Effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells / 中华烧伤杂志

Objective: To investigate the effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells (HUVECs). Methods: The experimental research methods were adopted. The third to the fifth passage of HUVECs in the logarithmic growth stage were used for the subsequent experiments. Three batches of cells were taken, with each batch of cells being divided into normal control group and negative pressure treatment alone group (both routinely cultured for 24 h), and 17-allylamino-17-demethoxy-geldanamycin (17-AAG) alone group and 17-AAG+negative pressure treatment group (both cultured with 17-AAG for 24 h). In addition, the intermittent negative pressure suction, with the negative pressure value of -5.33 kPa (suction for 30 s, pause for 10 s) was continuously applied for 8 h on cells in the two negative pressure treatment groups using an automatic three-dimensional cell gradient negative pressure loading device designed and developed by ourselves. After the treatment of the first batch of cells, the cell proliferation level was detected by cell counting kit 8 method at 0 (immediately), 24, 48, and 72 h of culture, with the number of samples being 6. After the treatment of the second batch of cells, the scratch experiment was performed. At 12 h after scratching, the cell migration was observed under an inverted phase contrast microscope and the cell migration rate was calculated, with the number of samples being 3. After the treatment of the third batch of cells, the tubule formation experiment was conducted. After 6 h of culture, the tubulogenesis was observed under an inverted phase contrast microscope and the total tubule length and the number of branch nodes of cells were calculated, with the number of samples being 3. The cells were taken and divided into normal control group, negative pressure treatment alone group, and 17-AAG+negative pressure treatment group. The cells were treated the same as in the previous corresponding group. After the treatment, Western blotting was used to detect the protein expressions of heat shock protein 90 (HSP90), caveolin 1, endothelial nitric oxide synthase (eNOS), and eNOS phosphorylation site 1177 in the cells, and the eNOS phosphorylation site 1177/eNOS ratio was calculated, with the number of samples being 3; co-immunoprecipitation (co-precipitating HSP90 and caveolin 1, caveolin 1 and eNOS) and Western blotting were used to detect the protein expressions of caveolin 1 and eNOS in the cells, with the number of samples being 3; the protein co-localization of HSP90 and caveolin 1 and that of caveolin 1 and eNOS in the cells was assessed by immunofluorescence double staining. The molecular docking prediction of caveolin 1 and eNOS was processed by HADDOCK 2.4 protein-protein docking program. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and least significant difference method. Results: Compared with that in normal control group, the cell proliferation level in 17-AAG alone group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01), while the cell proliferation level in negative pressure treatment alone group was significantly increased at culture hour of 24, 48, and 72 after the treatment (P<0.01). Compared with that in 17-AAG alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly increased at culture hour of 48 and 72 after the treatment (P<0.05 or P<0.01). Compared with that in negative pressure treatment alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01). At 12 h after scratching, compared with (39.9±2.7)% in normal control group, the cell migration rate in 17-AAG alone group was significantly decreased ((10.7±2.7)%, P<0.01), while the cell migration rate in negative pressure treatment alone group was significantly increased ((61.9±2.4)%, P<0.01). Compared with those in 17-AAG alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly increased ((37.7±3.7)%, P<0.01). Compared with that in negative pressure treatment alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly decreased (P<0.01). At culture hour of 6 after the treatment, compared with those in normal control group, the total length of the tube formed by the cells in 17-AAG alone group was significantly shortened (P<0.05) and the number of branch nodes was significantly reduced (P<0.05), while the total length of the tube formed by the cells in negative pressure treatment alone group was significantly prolonged (P<0.01) and the number of branch nodes was dramatically increased (P<0.01). Compared with that in 17-AAG alone group, the number of branch nodes of the tube formed by the cells was significantly increased in 17-AAG+negative pressure treatment group (P<0.05). Compared with those in negative pressure treatment alone group, the total length of the tube formed by the cells in 17-AAG+negative pressure treatment group was significantly shortened (P<0.01) and the number of branch nodes was significantly reduced (P<0.01). Western blotting detection showed that after treatment, the overall comparison of eNOS and caveolin 1 protein expressions among the three groups of cells showed no statistically significant differences (P>0.05). The expression of HSP90 protein and the eNOS phosphorylation site 1177/eNOS ratio in the cells of negative pressure treatment alone group were significantly increased (P<0.01) compared with those in normal control group. Compared with those in negative pressure treatment alone group, the HSP90 protein expression and the eNOS phosphorylation site 1177/eNOS ratio in the cells of 17-AAG+negative pressure treatment group were significantly decreased (P<0.01). Co-immunoprecipitation and Western blotting detection after the treatment showed that compared with those in normal control group, the expression of caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased (P<0.01), while the protein expression of eNOS was significantly decreased (P<0.05). Compared with those in negative pressure treatment alone group, the expression of caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased (P<0.01), while the protein expression of eNOS was significantly increased (P<0.01). After the treatment, compared with those in normal control group, the co-localization of HSP90 and caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased, while the co-localization of caveolin 1 and eNOS protein was significantly decreased. Compared with those in negative pressure treatment alone group, the co-localization of HSP90 and caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased, while the co-localization of caveolin 1 and eNOS protein was significantly increased. Molecular docking prediction suggested that caveolin 1 interacted strongly with eNOS and inhibited the 1177 site phosphorylation of eNOS. Conclusions: The negative pressure microenvironment may inhibit the binding of caveolin 1 to eNOS by promoting the binding of HSP90 to caveolin 1 in HUVECs, so as to relieve the inhibition of 1177 site phosphorylation of eNOS by caveolin 1, thereby promoting the proliferation, migration, and tubulogenesis of HUVECs, and ultimately promoting the neogenesis of HUVECs.

