Correction: Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

Imatinib mesylate (IM) therapy has been shown to induce lower T cell counts in chronic myelogenous leukemia (CML) patients and an interference of IM with T cell receptor (TCR) signaling has been invoked to explain this observation. However, IL-7 and TCR signaling are both essential for lymphocyte survival. This study was undertaken to determine whether IM interferes with IL-7 or TCR signaling to explain lower T cell counts in patients. At diagnosis, CML patients have typically lower CD4+ counts in their blood, yet CD8+ counts are normal or even increased in some. Following the initiation of IM treatment, CD4+ counts were further diminished and CD8+ T lymphocytes were dramatically decreased. In vitro studies confirmed IM interference with TCR signaling through the inhibition of ERK phosphorylation and we showed a similar effect on IL-7 signaling and STAT5 phosphorylation (STAT5-p). Importantly however, using an in vivo mouse model, we demonstrated that IM impaired T cell survival through the inhibition of IL-7 and STAT5-p but not TCR signaling which remained unaffected during IM therapy. Thus, off-target inhibitory effects of IM on IL-7 and STAT5-p explain how T cell lymphopenia occurs in patients treated with IM.

Clinical impact of positive Propionibacterium acnes cultures in orthopedic surgery.

BACKGROUND: The clinical significance of a positive culture to Propionibacterium acnes in orthopedic specimens remains unclear, whether about its role as a contaminant or a pathogen, or its impact as a coinfectant. Therefore, we performed a retrospective study to provide a more accurate description of the clinical impact of P. acnes in an orthopedic population aiming to determine: 1) if there is a clinical difference between P. acnes infection and contamination? 2) If there is a clinical difference between P. acnes monoinfection, and coinfection. HYPOTHESIS: There is a clinical difference between P. acnes infection and contamination. MATERIALS AND METHODS: Patients were selected over a five-year period, and those with a minimum of one positive culture for P. acnes, from any intraoperative orthopedic tissue sample, were included in the study. P. acnes infection was defined as the isolation of P. acnes from≥2 specimens, or in only one specimen, in the presence of typical perioperative findings and/or local signs of infection. RESULTS: A total of 68 patients had a positive P. acnes culture, 35 of which were considered to be infected. The infections affected mostly males (29/35-83%), occurred mostly in shoulders (22/35-63%), and at a site already containing an orthopedic implant (32/35-91%). Local inflammatory signs were present in half of the cases when an infection was diagnosed. Coinfection with other pathogens was present in 31% of patients (11/35). When comparing patients coinfected with P. acnes, and those who were monoinfected, the latter presented less often with local inflammatory signs. Recurrence rate was 24% (8/35) and the only risk factor for recurrence was the presence of a monoinfection. DISCUSSION: This study confirms the pathogenicity of P. acnes in an orthopedic population, as it is present in multiple samples in the same patient, and because it is present in cultures from cases with clinical recurrence. Our study showed that monoinfections differ from coinfections mainly by their higher risk of recurrence. LEVEL OF EVIDENCE: Level IV retrospective case series.

Securing the data economy: translating privacy and enacting security in the development of DataSHIELD.

Contemporary bioscience is seeing the emergence of a new data economy: with data as its fundamental unit of exchange. While sharing data within this new 'economy' provides many potential advantages, the sharing of individual data raises important social and ethical concerns. We examine ongoing development of one technology, DataSHIELD, which appears to elide privacy concerns about sharing data by enabling shared analysis while not actually sharing any individual-level data. We combine presentation of the development of DataSHIELD with presentation of an ethnographic study of a workshop to test the technology. DataSHIELD produced an application of the norm of privacy that was practical, flexible and operationalizable in researchers' everyday activities, and one which fulfilled the requirements of ethics committees. We demonstrated that an analysis run via DataSHIELD could precisely replicate results produced by a standard analysis where all data are physically pooled and analyzed together. In developing DataSHIELD, the ethical concept of privacy was transformed into an issue of security. Development of DataSHIELD was based on social practices as well as scientific and ethical motivations. Therefore, the 'success' of DataSHIELD would, likewise, be dependent on more than just the mathematics and the security of the technology.

