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J Landry

Publications and source records attributed to J Landry.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cholecystokinin stimulates heat shock protein 27 phosphorylation in rat pancreas both in vivo and in vitro.

BACKGROUND & AIMS: Mammalian heat shock protein 27 (hsp27) is believed to function under normal physiological conditions and during cellular stress. Recent studies indicate a role for hsp27 in regulating actin-cytoskeletal dynamics. In the present study, secretagogue-regulated phosphorylation of hsp27 in rat exocrine pancreas was investigated both in vivo and in isolated acinar cells. METHODS: Western analysis after two-dimensional electrophoresis was used to measure the phosphorylation of hsp27 after treatment of rats or acinar cells with secretagogues. Cholecystokinin-stimulated mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 activity was measured after immunoprecipitation of the kinase. RESULTS: hsp27 exists as three isoforms in acini: one nonphosphorylated (pI 6.2) and two phosphorylated (pIs 5.9 and 5.7) forms. Infusion of rats with a secretory or supermaximal dose of cerulein produced an acidic shift in hsp27, indicating an increase in its phosphorylation; the higher dose, known to cause pancreatitis, had a twofold greater effect. In isolated acini, increases in hsp27 phosphorylation were evident at 10 pmol/L and maximal at 1 nmol/L cholecystokinin. The hsp27-specific kinase MAPKAP kinase 2 was activated 2.4-fold with 1 nmol/L cholecystokinin treatment. CONCLUSIONS: hsp27 phosphorylation was stimulated by low and high concentrations of cholecystokinin, both in vivo and in vitro. Phosphorylation was potentially mediated via the MAPKAP kinase 2 intracellular signaling pathway.

Animals↗
PubMed1997

p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent chemotactic agent for endothelial cells. Yet the signalling pathways that modulate the motogenic effects of VEGF in vascular endothelial cells are still ill defined. In the present study, we found in primary cultures of human umbilical vein endothelial cells (HUVEC) that VEGF increased cell migration and induced a marked reorganization of the microfilament network that was characterized by the formation of stress fibers and the recruitment of vinculin to focal adhesions. VEGF also stimulated the mitogen activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and p38 (stress activated protein kinase-2), but not SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). Activation of p38 resulted in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). Inhibiting the VEGF-induced activation of ERK with PD098059 did not influence actin organization or cell migration but totally inhibited the VEGF-induced incorporation of thymidine into DNA. Inhibition of p38 activity by the specific inhibitor SB203580 led to an inhibition of HSP27 phosphorylation, actin reorganization and cell migration. The results indicate that the p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis.

Actins↗
PubMed1997

Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells.

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.

Actins↗
PubMed1997

Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.

We have studied the contribution of the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase), JNK/SAPK (c-JUN NH2-terminal kinase/stress-activated protein kinase) and p38, to activation of the HSP27 (heat shock protein 27) kinase MAPKAP kinase-2/3 and to HSP27 phosphorylation in Chinese hamster CCL39 cells stimulated by either growth factors, cytokines or stressing agents. In vitro assays using fractionated cell extracts or immunoprecipitates indicated that only fractions containing ERK or p38, and not those containing JNK/SAPK, had the capacity to activate MAPKAP kinase-2/3. In vivo, however, it appeared that only p38 is an upstream activator of HSP27 phosphorylation after both stress or growth factor stimulation: expression of an interfering mutant of ras, which blocked the activation of ERK by both types of inducers, had no effect on HSP27 phosphorylation and p38 activation; and the cell-permeant specific inhibitor of 038, SB203580, blocked MAPKAP-kinase2/3 activation and HSP27 phosphorylation. HSP27 has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. This raises the possibility that p38 might be directly involved in mediating actin responses to external stimuli. Accordingly, we observed that a prior activation of p38 increased the stability of the actin microfilaments in cells exposed to cytochalasin D. The effect was dependent on the expression of HSP27 and was totally annihilated by blocking the p38 activity with SB203580. The results provide strong support to the idea that activation of p38 during adverse environmental conditions serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. Its activation during normal agonist stimulation may constitute an additional actin signaling pathway, the importance of which depends on the level of expression of HSP27.