The Relationship between PPP2R5C and Molt-4 Cell Viability, HSP90-GR Signal in Childhood Acute T Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of PPP2R5C to the activity of Molt-4 cells in childhood acute T lymphocytic leukemia and its mechanism.@*METHODS@#The small interfering RNA (siRNA) technology targeting PPP2R5C gene was used to down-regulate the expression of PPP2R5C in Molt-4 cells. At the same time, a blank control group, a negative control group and a 17-DMAG group were set up. The cells in the negative control group were transfected with siRNA-NC, the cells in 17-DMAG group were treated with the HSP90 inhibitor 17-DMAG at a final concentration of 6.4 μmol/L for 48 h. Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot were used to detect transfection efficiency; CCK-8 method was used to detect the proliferation activity of the cells in each group, EdU was used to detect the proliferation level of the cells in each group, flow cytometry was used to detect the cell cycle distribution ratio of the cells in each group, Annexin V-FITC/PI staining was used to detect the apoptosis of the cell, RT-qPCR and Western blot were used to detect the expression changes of heat shock protein 90 (HSP90) and glucocorticoid receptor (GR) of the cells in each group.@*RESULTS@#After Molt-4 cells were transfected with siRNA-PPP2R5C, the expression of PPP2R5C mRNA and protein in the cells were down-regulated significantly compared with those in the blank control group and the si-NC group (P<0.05); compared with cells in the blank control group and the si-NC group, the proliferation activity of the cells in the siRNA-PPP2R5C group and the 17-DMAG group significantly decreased (P<0.05), and the rate of EdU positive cells was significantly reduced (P<0.05); the proportion of the cells in G1 phase decreased while the proportion of the cells in G2 phase increased (P<0.05), the apoptosis rate of the cells also increased significantly (P<0.05); in addition, the expression of PPP2R5C mRNA and protein of the cells in siRNA-PPP2R5C group was significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05). The expressions of PPP2R5C mRNA and protein in the 17-DMAG group were also significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05).@*CONCLUSION@#Down-regulation of PPP2R5C gene expression can inhibit Molt-4 cell activity in childhood acute T lymphocytic leukemia, block the cells in G2 phase, and promote cell apoptosis, the mechanism may be related to the inhibition of HSP90-GR signaling pathway.

Histological investigation of experimentally induced diabetes effects on the distribution of transforming growth factor (TGF beta), nuclear factor kappa b (Nf-KB), heat schock 90 beta (Hsp beta) and E-cadherin proteins in testicular tissue / Investigación histológica de los efectos de la diabetes inducida experimentalmente en la distribución del factor de crecimiento transformante (TGFß), nuclear factor kappa b (Nf-KB), y proteinas heat schock 90ß (Hsp90ß) y E-cadherina en tejido testicular

