Day/night variations of high-molecular-weight adiponectin and lipocalin-2 in healthy men studied under fed and fasted conditions.

AIMS/HYPOTHESIS: Adiponectin and lipocalin-2 are adipocyte-derived plasma proteins that have been proposed to have opposite effects on insulin sensitivity. Given the epidemiological, physiological and molecular links between sleep, the circadian timing system and glucose metabolism, the aim of this study was to assess effects of the sleep/wake cycle and the fasting/feeding cycle on high-molecular-weight adiponectin (HMW-adiponectin; the biologically active form) and lipocalin-2. We also aimed to compare the 24 h rhythms in the levels of these proteins with those of cortisol, leptin, leptin-binding protein and total adiponectin. METHODS: Lean men underwent a 3 day in-laboratory study, either in the fed state (n = 8, age: 20.9 ± 2.1 years, BMI: 22.8 ± 2.3 kg/m²) or fasting state (3 day fast, n = 4, age: 25.3 ± 3.9 years, BMI: 23.3 ± 2.2 kg/m²). The sleep episode was scheduled in darkness from 23:00 to 07:00 hours. Blood was sampled every 15 min for 24 h on the third day of each study. RESULTS: While fed, HMW-adiponectin and lipocalin-2 had large daily rhythms with troughs at night (HMW-adiponectin: ~04:00 hours, peak-to-trough amplitude 36%, p < 0.0001; lipocalin-2: ~04:00 hours, 40%, p < 0.0001). On the third day of fasting, the timing and relative amplitudes were unchanged (HMW-adiponectin: ~04:00 hours, 38%, p = 0.0014; lipocalin-2: ~05:00 hours, 38%, p = 0.0043). CONCLUSIONS/INTERPRETATION: These data show that HMW-adiponectin and lipocalin-2 both have significant day/night rhythms, both with troughs at night, that these are not driven by the feeding/fasting cycle, and that it is important to report and/or standardise the time of day for such assays. Further studies are required to determine whether the daily rhythm of HMW-adiponectin levels influences the daily rhythm of insulin sensitivity.

[Single-use laryngoscope blade assessment]. / Evaluation des lames de laryngoscopes à usage unique.

OBJECTIVES: To assess the feasibility of switching disposable laryngoscope blades and to compare the disposable blades available on the market to reusable blades within the context of a new variant of Creutzfeldt-Jakob disease. STUDY DESIGN: Comparative prospective study. MATERIAL AND METHODS: Study conducted on patients intubated for surgical procedures in all operating theatres of a university hospital. The anaesthetic practitioner filled in an assessment form giving a score on nine criteria for each blade used. Data were recorded on Epi Info software. Satisfaction scores of each criterion were compared for both disposable blades and reusable blades. RESULTS: Six brands of blades were tested with 225 blades. Disposable blades were evaluated as inferior to the reusable blades in 62% of cases. Two blades were reported as more satisfactory: the 670166 Rusch-Pilling and Vital View blades. CONCLUSION: The disposable blades were not easily accepted by the anaesthetists particularly for difficult intubations, which is why reusable blades should not be totally removed from practice. Single-use blades proposed by different manufacturers are not identical. We chose 670166 Rusch-Pilling blades, the best adapted to our institution. The switch to disposable blades would require that blade manufacturers improve the quality of the blades.

Inhibition of angiogenesis and metastasis in two murine models by the matrix metalloproteinase inhibitor, BMS-275291.

BMS-275291 is an p.o. bioavailable, sulfhydryl-based matrix metalloproteinase (MMP) inhibitor currently in clinical development for the treatment of cancer. This inhibitor was designed to potently inhibit MMP activities while minimally affecting those of other metalloproteases (e.g., sheddases) involved in the release of cell-associated molecules such as tumor necrosis factor-alpha, tumor necrosis factor-alpha receptor, interleukin-6 receptor, or L-selectin. In vitro, BMS-275291 is a potent inhibitor (nM) of the activities of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14. BMS-275291 inhibits tumor growth in a B16BL6 model of experimental metastasis, and in this model, BMS-275291 treatment results in a dose-dependent reduction in the number of lung metastases compared with vehicle controls. BMS-275291 also inhibits angiogenesis in a murine angiogenesis model, where once daily treatment with BMS-275291 results in a dose-dependent inhibition of endothelial cell migration into s.c. implanted Matrigel plugs. Pharmacokinetic studies demonstrated that the plasma concentrations of parent BMS-275291 in mice exceeds the in vitro IC(50) values for MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 for at least 4 h after the administration of a therapeutic dose of BMS-275291. Taken together, these data demonstrate that BMS-275291 inhibits MMP activities that contribute to tumor metastasis and angiogenesis.