Actin Cytoskeleton↗
PubMed1996

Tissue-specific posttranslational modification of the small heat shock protein HSP27 in Drosophila.

Drosophila sHSPs (small heat shock proteins) are expressed in the absence of stress in specific regions of the central nervous system and in gonads of young adults flies. In these two organs, the sHSPs show a cell-specific and developmental stage-specific pattern of expression suggesting distinct regulation and function(s) of each individual sHSP (R. Marin et al., Dev. Genet. 14, 69-77, 1993). Since mammalian HSP27 has been reported to be phosphorylated through a complex novel cascade implicating distinct kinases, we examined whether two of the sHSPs (HSP27 and HSP23) exist in different isoforms as a result of posttranslational modification in vivo. HSP27 and HSP23 were analyzed in various tissues in unstressed and heat-shocked flies. Four isoforms of HSP27 were found to be constitutively expressed in the nervous system and in testes and two in ovaries. The proportion of these isoforms relative to each other was specific to a given tissue. In the case of HSP23, two isoforms were expressed in the heads and in testes of unstressed flies. In ovaries, a low level of a single isoform of HSP23 was found. Heat shock caused an increase in the amount of preexisting HSP27 and HSP23 and the appearance of additional isoforms in ovaries. Susceptibility to phosphatase treatment indicated that isoforms of HSP27 were phosphoproteins. This was further supported by in vitro experiments in which Drosophila sHSPs were incubated with purified Chinese hamster HSP27 kinase. Only HSP27 was shown to be a substrate of this mammalian HSP27 kinase. The present data suggest that tissue- and HSP-specific posttranslational modification systems may modulate the function of these proteins in different cell types. Furthermore, the signal transduction pathways leading to phosphorylation of the sHSPs are conserved between mammals and Drosophila, and the sHSP kinase cascade may be developmentally regulated.

Animals↗
PubMed1996

HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress.

Phosphorylation of heat shock protein 27 (HSP27) has been suggested to play an important role in the regulation of F-actin dynamics in response to growth factors and stress. Because the microfilament network is one of the earliest targets of oxidative stress and because phosphorylation of HSP27 is strongly induced by reactive oxygen metabolites, we have investigated the role of HSP27 phosphorylation in regulating actin dynamics in response to oxidative stress. Experiments were done in Chinese hamster CCL39 cells overexpressing various levels of the wild-type or a nonphosphorylatable form of human HSP27 (pm3 HSP27). In control cells, both H2O2 and menadione induced fragmentation of F-actin, which forms aggregates and patches concentrated around the nucleus. Stable overexpression of wild-type HSP27, but not of pm3 HSP27, conferred resistance against actin fragmentation, suggesting that HSP27 has a phosphorylation-activated protective function against actin disruption by oxidative stress. Cell lines that overexpressed the highest levels of the wild-type form of human HSP27 also showed an increased cell survival following exposure to H2O2. In contrast, cells expressing pm3 HSP27 were as sensitive as the controls to the lethal effect of H2O2. These results suggest that phosphorylation of HSP27 is causally related to the regulation of microfilament dynamics following oxidative stress and may be involved in mediating an adaptive response to oxyradical-generating agents such as carcinogens, anticancer drugs, and other xenobiotics.

Actins↗
PubMed1996

The tryptophan content in protein of cereal grains and legume seeds as a function of nitrogen content. A reappraisal of tryptophan score.

UNLABELLED: The tryptophan content ([Trp]) in the dry matter (DM) of wheat, maize, barley, sorghum, rice, pearl millet grains, pea and broad bean seeds is evaluated from samples, with various nitrogen contents ([N]DM) and genotypes, using a procedure that has been shown to be strictly quantitative. The determination of linear correlations between [Trp]DM and [N]DM, and between the tryptophan content in protein ([Trp]N) and [N]DM or 1/[N]DM for every species leads to the following observations: (1) [Trp]DM and [N]DM are linearly related. The data show that previous reports of similar relationships underestimate tryptophan by 10 +/- 5% owing to tryptophan degradation during alkaline hydrolysis preparatory to analysis; (2) the linear correlations between [Trp]N and 1/[N]DM, resulting from linear relationships [Trp]DM and [N]DM display coefficients of determination (r2) far lower than 1 and similar to those found for linear correlations between [Trp]N and [N]DM; (3) [Trp]N increases with [N]DM increasing for rice and pearl millet while it decreases for all other species. IN CONCLUSION: (1) linear relationships between tryptophan and nitrogen have a low predictive value; (2) the nutritional score of tryptophan of foods and feeds, as calculated from the determination of tryptophan using a procedure involving alkaline hydrolysis, is generally underestimated by 10%.

