory surgery (laboratory investigations and preoperative endoscopy negative). The "providential" double ureter made it possible to combine excision of the pathological ureter with a termino-lateral anastomosis between the lower renal pelvis and the ureter of the upper renal pelvis. The result remains very satisfactory with a follow-up of 13 months.