On the validation of magnetic resonance velocimetry in single-phase turbulent pipe flows.

A nuclear magnetic resonance imaging technique is used to measure velocity distributions in turbulent pipe flows up to Re=24580. While turbulent intensity is usually determined from signal attenuation, we deduce turbulent intensity from velocity distribution with no need to suppose a gaussian distribution for velocity fluctuations. Skewness and flatness measurements are also presented in this paper. Comparison with DNS show good agreement and we show that NMR data is sufficently accurate to provide turbulent viscosity profile. The low field system used in this study allow the suppression of susceptibility artifacts and thus open its use for studying two-phase flows. We postulate that the method used here could be applied to two-phase flows and would thus provide valuable information on turbulent viscosity models.

CSF 5-HIAA levels are lower in impulsive as compared to nonimpulsive violent suicide attempters and control subjects.

BACKGROUND: We studied CSF 5-HIAA and HVA concentrations in violent suicide attempters and examined their relationship with depression, anxiety, and impulsivity. METHODS: CSF 5-HIAA and HVA concentrations were determined very shortly after hospital admission and compared to those of a matched control population. Clinical evaluation was performed concomitantly; the level impulsivity was evaluated by the Impulsivity Rating Scale (IRS). RESULTS: Twenty-three patients and 23 control subjects were included. According to the IRS, 14 patients were classified as impulsive, including all patients suffering from personality disorders, and 9 as nonimpulsive, with a main DSM-IIIR diagnosis of melancholia. CSF 5-HIAA concentrations in the suicide group were significantly lower than in control subjects. This difference was entirely due to the impulsive suicide attempters. There was an inverse correlation between the IRS score and CSF 5-HIAA (r = -.47, p = .02) and only a trend for HVA (r = -.41, p = .078) levels in the suicide group. CONCLUSIONS: This study of a group of violent suicide attempters distinguished a subgroup of patients diagnosed with personality disorder with high impulsivity scores and a subgroup of patients with the main diagnosis of severe depression. CSF 5-HIAA was significantly lower in impulsive violent attempters than in nonimpulsive violent attempters, therefore desintangling violence from impulsivity and linking this biologic abnormality to impulsivity.

Growth stimulation of murine fibroblasts by TGF-beta1 depends on the expression of a functional p53 protein.

Transforming Growth Factor-beta1 (TGF-beta1) inhibits the proliferation of most cells, but stimulates some mesenchymal cell types, including murine NIH3T3 fibroblasts. We show here that TGF-beta1 growth stimulation of NIH3T3 fibroblasts is reversed when these cells are transformed by SV40 or are transfected with a plasmid encoding the SV40 Large T antigen. Inversion of the TGF-beta1 growth stimulation of NIH3T3 cells is not observed when these cells are transfected with plasmids expressing either a mutant Large T, unable to bind P53, or the E1A adenovirus oncoprotein which binds the retinoblastoma protein pRB but not P53. But when the TGF-beta1-growth stimulated cells are transfected with a plasmid expressing a mutant form of Large T capable of binding to P53, but not to pRB, or with one expressing the E1B-55 kD adenovirus oncoprotein, which also binds to P53 but not to pRB, the cells are growth-inhibited by TGF-beta1. The cdk inhibitor p21Waf is decreased in TGF-beta1-stimulated NIH3T3 fibroblasts and increased in TGF-beta1-inhibited SV40-transformed cells. Finally, we show that T12 fibroblasts, from a P53 knockout mouse, are growth inhibited by TGF-beta1 and that they remain so upon transfection with a P53 which is mutant at restrictive temperature, but become growth-stimulated by this factor at permissive temperature when P53 is functional. These data strongly suggest that growth-stimulation of fibroblasts by TGF-beta1 depends on the presence of a functional P53 protein and that inversion of this response occurs if P53 is absent or inactivated.

Transforming growth factor-beta1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line.

In tissue culture conditions, exogeneous active transforming growth factor-beta1 (TGF-beta1) enhances the lethal effect of DNA-damaging agents (UV-C, gamma rays, cisplatin, methotrexate and 5-fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild-type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF-beta1 and UV-C was studied on mink and human cell lines, and the biomodulation by TGF-beta1 of cell killing by cisplatin, gamma rays, 5-fluorouracil or methotrexate was tested only on human cells. As investigated with UV-C-irradiated human A549 cells, TGF-beta1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF-beta1 might affect the curative and/or undesirable secondary side effects of cancer therapy.

Detection of a physical and functional interaction between Csk and Lck which involves the SH2 domain of Csk and is mediated by autophosphorylation of Lck on tyrosine 394.