Edible Grain↗
PubMed1996

Tryptophan content of foods and feeds. Conditions for a reliable quantitation.

A procedure for the quantitative determination of tryptophan from complex material was developed. It involved the hydrolysis of samples in the presence of baryta. Data were presented, revealing its quantitativeness and pointing out its efficiency when it was compared with diverse methods developed by other laboratories as a part of collaborative studies. The influence of the way of treating and heating of (sample+baryta) mixture upon tryptophan recovery was also investigated. The placement of (sample+baryta) mixture in an autoclave, when water was boiling, or the careful deaeration of the mixture when it was heated in an oven was essential for quantitative results. These specific conditions can be explained by differences of the protein solvatation, related to the way by which the (sample+baryta) mixture was heated.

Animal Feed↗
PubMed1996

Immediate breast reconstruction for stage III breast cancer using transverse rectus abdominis musculocutaneous (TRAM) flap.

BACKGROUND: The management of state III breast cancer is challenging; it often includes multimodal treatment with systemic therapy and/or radiation therapy and surgery. Immediate breast reconstruction has not traditionally been performed in these patients. We review the results of immediate transverse rectus abdominis musculocutaneous (TRAM) flap in 21 patients treated for stage III breast cancer. METHODS: Data have been collected retrospectively on 21 patients diagnosed with stage III breast cancer between 1987 and 1994. All patients had mastectomy and immediate TRAM reconstruction. Thirteen patients received primary systemic therapy, 10 patients received postoperative consolidation radiotherapy to the operative site, and 3 patients received preoperative radiation. RESULTS: Mean follow-up for the group was 26 months. Two patients died with disseminated disease: neither of them developed local disease recurrence in the operative site; 82% of the patients followed for at least two years are free of disease. Sixty-two percent of the patients received preoperative chemotherapy, the remaining patients received postoperative multiagent chemotherapy and/or radiation therapy. Two of the patients received autologous bone marrow transplants after their adjuvant therapy. Ten patients had postoperative radiotherapy for consolidation; three patients received preoperative radiation. CONCLUSIONS: Immediate TRAM reconstruction for stage III breast cancer is not associated with a delay in adjuvant therapy or an increased risk of local relapse. It facilitates wide resection of involved skin without skin grafting. Radiation therapy can be delivered to the reconstructed breast when indicated without difficulty. Breast reconstruction facilitates surgical resection of stage III breast cancer with primary closure and should be considered if the patient desires immediate breast reconstruction.

Breast Neoplasms↗
PubMed1996

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat.

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.

Animals↗
PubMed1995

Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27.

Heat-shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation-regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15-fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N-acetyl-L-cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by heat shock, H2O2, sodium arsenite, TNF or growth factors was identified by in-gel renaturation and purified approximately 8000-fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen-activated-protein kinase-activated protein (MAPKAP) kinase-2 based on its reactivation in vitro by 42/44-kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54-kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild-type HSP27 but not of a nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54-kDa HSP27 kinase is a common mechanism of HSP27 phosphorylation to which converge both oxyradical-dependent and oxyradical-independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.

Actin Cytoskeleton↗
PubMed1995

Prognostic significance of heat-shock protein-27 in node-positive breast carcinoma: an immunohistochemical study.

Immunostaining for heat-shock protein-27 (HSP-27) was performed on formalin fixed-paraffin embedded sections of 890 node-positive breast carcinomas resected between 1980 and 1986. The follow-up ranged from 2.5 to 10.5 years. A polyclonal antibody (Hu27, dilution: 1/200) was used. A positive cytoplasmic staining was obtained in 383 cases (43%). No difference in distant metastasis-free survival (DMFS) or overall survival (OS) was noted between cases with positive or negative immunostaining. This study suggests that HSP-27 expression is not predictive of the outcome in node-positive breast cancer.