SUMMARY: Diabetes is a metabolic disorder characterized by high blood sugar levels and it causes complications in many systems, including the reproductive system. As a result of diabetic conditions, one of the mechanisms that can cause repression of reproductive activity is testicular oxidant stress. The identification of diabetes on the cell signaling molecules axis is still under discussion. The aim of this study was to determine the effect of Transforming Growth Factor (TGFβ), Nuclear Factor kappa B (NF-kB), Heat-schock 90β (HSP90β) signal pathways and E-cadherin cell adhesion molecule on infertility in diabetic rat testicular tissue. In our study, includes histological, molecular and biochemical analysis of testicular tissue removed at the end of the 2 weeks experiment period. A total of 14 adult male rats were divided as control and diabetes. No intervention was given to 7 male rats in the control group. For the diabetic group, 7 male rats were injected by intraperitoneal with a single dose of 55 mg/kg streptozotocin (STZ). TGFβ, NF-kB, HSP90β and E-cadherin proteins were immunohistochemically studied to investigate possible tissue damage, inflammatory process, cell stabilization and integrity due to diabetes. In order to determine oxidant stress, lipid peroxidation product malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx) analyzes were performed. Fibrosis, inflammatory changes and loss of spermatogenetic series are prominent findings in the diabetic group. On analysis of all the samples with immunostaining, in the diabetic group, TGFβ and NF-kB immunoexpression significantly increased, while Hsp90β and E-cadherin immunoexpression significantly decreased compared with control groups. Experimental diabetes was found to cause fibrosis, inflammation, disrupting cell adhesion and stabilization in testicular tissue. These results suggest that cellular therapy studies are needed for possible damage.

RESUMEN: La diabetes es una enfermedad metabólica caracterizada por niveles altos de azúcar en sangre y causa complicaciones en muchos sistemas, incluido el sistema reproductivo. Como resultado de las condiciones diabéticas, uno de los mecanismos que puede causar alteraciones en la actividad reproductiva es el estrés oxidativo testicular. La identificación de la diabetes en el eje de las moléculas de señalización celular aún está en discusión. El objetivo de este estudio fue determinar el efecto del factor de crecimiento transformante (TGFβ), el factor nuclear kappa B (NF-kB), las vías de señalización de Heat-Schock 90b (HSP90β) y la molécula de adhesión celular de E-cadherina sobre la infertilidad en testículo de rata diabética. Al término de dos semanas se realizaron análisis histológico, molecular y bioquímico del tejido testicular extraído. Las 7 ratas macho del grupo control no fueron intervenidas. Para el grupo de diabéticos, 7 ratas macho fueron inyectadas por vía intraperitoneal con una dosis única de 55 mg / kg de estreptozotocina (STZ). Se estudiaron inmunohistoquímicamente las proteínas TGFβ, NF-kB, HSP90β y E-cadherina para investigar el posible daño tisular, el proceso inflamatorio, la estabilización celular y la integridad debido a la diabetes. Para determinar el estrés oxidativo, se realizaron análisis del producto de peroxidación lipídica malondialdehído (MDA), glutatión (GSH) y glutatión peroxidasa (GPx). La fibrosis, los cambios inflamatorios y la pérdida de series espermatogenéticas son hallazgos destacados en el grupo de ratas diabéticas. En el análisis de todas las muestras con inmunotinción, en el grupo diabético, la inmunoexpresión de TGFβ y NF-kB aumentó significativamente, mientras que la inmunoexpresión de Hsp90β y e-cadherina disminuyó significativamente en comparación con los grupos control. Se encontró que la diabetes experimental causa fibrosis, inflamación, alteración de la adhesión celular y estabilización en el tejido testicular. Estos resultados sugieren que son necesarios estudios de terapia celular para verificar posibles daños.

Expression and Clinical Significance of Heat Shock Protein 90 in Multiple Myeloma Patients / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of HSP90 in bone marrow samples of multiple myeloma (MM) patients and explore its clinical significance.@*METHODS@#Maxvision immunohistochemistry technique was used to detect the protein expression level of HSP90 76 MM patients and 29 normal healthy donors. The clinical characteristics of the patients were collected, and the correlation between the HSP90 expression and the clinical characteristics was analyzed.@*RESULTS@#The count of MM patients with positive HSP90 protein was significantly higher than that of normal healthy donor, and there were no significant correlation between HSP90 expression and age, sex, hemoglobin (Hb), creatinine (CREA), blood calcium, lactate dehydrogenase (LDH), bone marrow plasma cell proportion and MM subtypes (P>0.05), but HSP90 expression was correlated with β@*CONCLUSION@#HSP90 protein was over-expressed in MM patients, and was correlated with β

Involvement of HSP90 in ischemic postconditioning-induced cardioprotection by inhibition of the complement system, JNK and inflammation