Inhaled nitric oxide: effects on hemodynamics, myocardial contractility, and regional blood flow in dogs with mechanically induced pulmonary artery hypertension.

BACKGROUND: Pulmonary artery hypertension with right ventricular failure is a frequent complication that occurs immediately after heart transplantation in which the use of inhaled nitric oxide may be effective. METHODS: The effects of pulmonary artery hypertension and nitric oxide on myocardial function and on pulmonary and systemic hemodynamic parameters were evaluated in eight anesthetized dogs. Pulmonary artery hypertension was induced by successive microbead injections into the pulmonary circulation. RESULTS: Microbead injections resulted in overt pulmonary artery hypertension (pulmonary artery pressure, + 190%; pulmonary vascular resistance, + 389%; ratio of pulmonary vascular resistance to systemic vascular resistance, 0.41). RESULTS: The end-diastolic length of the right ventricular outflow tract increased significantly along with an increase in right ventricular contractility (peak first derivative of left ventricular pressure as a function of time, + 100%; outflow tract systolic shortening, + 19%). Despite this compensatory mechanism, the increased pulmonary barrier resulted in a decrease in stroke volume (-31%). Systemic effects were observed, such as an increase in heart rate that maintained the cardiac output despite a decrease in left ventricular end-diastolic length (end-diastolic length in region of left anterior descending artery, - 9%). Right myocardial and septal blood flows were also significantly increased. CONCLUSIONS: Nitric oxide administration restored the stroke volume with a decrease in pulmonary artery hypertension and an improvement of the pulmonary vascular resistance to systemic vascular resistance ratio. Systemic blood pressure and coronary perfusion remained unaffected. This selective effect on the pulmonary circulation should be considered a major advantage of nitric oxide inhalation in the treatment of right ventricular dysfunction in acute pulmonary hypertension.

The SH3 domain of p56lck binds to proline-rich sequences in the cytoplasmic domain of CD2.

CD2, a cell surface glycoprotein expressed on T cells and natural killer cells, can couple to signaling pathways that result in T cell proliferation. An Src-like protein tyrosine kinase, p56lck, coprecipitates with CD2, and perturbation of CD2 by monoclonal antibodies results in an increase in the activity of p56lck, suggesting that an interaction with p56lck contributes to CD2-mediated signaling. Herein, we investigate the mechanism by which CD2 associates with p56lck. We demonstrate that CD2 and p56lck associate when coexpressed in nonlymphoid cells, that this association requires the cytoplasmic domain of CD2, and that the SH3 domain of p56lck mediates its interactions with CD2. Using truncation mutants of CD2, we identify two regions in the cytoplasmic domain of CD2 involved in binding p56lck. Each region contains a proline-rich sequence that, in the form of a synthetic peptide, directly binds p56lck. Thus, proline-rich sequences in the cytoplasmic domain of CD2 allow this transmembrane receptor to bind to the SH3 domain of p56lck.

Syk mutation in Jurkat E6-derived clones results in lack of p72syk expression.

The human leukemic Jurkat cell line is commonly used as a model cellular system to study T lymphocyte signal transduction. Various clonal derivatives of Jurkat T cells exist which display different characteristics with regard to responses to external stimuli. Among these, the E6-1 clone of Jurkat T cells has been used as a parental line from which numerous important somatic mutant clones have been generated. During the course of experiments examining signals initiated by the T cell antigen receptor in an E6-1-derived Jurkat cell clone J.CaM1, we observed that the 72-kilodalton Syk protein tyrosine kinase previously found in other Jurkat cells was not detected. Upon further analysis it was determined that Syk transcripts from the J.CaM1 cells as well as the parental E6-1 cells contain a single guanine nucleotide insertion at position 92. This nucleotide insertion results in a shift in the Syk open reading frame leading to alternate codon usage as well as the generation of a termination codon at position 109. Thus, Syk transcripts in E6-1 cells and E6-1-derived clones are predicted to be capable of encoding only the first 33 amino acids of the 630-amino acid wild type Syk. These findings are incompatible with a recently proposed model of T cell antigen receptor signal transduction based, in part, on experiments conducted using E6-1-derived cells, suggesting that Syk might play a role upstream of Lck and Zap70.

Reconstitution of the B cell antigen receptor signaling components in COS cells.

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.

A computer program for automatic measurement of respiratory mechanics in artificially ventilated patients.

A program for automatic and periodic determination of respiratory mechanics in artificially ventilated patients is described. Airway pressure and flow signals are obtained from the ventilator in the controlled ventilation mode with constant flow inflation and end-inspiratory pause. Periodically, the program records both signals for a given time and it delimits a ventilatory cycle and its components out of this record. Then, four mechanical parameters of the respiratory system are calculated: (1) Rinit, the resistance obtained with the end-inflation occlusion technique; (2) Ers, the elastance (inspiratory) calculated from the slope of the airway pressure profile during inflation; (3) tau, the expiratory time constant; (4) PEEP, the global positive end expiratory pressure. All parameter measurements have been evaluated in experimental conditions, and are in good agreement with reference values. The complete software includes the display of the signals and of the trends together with automatic disk file backups. An additional program allows one to display the trends again and to create table text files containing all the recorded data for further analysis. The system proved to work in ICU and anaesthesia patients with various ventilators.