The COOH-terminal Src kinase (Csk) is responsible for the phosphorylation of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases. Up to now, no stable binding of Csk to Src kinases has been detected. We therefore decided to analyze this interaction using two systems which allow detection of transient interaction. We produced and purified recombinant proteins in the glutathione S-transferase prokaryotic expression system. First, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its substrates Lck, a lymphocyte-specific member of the Src family. This interaction requires the autophosphorylation of Lck on tyrosine 394 (the phosphorylation of which is correlated with an increase of the kinase activity) and involves a functional Csk SH2 domain. Second, using the yeast two-hybrid system, we confirmed in vivo the physical interaction between Csk and Lck. Furthermore, in vitro we showed that autophosphorylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk. These findings might explain the mechanism(s) by which Csk interacts with most of Src kinases to down-regulate their kinase activity.

Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli.

The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates p56lck activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient p56lck with a mutation of the ATP-binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase-deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that p56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck.

Transforming growth factor-beta 1 enhances serum-induced dephosphorylation of the P53 protein in cell lines growth-inhibited by this factor.

A 24 hr TGF-beta 1 treatment (4 ng/ml) of SV40-transformed WI38 embryonic fibroblasts (VA13 cells) causes a moderate but reproducible inhibition of their serum-stimulated growth. By immunoprecipitation with the PAb122 antibody, we show that serum stimulation of previously serum-deprived cells causes a dephosphorylation of the wild type P53 protein, which is accentuated by the TGF-beta 1 treatment. The TGF-beta 1-enhanced dephosphorylation effect is also observed in two other cell lines growth-inhibited by TGF-beta 1, but which do not contain Large T (mink lung CCL64 and human KHOS cells). On the contrary, TGF-beta 1 treatment of the untransformed WI38 fibroblasts stimulates their growth, without affecting the phosphorylation of P53. Such treatment did not affect the expression of the corresponding mRNA nor the level of synthesis of the protein. The results suggest that the P53 protein could be a downstream target of TGF-beta 1 action on those cells growth-inhibited by the factor.

Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).

The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family. This phosphorylation site has a negative regulatory function. Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity. Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein. We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s). GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro. The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase. This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro. These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

Contrasting effect of transforming growth factor type beta 1 (TGF-beta 1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3-CD4-CD8-) thymocytes.

Transforming growth factor beta (TGF-beta) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4-6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4-6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-beta 1 on the IL-2R 55kD alpha chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-beta 1 was able to increase both the percentage of CD3-DN cells expressing IL-2R alpha chains and the expression of IL-2R alpha chain in these cells. This stimulatory effect of TGF-beta 1 was distal from early transduction events. In addition, TGF-beta 1 was found to modulate CD3-DN cell proliferation. During differentiation in the thymus, CD3-DN cells transiently express the IL-2R alpha chain of the IL-2R and these IL-2R+ CD3-DN cells are preprogrammed to down-regulate the IL-2R alpha chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-beta 1 on IL-2R alpha chain expression in these in vitro differentiating CD3-DN cells. We found that TGF-beta 1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3-DN cells, the effect of TGF-beta 1 on IL-2R expression seems to be restricted to proliferating cells.

Temperature monitoring during epidural anesthesia for cesarean delivery.

Tympanic, rectal, and axillary temperatures were measured and compared in 12 ASA Physical Status I and II parturients during epidural anesthesia for cesarean delivery. Measurements were performed before (T0) and at 15 (T1), 30 (T2), 45 (T3), and 60 (T4) minutes after epidural anesthesia. At birth, rectal neonatal and maternal temperatures were measured. Before anesthesia, maternal tympanic and rectal temperatures were statistically not different but higher than axillary temperature (difference, 0.5 degrees C). During anesthesia, all three maternal temperatures decreased. There was no difference for the first 45 minutes between rectal and tympanic membrane temperatures and no difference between tympanic and axillary temperatures after 30 minutes. The difference between rectal and tympanic temperatures became significant at T4. During the same period, the difference between axillary and tympanic temperatures became nonsignificant at T3 and T4. At birth, both maternal and newborn rectal temperatures were similar at 36.0 +/- 0.2 degrees C. The relative hypothermia observed in the newborns at birth after regional anesthesia was well correlated with the decrease in maternal temperature. A decrease in tympanic temperature of 1.4 degrees C developed during the course of epidural anesthesia for cesarean delivery. This decrease was underestimated by the measurement of rectal and axillary temperatures.

TGF-beta-like activity produced during regression of exacerbations in multiple sclerosis.