The expanding universe of alkaloid biosynthesis.

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase.

The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.

Glucose tolerance and lipid profiles in dogs fed different fiber diets.

Increased dietary fiber is thought to have beneficial effects on health in humans. In diabetic dogs, a beneficial effect of fiber on glycemia has been suggested, while its effect on lipid profiles in dogs has not been described. The effects of different amounts and types of fiber on glucose tolerance and lipid concentrations were evaluated and compared with those of a standard maintenance ration in 30 healthy dogs. It was concluded that increased fiber intake had no influence on glucose in healthy dogs but it may have modulated lipid homeostasis.

The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer.

The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of approximately 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.

[Hockey injuries: consultations in a hospital center in Quebec]. / Blessures liées à la pratique du hockey: consultations dans un centre hospitalier de la région de Québec.

OBJECTIVES: Assess the context and characteristics of hockey injuries, and evaluate the probable effects of regulations concerning mandatory use of head and neck protective equipment. DESIGN: Descriptive study of 247 patients suffering from hockey injuries. Cases were recorded at the emergency room of the Hôpital de l'Enfant-Jésus, in Quebec City, from October 1 1991 to April 30 1992. Injury characteristics are presented by categories, and "Organized hockey on skates" (HPO) is the only category where protective equipment is mandatory. RESULTS: Nearly 42% of consultations were related to non-HPO. In the HPO injuries 15.4% were head injuries whereas in other categories, head injuries represented 31.4%, 33.3% and 44.0% of total injuries. DISCUSSION: Data suggest that regulation imposing mandatory head and neck protection should be maintained in the HPO category because of its apparent preventive effect. Accordingly, implementation could also be considered in other categories.

Effect of occlusive pressure patching on the rate of epithelial wound healing after photorefractive keratectomy.

PURPOSE: This study measured the effect of pressure patching on the rate of epithelial wound healing in 41 myopic patients after photorefractive keratectomy (PRK). METHODS: On day 0, mechanical debridement was done to remove the central 6.5 mm diameter of epithelium. Subsequently, a 5.0 mm diameter photoablation was performed, the depth of ablation being proportional to the degree of myopia (-1.0 to -6.0 diopters). The patients were discharged with an application of antibiotic/steroid ointment (A/S) followed by pressure patching. On day 1, the epithelial defects were stained with fluorescein and photographed. The patients were then randomized prospectively into two groups: Group 1 was pressure patched after application of A/S ointment; Group 2 received A/S ointment twice a day without patching. A photograph of the remaining epithelial defect was taken on day 2. The area of the epithelial defect was measured with a computerized image analyzer. Wound diameter (radius) was used to calculate the epithelial healing rate (EHR). RESULTS: Between days 1 and 2, the mean EHR in Group 1 was 0.072 +/- 0.005 mm/hr and in Group 2, 0.056 +/- 0.004 mm/hr (P < 0.05). There was no correlation between the EHR and patient gender or the degree of myopia. However, a correlation was noted between patient age and wound size on day 1. CONCLUSION: Pressure patching significantly accelerated the EHR following PRK.

Modification of the electrostatic environment is tolerated in the oxyanion hole of the cysteine protease papain.