Molecular cloning of rodent p72Syk. Evidence of alternative mRNA splicing.

Northern blot analysis of polyadenylated RNA prepared from RBL-2H3 cells revealed the presence of three distinct mRNAs encoding p72Syk, a protein-tyrosine kinase previously shown to be associated with the high affinity IgE receptor present on the surface of these cells (Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9107-9111). Here we report the full-length nucleotide sequence of two of these messages, as well as the complete predicted amino acid sequence of the rodent p72Syk protein-tyrosine kinase. In addition, we report evidence indicating alternative splicing of p72Syk mRNAs within RBL-2H3 cells. This splicing event results in the expression of two distinct protein isoforms that differ with respect to the presence of a 23-amino acid insert located within the region of the protein that separates the two SH2 domains from the catalytic domain. Both mRNAs arising from this splicing event appear to encode functional protein-tyrosine kinases, as expression of the corresponding cDNAs in COS cells results in the production of proteins of the expected sizes that possess intrinsic tyrosine specific kinase activity.

Comparison between acute hypoxia-induced and mechanically-induced pulmonary artery hypertension on the hemodynamics, myocardial contractility and regional blood flow in dogs.

Two groups of eight anesthetized dogs with pulmonary artery hypertension (PAH) were compared. PAH was induced by submitting one group (HP) to hypoxia (FiO2 range: 6-10%) and the other group (ME) to microemboli through glass microbead injection into the pulmonary circulation. Hypoxia-induced PAH was moderate (PAP: +65%; PVR: +152%) contrasting with marked PAH after microbead injection (PAP: +190%; PVR: +389%). For similar effects on left ventricular contractility (LV dP/dt max and segmental myocardial shortening), heart rate and systemic vascular resistance, left ventricular end-diastolic pressure showed significant differences between the two groups (HP group: +75%, ME group: -9%), and so did left ventricular end-diastolic length (HP: +9%, ME: -11%). Thus, contrary to the injection of microbeads, hypoxia did not give rise to any pulmonary barrier, and consequently the changes in cardiac output (HP: +19%, ME: -15%) and hepatic blood flow (HP: +383%, ME: -77%) were significantly different. Hypoxia, and not microbead injection, was responsible for systemic hypertension (MAP: +34% and -4%, respectively). The microbead model resulted in a significantly higher PVR/SVR ratio compared to the hypoxic model (HP: 0.14, ME: 0.41). Hypoxia increased left and right myocardial blood flows whereas microbead injection affected only right ventricular blood flow, leading to significantly different RV/LV endocardial perfusion ratios (HP: +10%, ME: +98%). We conclude that microbead-induced PAH is more appropriate than hypoxia-induced PAH for hemodynamic and pharmacological studies.

Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement.

We evaluated in Jurkat T cells the time-dependent responses of Fyn, Lck, Syk, and Zap following antibody-mediated cross-linking of the T cell antigen receptor. Our results show that the protein kinase activities of Fyn and Lck were activated within seconds of receptor cross-linking. Fyn activity, as measured by autophosphorylation and tyrosine phosphorylation of an exogenous substrate, was maximal 5 s to 1 min following receptor cross-linking. Lck was also found to be activated within 5 s of antigen receptor cross-linking but differed from Fyn in that Lck activity was elevated for at least 30 min. Syk and Zap protein kinase activities were found to peak between 5 and 10 min following receptor cross-linking, returning to approximately basal activity levels by 60 min. The protein kinase activities of both Syk and Zap were found to parallel their reactivity in immunoblotting experiments with anti-phosphotyrosine antibodies. Both Syk and Zap were found to associate with the tyrosine-phosphorylated zeta subunit of the T cell antigen receptor. These observations imply that T cell antigen receptor signal transduction involves the activation of multiple members of at least two different families of non-transmembrane protein tyrosine kinases.

Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction.

Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family.

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.

Interleukin-2-induced tyrosine phosphorylation of Shc proteins correlates with factor-dependent T cell proliferation.