Abstract Purpose To investigate whether heat shock protein 90 (HSP90) is involved in complement regulation in ischemic postconditioning (IPC). Methods The left coronary artery of rats underwent 30 min of occlusion, followed by 120 min of reperfusion and treatment with IPC via 3 cycles of 30s reperfusion and 30s occlusion. The rats were injected intraperitoneally with 1 mg/kg HSP90 inhibitor geldanamycin (GA) after anesthesia. Eighty rats were randomly divided into four groups: sham, ischemia-reperfusion (I/R), IPC and IPC + GA. Myocardial infarct size, apoptosis index and the expression of HSP90, C3, C5a, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1β and c-Jun N-terminal kinase (JNK) were assessed. Results Compared with the I/R injury, the IPC treatment significantly reduced infarct size, release of troponin T, creatine kinase-MB, and lactate dehydrogenase, and cardiomyocyte apoptosis. These beneficial effects were accompanied by a decrease in TNF-α, IL-1β, C3, C5a and JNK expression levels. However, all these effects were abrogated by administration of the HSP90 inhibitor GA. Conclusion HSP90 exerts a profound effect on IPC cardioprotection, and may be linked to the inhibition of the complement system and JNK, ultimately attenuating I/R-induced myocardial injury and apoptosis.

Regulatory mechanism of heat shock protein 90 on autophagy-related transcription factor EB in human hepatocellular carcinoma cells / 生理学报

This study was aimed to investigate the regulatory mechanism of heat shock protein 90 (Hsp90) on transcription factor EB (TFEB) during autophagy in liver cancer cells. Human hepatocellular carcinoma cell line HepG2 was treated with Hsp90 N- and C-terminal inhibitors (STA9090 and Novobiocin), respectively. Western blot and RT-PCR were used to detect the expression levels of TFEB and autophagy-related proteins. Chromatin immunoprecipitation (ChIP) assay was used to observe the ability of Hsp90α binding to the TFEB proximal promoter region. The double-luciferase gene reporter experiment was used to determine the activity of TFEB promoter. The results showed that hypoxia induced up-regulation of TFEB protein and mRNA expression levels in the HepG2 cells. The protein expression levels of TFEB, LC3 and P62 were down-regulated significantly by either STA9090 or Novobiocin, under both normoxic and hypoxic conditions. Transfection of Hsp90α-overexpressing plasmids up-regulated TFEB protein levels in either wild-type or Hsp90α knockout HepG2 cells. Hsp90 bound to the TFEB proximal promoter region and was involved in regulating TFEB transcriptional process. Whereas both STA9090 and Novobiocin inhibited Hsp90 to bind to the TFEB proximal promoter region, and decreased the activity of TFEB promoter. These results suggest that Hsp90 promotes TFEB transcription in human hepatocellular carcinoma cells by binding to the proximal promoter region, thereby up-regulating the expression levels of autophagy-related proteins.

Avaliação de mecanismos de modificação pós-traducional da óxido nítrico sintase endotelial (eNOS) associados a biodisponibilidade do óxido nítrico em artérias de ratas espontaneamente hipertensas (SHR) ao final da prenhez / Evaluation of post-translational modification mechanisms of endothelial nitric oxide synthase (eNOS) associated with the nitric oxide bioavailability in arteries from spontaneously hypertensive rats (SHR) in the end of pregnancy

A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas endoteliais da aorta e da artéria mesentérica na presença ou ausência da metil-ß-ciclodextrina (dextrina, 10 mmol/L) por microscopia eletrônica. Em estudos funcionais, avaliamos a participação da enzima OGA, pela inibição com PugNAc (100 µmol/L) e das cavéolas, utilizando um desorganizador de cavéolas, a dextrina (10 ou 20 mmol/L), na menor reatividade vascular à PE observada em aortas de ratas P. Observamos que o conteúdo de proteínas O-GlcNAciladas estava diminuído em aorta e em leito mesentérico de ratas Wistar P e SHR P. Apesar da expressão da OGT e da OGA não estar alterada, a atividade da OGA foi aumentada em aorta e leito mesentérico de ratas Wistar P, mas, encontra-se diminuída em aorta e aumentada em leito mesentérico de SHP P. A incubação com PugNAc reverteu a reduzida reatividade à PE em aorta e artéria mesentérica de ratas Wistar P mas este efeito não foi observado em vasos SHR P, demonstrando que a OGA parece ter um papel importante na redução da O-GlcNAcilação de proteínas vasculares em Wistar P. Em vasos incubados com PugNAc, a remoção do endotélio ou a incubação com L-NAME, não alterou significativamente a reatividade à PE. Juntos estes resultados sugerem que a maior atividade da eNOS observada em vasos de Wistar P, fica prejudicada na presença do PugNAc, e depende da atividade da OGA. Como não houve alteração da resposta contrátil à PE em vasos de SHR P incubados com PugNAc, possivelmente um mecanismo diferente, envolvendo a menor atividade da OGT, ocorre nestas artérias para a redução da O-GlcNAcilação da eNOS. A desorganização das cavéolas por meio da dextrina causou aumento de contração à PE e redução de potência da ACh em aortas de Wistar NP e SHR NP, porém não houve alteração em aortas de ratas Wistar P e SHR P. A dextrina não alterou o número de cavéolas em artérias de Wistar P e SHR P quando comparado com ratas NP. SHR NP apresentam um reduzido número de cavéolas das aortas em relação a Wistar NP bem como expressão reduzida de Cav-1, p-Cav-1 e CaM. A prenhez não foi capaz de alterar a expressão da Cav-1, CaM e Hsp90 em aorta e leito mesentérico de ratas normotensas e hipertensas. Estes resultados sugerem que a prenhez não altera a expressão das proteínas Cav-1, CaM e Hsp90 e possivelmente a interação com a eNOS em aorta e artérias mesentéricas de ratas normotensas e hipertensas. Em conclusão, entre os mecanismos estudados de modificação pós-traducional da eNOS, a redução da O-GlcNAcilação da eNOS, por mecanismos que envolvem a atividade da OGA e possivelmente da OGT, favoreceria a fosforilação da eNOS e consequente maior biodisponibilidade de NO, contribuindo desta forma para modulação da resposta contrátil da PE nas artérias de ratas P(AU)