Samples representative of different stages of disease from a longitudinal study of multiple sclerosis patients were tested in the anchorage-independent growth assay for TGF-beta and an increased activity was detected in the supernatants from 2-day blood cell cultures from patients with active disease compared to patients without active disease and healthy donors. Within the group of patients with active disease, the TGF-beta like activity was significantly increased in the subgroup of patients tested during the period of regression of the symptoms where it appeared in 86.9% of the samples. These results suggest that TGF-beta or a TGF-beta like factor may play a role in regeneration processes in multiple sclerosis.

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line.

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.

Alkalinization of 0.5% lidocaine for intravenous regional anesthesia.

Although remaining a controversial issue, alkalinization of lidocaine or bupivacaine may shorten the time to onset and increase the duration of the sensory block. The aim of this study was to evaluate the effect of pH adjustment on the sensory and motor blocks during intravenous regional anesthesia (IVRA) with lidocaine. Thirty-one patients scheduled for minor hand surgery performed under IVRA were randomized into two groups: Group 1 (n = 14): 1% lidocaine, 3 mg/kg, diluted with the same volume of physiological saline solution (pH = 6.63 +/- 0.05), and Group 2 (n = 17): 1% lidocaine, 3 mg/kg, diluted with the same volume of 1.4% sodium bicarbonate (pH = 7.34 +/- 0.05). final concentration of lidocaine was thus 0.5% in both groups. Sensory block was assessed by pinprick every 2 minutes in areas corresponding to six terminal nerves: ulnar, median, radial, musculocutaneous, medial cutaneous nerve of arm and intercostobrachial, and medial cutaneous nerve of forearm. The time between release of tourniquet (at the end of surgery) and appearance of pain was recorded. Motor blockade was evaluated by asking the patient to squeeze strongly a blood pressure cuff previously inflated to 40 mmHg. This maneuver was performed before and every 2 minutes after injection. No statistical differences were found between the two groups whatever the parameter studied. In conclusion, there is no advantage (over plain solutions) to using pH-adjusted lidocaine during IVRA for hand surgery.

Modification of the gluconeogenic/glycolytic balance in isolated perfused liver of myeloproliferative leukemia infected mice: a 13C NMR study.

1. Isolated perfused livers from starved mice inoculated with myeloproliferative leukemia virus exhibited similar rates of consumption of [3-13C]alanine and of synthesis of glutamine and glutamate labeled at the C-2 or C-3 positions as livers from uninfected mice. 2. Leukemic livers also formed glutamine and glutamate labeled at the C-4 position. This is related to their lower content of triglyceride as compared to that of control livers which do not produce these isotopomers. 3. The glucose synthesis rate was much lower in livers from leukemic mice. This is explained by the glycolytic properties of the leukemic infiltrating cells.

A continuous line of chicken embryo cells derived from a chondrocyte culture infected with RSV.

A continuous line of chicken embryo cells was derived from a culture of chondrocytes infected with Rous sarcoma virus (RSV). These cells, designated as NA101, have reduced serum requirements and are able to grow in a semisolid medium. NA101 cells show the same phenotype as freshly RSV infected chondrocyte cultures, i.e. synthesis of fibronectin and type I collagen, elongated bipolar shape and absence of tumorigenicity following grafting onto the chorioallantoic membrane of embryonated duck eggs; thus they appear to be distinct from transformed fibroblasts. Neither NA101 cells nor freshly infected chondrocyte cultures synthesize type II or type X collagen, which are the differentiation markers of normal chicken chondrocytes in culture.

Acidic cellular environments: activation of latent TGF-beta and sensitization of cellular responses to TGF-beta and EGF.

Transient (about 2 hr) acidification to approx. pH 5.0 of agar-gelled overlayers containing untransformed NRK-49F or KiMSV-transformed NRK-49F cells in the presence of fetal calf serum or crude 49F-cell conditioned medium, as sources of latent TGF-beta, elicited EGF-dependent colony formation of 49F cells and inhibited spontaneous growth of transformed cells. Pure, active TGF-beta (porcine, type I) had the same effects on these respective cell types, suggesting that the above results were due to activation of latent TGF-beta in the transiently acidic cellular environment. Similar acidifications in the absence of a source of latent TGF-beta enhanced the positive growth response of 49F and AKR-2B cells to EGF and active TGF-beta and also the negative growth response of KiMSV-transformed 49F cells to active TGF-beta. These results are compatible with the idea that acidic cellular environments, particularly in tumor tissues, are conducive to activation of latent TGF-beta, perhaps in conjunction with other activating mechanisms, and to an enhanced response to some growth factors. However, the heterogeneity of cell populations within tumoral masses presents an obstacle to a clear understanding of the consequences of such activation.

Evidence for a novel growth factor in Xenopus oocytes.

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.