Interleukin-2 (IL-2) is a growth factor involved in the clonal expansion of antigen-activated T lymphocytes. Interaction of IL-2 with its receptor triggers tyrosine phosphorylation of a series of proteins and results in the activation of p21ras. We report here that Shc, an SH2-containing adaptor protein, is tyrosine-phosphorylated following IL-2 stimulation. IL-2-induced tyrosine phosphorylation of Shc was detectable within seconds following IL-2 addition, reaching its highest level by 15 min. Tyrosine phosphorylation of Shc was induced in multiple IL-2-dependent T cell lines and was found to correlate with IL-2-dependent cell proliferation. Tyrosine-phosphorylated Shc was found to be capable of associating with the SH2 domain of Grb2 following IL-2 stimulation. These results indicate that tyrosine phosphorylation of Shc and its association with Grb2 may be important events in IL-2-initiated signal transduction events in T cells.

Analysis of the tyrosine protein kinase p56lck expressed as a glutathione S-transferase fusion protein in Spodoptera frugiperda cells.

A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.

Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase.

The cellular yes (c-yes) gene is a member of the class of proto-oncogenes that encode non-receptor tyrosine protein kinases. In this report we describe the isolation of cDNAs that encode the murine c-yes gene product and analysis of the nucleotide sequence of the murine c-yes cDNA clones. The reading frame encodes a protein of 541 amino acids with a calculated molecular mass of 60.63 kDa that is reactive with anti-Yes antisera and possesses protein kinase activity.

Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking.

Activation of T cells by specific antigens in the context of major histocompatibility complex encoded proteins is mediated by the T cell antigen receptor (TcR), consisting of a variable (Ti) and an invariant (CD3) subunits. Tyrosine phosphorylation is considered to be one of the earliest steps in TcR-mediated signal transduction. There are indications that the p60fyn protein tyrosine kinase is involved in signaling via TcR. However, enzymatic activation of the Src-related tyrosine kinases upon TcR triggering has not been shown yet, therefore the identity of TcR-activated tyrosine kinase(s) remains unclear. We demonstrate that cross-linking of CD3 activates p60fyn and induces tyrosine phosphorylation of cellular proteins in human T cells (resting peripheral T cells, a helper T cell clone, a helper T cell clone immortalized with Herpesvirus saimiri, and a leukemic T cell line). Activation of p60fyn was fast, and its maximum (2-4-fold activation as compared with the basal activity) was followed by a decline. The amount of p60fyn in the cells remained unchanged. None of the other T cell Src-related tyrosine kinases was activated after cross-linking of CD3. Activation of p60fyn was induced by anti-CD3, but not by anti-CD4, anti-CD2, or anti-CD28. The activation was correlated with an increase of the phosphotyrosine content of p60fyn. These studies provide direct proof for the functional association between p60fyn and the TcR.

Cross-linking of surface immunoglobulin activates src-related tyrosine kinases in WEHI 231 cells.

Crosslinking of sIgM on the B cell line WEHI 231 with anti-sIgM antibody induces protein tyrosine phosphorylation, implicating protein tyrosine kinases (PTKs) in sIg-mediated signal transduction. We have analyzed this cell line for members of the src family of PTKs and have evaluated whether these PTKs might be involved in the process of sIgM-mediated signaling. Our results show that Blk, Lyn, Lck, and Hck are detectable in WEHI 231 cells. Addition of antibodies to sIgM were found to variably stimulate the activities of Blk, Lyn, Lck, and Hck as measured by immune-complex protein kinase assays. Autophosphorylation of these src PTKs, as assessed by reaction with anti-phosphotyrosine antibodies, increased over the time course of sIgM-mediated activation. Co-immunoprecipitation studies to investigate the potential physical interaction of src PTKs with the sIgM receptor complex revealed that, under digitonin and Brij 96 lysis conditions Lyn, Lck, Hck, but not Blk associated with sIgM.

Isolation, characterization and chromosomal localization of the human GADD153 gene.

We report the isolation and characterization of the growth arrest and DNA-damage-inducible gene, GADD153, from human cells and show that it is localized in the region 12q13.1-q13.2 on chromosome 12. Comparison of the human gene with the previously described hamster gene revealed a high level of conservation in both the structural and regulatory regions of the genes. Each is composed of four exons with intron/exon junctions maintained at the identical positions. The human Gadd153 protein shares 91% identity with the hamster protein in amino acid sequence, and 78% identity in nucleotide sequence. A 900-bp fragment of 5' flanking sequence from the human gene, when linked to the bacterial cat reporter gene, was found to exhibit promoter activity in HeLa cells which could be further activated by treatment with the DNA alkylating agent, methyl methanesulfonate. Sequence analysis indicated that the human promoter region is relatively G+C-rich and contains putative binding sites for multiple transcription factors, including recognition sites for TATA- and CAAT-binding proteins, six Sp1-binding sites, an activator protein-1 binding site, an E-26-specific sequence-binding protein-1 DNA-binding site, and four interleukin-6 response elements. Many of these sites are also present in an identical position in the hamster gene suggesting they may play an important role in regulating GADD153 expression.