Reduction of vascular reactivity to phenylephrine (PE) in aorta of spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higher production and/or higer bioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation, by PI3K/Akt. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOS on serine site leads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this study was to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteries of pregnant rats. The O-GlcNAc-protein content and also the enzymes expression that participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2nd or 3rd branch) of non-pregnant (NP) and pregnant (P), normotensive rats (Wistar) and SHR. Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolae in the aorta and the mesenteric artery in the presence or absence of methyl-ß-cyclodextrin (dextrin, 10 mmol/L) by electronic microscopy. In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 µmol/L) and of the caveolae, using a caveolae disassembler, dextrin (10 or 20 mmol/L), in the reduced vascular reactivity observed in aortas or mesenteric arteries of P rats. We observed that the content of O-GlcNAcylated proteins was decreased in the aorta and in the mesenteric bed of Wistar P and SHR P rats. Although OGT and OGA expression is not altered, OGA activity was increased in the aorta and mesenteric bed of Wistar P rats but was decreased in the aorta and increased in the mesenteric bed of SHP P. Incubation with PugNAc reversed the reduced reactivity to PE in the aorta and mesenteric artery of Wistar P but this effect was not observed in SHR P arteries, demonstrating that OGA appears to play an important role in reducing O-GlcNAcylation of vascular proteins in Wistar P. In arteries incubated with PugNAc, endothelial removal or incubation with L-NAME did not significantly alter reactivity to PE. Together, these results suggest that the greater eNOS activity observed in Wistar P vessels was impaired in the presence of PugNAc, and it depends on OGA activity. As there was no change in the contractile response to PE in SHR P arteries incubated with PugNAc, possibly a different mechanism, involving the lower activity of OGT, occurs in these vessels for the reduction of O-GlcNAcylation of eNOS. Dextrin caused increased contraction of PE and decreased ACh potency in Wistar NP and SHR NP aortas, but there was no change in aortas of Wistar P and SHR P. Dextrin did not alter the number of cavelae in Wistar P and SHR P arteries compared to NP rats. SHR NP showed a lower number of caveolae than to NP Wistar as well reduced expression of Cav-1 and CaM. Pregnancy was not able to alter the expression of Cav-1, CaM and Hsp90 in the aorta and mesenteric bed of normotensive and hypertensive rats. These results suggest that pregnancy does not alter the expression of Cav-1, CaM and Hsp90 proteins and possibly interaction with eNOS in the aorta and mesenteric arteries of normotensive and hypertensive rats. In conclusion, among the studied mechanisms of post-translational modification of eNOS, the reduction of O-GlcNAcylation of eNOS, by mechanisms that involve OGA activity and possibly OGT, would favor eNOS phosphorylation and consequent greater NO bioavailability, contributing in this way for modulation of the contractile response to PE in the arteries of P rats(AU)

Identification of the targets of hematoporphyrin derivative in lung adenocarcinoma using integrated network analysis

BACKGROUND: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. METHODS: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using "BioCloud" online tool. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. RESULTS: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated-treated with X-ray-X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. CONCLUSION: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.