The oxyanion hole in cysteine and serine proteases can be viewed as an arrangement of prealigned dipoles that complements the changes in charge distribution during the enzymatic reaction. Because of the electrostatic nature of the interaction involved in the oxyanion hole, the introduction of charged residues in that region could have a major effect on the catalytic properties of the enzyme. In this study, residue Gln19, which contributes to one of the hydrogen bonds in the oxyanion hole of papain, has been replaced by glutamic acid, histidine, and asparagine residues. These mutations result in 65-315-fold decreases in kcat/KM, supporting our previous finding that the side chain of Gln19 contributes to transition state stabilization in the oxyanion hole of papain (Ménard et al., 1991a). Since papain is active over a wide range of pH values, the influence of side chain ionization on activity could be measured quantitatively with the mutant Gln19Glu. The pH dependency of kcat/KM for Gln19Glu is not of the classical bell-shaped form normally observed for papain, but instead is modulated by ionization of the Glu19 side chain with a pKa of 6.02. The Gln19Glu mutant at low pH, where the Glu19 side chain is neutral, is the enzyme that displays activity closest to that of wild-type enzyme, with a (kcat/KM)1lim value only 20-fold lower than that for papain. As expected, the activity of the Gln19Glu mutant decreases when the Glu19 side chain ionizes. However, introduction of the negatively charged glutamate into the oxyanion hole of papain leads to a further reduction in activity of only 12-fold, and this mutant is still more active than the Gln19Ser enzyme and only 3-fold less active than Gln19Asn.(ABSTRACT TRUNCATED AT 250 WORDS)

Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing.

The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).

Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain.

The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in kcat/KM for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464,000 M-1 s-1 in wild-type papain. With a Gln19Ser variant, the activity is even lower, with a kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in kcat, while KM values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis.

The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and Glu, respectively, as well as a triple mutant (Val133Ala/Val157Gly/Ser205Glu), where Val157 is also replaced by Gly. Three synthetic substrates were used for the kinetic characterization of the mutants, as well as wild-type papain and cathepsin B: CBZ-Phe-Arg-MCA, CBZ-Arg-Arg-MCA, and CBZ-Cit-Arg-MCA. The ratio of kcat/KM obtained by using CBZ-Phe-Arg-MCA as substrate over that obtained with CBZ-Arg-Arg-MCA is 8.0 for the Val133Ala/Ser205Glu variant, while the equivalent values for wild-type papain and cathepsin B are 904 and 3.6, respectively. This change in specificity has been achieved by replacing only two amino acids out of a total of 212 in papain and with little loss in overall enzyme activity. However, further replacement of Val157 by Gly as in Val133Ala/Val157Gly/Ser205Glu causes an important decrease in activity, although the enzyme still displays a cathepsin B like substrate specificity. In addition, the pH dependence of activity for the Val133Ala/Ser205Glu variant compares well with that of cathepsin B. In particular, the activity toward CBZ-Arg-Arg-MCA is modulated by a group with a pKa of 5.51, a behavior that is also encountered in the case of cathepsin B but is absent with papain.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain.

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.

Passive and active inspired gas humidification in infants and children.

The hypothesis that both active and passive airway humidification prevents hypothermia in infants and children, but that neither decreases the duration of postoperative recovery was tested. Twenty-seven ASA physical status 1 or 2 patients were studied who weighed between 5 and 30 kg, underwent superficial operations, were anesthetized with halothane and 70% N2O, and whose lungs were ventilated via a Rees modification of an Ayre's t-piece. The children were randomly assigned to receive active airway humidification and warming using an MR450 Servo airway heater and humidifier set at 37 degrees C (n = 10), passive airway humidification using the Humid-Vent 1 heat and moisture exchanger placed between the Ayre's t-piece and the endotracheal tube (n = 8), or no airway humidification and heating (control, n = 9). Distal tracheal and tympanic membrane temperatures and airway humidity were recorded during the first 90 min of surgery. Rectal temperature was measured during the postanesthetic recovery period. Relative humidity of inspired respiratory gases was approximately 30% in the control group and approximately 90% in the group given active airway humidification. Initial inspired humidity in the passive humidification group (50%) increased to approximately 80%, a level not significantly different from that in the active group after 80 min of anesthesia. Central body temperature increased 0.25 degrees C during active active airway humidification and heating, whereas temperature decreased 0.25 degrees C during passive humidification and 0.75 degrees C without airway humidification. Distal tracheal temperature was significantly higher in the groups given passive and active humidification than in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)