Breast Neoplasms↗
PubMed1995

TRAM flap breast reconstruction after radiation treatment.

OBJECTIVE: Patients with and without radiation treatment before their breast reconstruction were compared to study the relationship of radiation to flap-related complications. SUMMARY BACKGROUND DATA: The transverse rectus abdominis muscle (TRAM) flap for breast reconstruction involves a a vascular pedicle and recipient bed, both included in the radiated field of patients undergoing adjunctive therapy. Detailed reviews of flap-related complications in this subgroup of patients have been limited. METHODS: One hundred eight patients with radiation treatment who subsequently underwent a TRAM flap breast reconstruction were compared with 572 patients with no radiation treatment before similar reconstruction. Flap-related complications, radiation dosage, time, fields, relationships between risk factors, and complications were studied. RESULTS: Overall complication rates were comparable between the two groups. Only fat necrosis (> 10% of total reconstruction) was found to be statistically significant (17.6% vs. 10.1%, p = 0.03228). No difference was found for fat necrosis in unipedicled vs. bipedicled flaps controlled for radiation (17.7% vs. 17.4%). Obesity and radiation therapy were associated with fat necrosis and major infection in a logistic regression. Significant abdominal scarring was also associated with major infection (p = 0.0044). CONCLUSIONS: In this, the largest reported series, radiation therapy was associated with increased fat necrosis and major infection. The use of the TRAM flap was not found to be prohibitive in radiated patients and should still be the first choice in this subgroup of patients.

Breast Neoplasms↗
PubMed1995

Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

Actin Cytoskeleton↗
PubMed1994

Cells overexpressing Hsp27 show accelerated recovery from heat-induced nuclear protein aggregation.

Protein denaturation/aggregation upon cell exposure to heat shock is a likely cause of cell death. In the nucleus, protein aggregation has often been correlated to inhibition of nuclear located processes and heat-induced cell killing. In Chinese hamster O23 cells made thermotolerant by a prior heating (20'44 degrees C + 10h 37 degrees C) which induces the whole spectrum of heat shock proteins (hsps), the extent of nuclear protein aggregation during heat shock is reduced and the rate of recovery from aggregation after heat shock is enhanced. In contrast, a heat resistant Chinese hamster cell line overexpressing only hsp27 shows an unaltered sensitivity to formation of nuclear protein aggregates by heat, but shows the same enhanced rate of recovery from nuclear protein aggregation as thermotolerant cells. This suggests that accelerated recovery of protein aggregation could be partly responsible for hsp27-mediated thermoprotection.

Animals↗
PubMed1993

Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27.

We recently reported that overexpression of heat shock protein 27 (HSP27) in rodent fibroblasts increases the stability of stress fibers during hyperthermia and partially prevents actin depolymerization during exposure to cytochalasin D (Lavoie, J.N., Gingras-Breton, G., Tanguay, R. M., and Landry, J. (1993) J. Biol. Chem. 268, 3420-3429). Because HSP27 is a ubiquitous target of phosphorylation upon cell stimulation with a variety of growth factors and agents that affect cellular differentiation, we examined the role of HSP27 phosphorylation in regulating actin filament dynamics. Here we show that HSP27 is enriched at the leading edge of polarized fibroblasts. HSP27 is localized in lamellipodia and membrane ruffles where most actin polymerization occurs. We developed Chinese hamster cell lines that constitutively overexpressed either human HSP27 or a nonphosphorylatable mutant form of the protein. Overexpression of HSP27 caused an increased concentration of filamentous actin (F-actin) at the cell cortex and elevated pinocytotic activity. In contrast, overexpression of the non-phosphorylatable mutant form of HSP27 reduced cortical F-actin concentration and decreased pinocytosis activity relative to control cells. Mitogenic stimulation of fibroblasts resulted in a rapid polymerization of submembranous actin filaments. HSP27 enhanced growth factor-induced F-actin accumulation, whereas mutant HSP27 exerted a dominant negative effect and inhibited this response to growth factors. Thus, HSP27 is a component of a signal transduction pathway that can regulate microfilament dynamics.

Actins↗