Envolvimento de heat shock protein 90 na ativação de TLR4 em células de gliobastoma murino / Involvement of heat shock protein 90 in TLR4 activation in murine gliobastoma cells

O glioblastoma é um tumor cerebral altamente invasivo e de mau prognóstico, derivado da transformação maligna de células-tronco neurais, células precursoras de oligodendrócitos e astrócitos. As células de glioblastoma expressam o receptor de reconhecimento de padrões (PRR) do tipo toll (TLR)4, que reconhece ligantes endógenos presentes no microambiente tumoral. A ativação deste receptor induz a produção de mediadores inflamatórios envolvidos na sobrevivência, migração e invasão das células tumorais. A proteína de choque térmico (HSP do inglês, heat shock protein) 90 é superexpressa por células tumorais, sendo essencial para o crescimento tumoral. Há relatos que sugerem que a ativação do TLR4 depende da atividade da HSP90, uma vez que esta mantém a integridade do receptor durante o reconhecimento de seu ligante, assim como regula diversas proteínas envolvidas na via de sinalização deste receptor, incluindo oncoproteínas e quinases. Desta forma, o objetivo do presente estudo foi investigar o efeito da ativação de TLR4 em células de glioblastoma e o impacto da atividade da HSP90 sobre a ativação e crescimento tumoral in vitro. Dados obtidos neste trabalho demonstram que as células de glioblastoma murino GL261 expressam TLR4 funcional. A ativação deste receptor pelo seu ligante, lipopolissacarídeo (LPS), levou ao aumento da expressão da proteína ácida fibrilar glial (GFAP do inglês, glial fibrillary acidic protein) e da produção de fatores envolvidos na resposta inflamatória e no desenvolvimento tumoral, tais como interleucina (IL)-6 e -10, CCL2 (do inglês CC chemokine ligand 2) e fator de crescimento do endotélio vascular (VEGF do inglês, vascular endothelial growth factor)

A ativação de TLR4 também levou à translocação do fator nuclear κB (NFκB do inglês, factor nuclear κB) para o núcleo das células. Além disso, observamos que o inibidor seletivo da atividade de HSP90 (17-allylaminogeldanamycin, 17-AAG) modulou negativamente a produção dos mediadores inflamatórios supracitados, a translocação de NFκB, assim como reduziu proliferação celular, sugerindo que a HSP90 participa da resposta inflamatória induzida pela ativação de TLR4 e do crescimento de células GL261. Neste trabalho, demonstramos que a ativação de TLR4 em células GL261 induz a resposta inflamatória e que a inibição da atividade de HSP90 contribui para a redução deste fenômeno. Considerando a importância da inflamação para o desenvolvimento tumoral, nossos dados podem ainda contribuir para o desenvolvimento futuro de novas abordagens terapêuticas. (AU)

The Role of Serotonin in Ventricular Repolarization in Pregnant Mice

PURPOSE: The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy. MATERIALS AND METHODS: We measured current amplitudes and the expression levels of voltage-gated K⁺ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a(−/−)-NP). RESULTS: During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a(−/−)-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP. CONCLUSION: Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents.

Palmitoylation of heat shock protein 90 in mouse sperm / 生理学报

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

Gene Expression Profile of Apoptosis in Leukemia Cells Induced by Hsp90 Selective inhibitor 17-AAG / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism.</p><p><b>METHODS</b>CCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays.</p><p><b>RESULTS</b>The inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment.</p><p><b>CONCLUSION</b>The 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.</p>

Influence of Co-inhibiting mTORC2 and HSP90 on Proliferation Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

<p><b>UNLABELLED</b>Objective：To explore the influence of co-inhibiting mTORC2 and HSP90 on the proliferation and apoptosis of multiple myeloma(MM) cell line U266.</p><p><b>METHODS</b>During culture, the human MM cell line U266 were treated with 20 nmol/L of rapamycin, 600 nmol/L 17-AAG, 20 nmol/L of rapamycin + 600 nmol/L 17-AGG and phosphate-buffered saline (PBS), then the growth inhibition rate, morphologic changes, apoptosis rate and the expression of caspase 3 and ATK protein in U266 cells were compared and analyzed.</p><p><b>RESULTS</b>The rapamycin and 17-AAG both could inhibit the growth of U266 cells, while the inhibitory effect of rapamycin in combination with 17-AAG on growth of U266 cells was significantly higher them that of rapamycin and 17-AAG alone and control (PBS); the apoptosis rate of U266 cells treated with rapamycin, 17-AAG and their combination was higher than that of control PBS groups, and the efficacy of 2 drug conbination was higher than that of control PBS group, and the efficacy of 2 drug combination was superior to single drug. The expression levels of caspase 3 and ATK in U266 cells treated with rapamycin, 17-AAG and their combination were higher and lower than those in control group respectively, and the efficacy of 2 drug combination was superior to signle drug. There were significant difference between them (P<0.05).</p><p><b>CONCLUSION</b>The co-inhibition of mTORC2 and HSP90 can suppress the proliferation and induce the apoptosis of MM cells.</p>

Mechanism of heat shock protein 90 for regulating 26S proteasome in hyperthermia / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia.</p><p><b>METHODS</b>Hyperthermic HepG2 cell models established by exposure of the cells to 42 degrees celsius; for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed.</p><p><b>RESULTS</b>ROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001).</p><p><b>CONCLUSION</b>The intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.</p>

HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism.</p><p><b>METHODS</b>The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively.</p><p><b>RESULTS</b>The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P < 0.05 and r = -0.473, P < 0.05), while the culture medium without 17-AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P < 0.05); At same time of culture, the more high of 17-AAG concentration, the more high of cell ratio in G1 phase (P < 0.05), at same concentration of 17-AAG, the more long time of culture, the more high of cell ratio in G1 phase (P < 0.05).</p><p><b>CONCLUSION</b>The HSP90 inhibitory 17-AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.</p>

The association of Hsp90 expression induced by aspirin with anti-stress damage in chicken myocardial cells

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.

Social determinants of health, inequality and social inclusion among people with disabilities / Determinantes sociais da saúde, iniquidades e inclusão social entre pessoas com deficiência / Determinantes sociales de la salud, iniquidades e inclusión social entre personas con deficiencia

OBJECTIVE: to analyze the socio-familial and community inclusion and social participation of people with disabilities, as well as their inclusion in occupations in daily life. METHOD: qualitative study with data collected through open interviews concerning the participants' life histories and systematic observation. The sample was composed of ten individuals with acquired or congenital disabilities living in the region covered by a Family Health Center. The social conception of disability was the theoretical framework used. Data were analyzed according to an interpretative reconstructive approach based on Habermas' Theory of Communicative Action. RESULTS: the results show that the socio-familial and community inclusion of the study participants is conditioned to the social determinants of health and present high levels of social inequality expressed by difficult access to PHC and rehabilitation services, work and income, education, culture, transportation and social participation. CONCLUSION: there is a need to develop community-centered care programs in cooperation with PHC services aiming to cope with poverty and improve social inclusion. .

OBJETIVO: analisar a inclusão sociofamiliar e comunitária e a participação social de pessoas com deficiência, bem como sua inserção em ocupações na vida cotidiana. MÉTODO: estudo qualitativo, com coleta de dados por meio de entrevistas abertas sobre história de vida e observação sistemática. A amostra foi composta por dez pessoas com deficiência, adquirida ou congênita, moradoras de região adstrita a um Núcleo de Saúde da Família. O referencial teórico foi a concepção social da deficiência. Os dados foram analisados segundo abordagem interpretativa reconstrutiva, fundamentada no referencial da Teoria da Ação Comunicativa de Habermas. RESULTADOS: os resultados evidenciaram que a inclusão sociofamiliar e comunitária dos sujeitos do estudo condiciona-se a determinantes sociais da saúde, apresentando índices de iniquidades sociais, expressos pela dificuldade de acesso a serviços de Atenção Primária à Saúde e de reabilitação, trabalho e renda, educação, cultura, transporte e participação social. CONCLUSÃO: conclui-se a necessidade da elaboração de programas de atenção centrados na comunidade, voltados ao enfrentamento da pobreza e à inclusão social, em articulação com serviços de Atenção Primaria à Saúde. .

OBJETIVO: analizar la inclusión social familiar y comunitaria, y la participación social de personas con deficiencia, así como su inserción en ocupaciones en la vida cotidiana. MÉTODO: estudio cualitativo, con recolección de datos por medio de entrevistas abiertas sobre historia de vida y por observación sistemática. La muestra estuvo compuesta por diez personas con deficiencia, adquirida o congénita, habitantes de una región adscrita a un Núcleo de Salud de la Familia. El referencial teórico fue la concepción social de la deficiencia. Los datos fueron analizados según abordaje interpretativo reconstructivo, fundamentado en el referencial de la Teoría de la Acción Comunicativa de Habermas. RESULTADOS: los resultados evidenciaron que la inclusión social familiar y comunitaria de los sujetos del estudio se condiciona a determinantes sociales de la salud, presentando índices de iniquidades sociales, expresados por la dificultad de acceso a servicios de Atención Primaria de la Salud y de rehabilitación, trabajo y renta, educación, cultura, transporte y participación social. CONCLUSIÓN: se concluye que existe la necesidad de elaborar programas de atención centrados en la comunidad, dirigidos al enfrentamiento de la pobreza y a la inclusión social, en articulación con servicios de Atención Primaria a la Salud. .

Pontos de venda de alimentos e associação com sobrepeso/obesidade em escolares de Florianópolis, Santa Catarina, Brasil / Retail food outlets and the association with overweight/obesity in schoolchildren from Florianópolis, Santa Catarina State, Brazil / Puntos de venta de alimentos y asociación con sobrepeso/obesidad en escolares de Florianópolis, Santa Catarina, Brasil

O estudo descreve os pontos de venda de alimentos e sua associação com sobrepeso/obesidade em escolares de Florianópolis, Santa Catarina, Brasil. Desenho transversal com amostra probabilística de 2.506 escolares de escolas públicas (n = 19) e privadas (n = 11). O sobrepeso/obesidade foi classificado pela referência da Organização Mundial da Saúde de 2007. Foram realizadas análises brutas e ajustadas por meio de regressão de Poisson. A prevalência de sobrepeso/obesidade foi de 34,2%. Na rede pública, foram verificados 19,6% de sobrepeso e 13,5% de obesidade. Na rede privada, observaram-se 22,4% de sobrepeso e 11,1% de obesidade. Na rede pública, foi encontrada associação entre sobrepeso/obesidade e utilização da padaria (p = 0,004). Na rede privada, observou-se que os escolares de famílias que utilizaram o supermercado apresentaram 26% menos de sobrepeso/obesidade do que os escolares que não utilizam esses pontos de venda de alimentos (p = 0,003). Os dados encontrados evidenciam a existência de associação entre a utilização de alguns tipos de pontos de venda de alimentos (supermercado e padaria) e a prevalência de sobrepeso/obesidade na população escolar.

The study analyzes retail food outlets and their association with overweight/obesity in schoolchildren from Florianópolis, Santa Catarina State, Brazil. The study used a cross-sectional design with a random sample of 2,506 schoolchildren from public (n = 19) and private schools (n = 11). Overweight and obesity were classified according to World Health Organization guidelines for 2007, and crude and adjusted analyses were performed using Poisson regression. Prevalence of overweight/obesity was 34.2%. In public schools, 19.6% of the children were overweight and 13.5% were obese, as compared to 22.4% and 11.1% in private schools. An association was found in the public school system between overweight/obesity and the use of bakeries for food purchases (p = 0.004). In the private school system, children of families that bought groceries at the supermarket showed 26% less overweight/obesity compared to those who did not (p = 0.003). The data show an association between some types of food outlets (supermarkets and bakeries) and prevalence of overweight/obesity in the school-age population.

El estudio describe los puntos de venta de alimentos y su asociación con el sobrepeso/obesidad en escolares de Florianópolis, Santa Catarina, Brasil. Se trata de un estudio transversal con una muestra aleatoria de 2.506 escolares de las escuelas públicas (n = 19) y privadas (n = 11). El sobrepeso/obesidad se clasifica, en función de la OMS en 2007, con análisis ajustados y crudos que se realizaron mediante la regresión de Poisson. La prevalencia de sobrepeso/obesidad fue de un 34,2%. En el sistema público el resultado fue de un 19,6% sobrepeso y un 13,5% obesidad. En el privado se observó un 22,4% de sobrepeso y 11,1% obesidad. En el primero se encontró una correlación entre el sobrepeso/obesidad y el consumo de bollería (p = 0,004). En las escuelas privadas se observó que los escolares de familias que habían utilizado el supermercado tenían un 26% menos de sobrepeso/ obesidad que los niños en edad escolar que no utilizaron este punto de venta de alimentos (p = 0,003). En el momento del estudio existe una asociación entre el uso de algunos tipos de punto de venta de alimentos (supermercado y panadería) y la prevalencia de sobrepeso/obesidad en escolares.

Modulating heat shock proteins 70 and 90 expression by low power laser irradiation [635nm and 780nm] in jurkat E6.1 T-lymphocyte leukemia cell line

Heat shock proteins [HSPs] are molecular chaperones involved in protein folding, stability and turnover, and due to their role in cancer progression, the effect of low power laser irradiation [LPLI] on the expression of HSP70 and HSP90 in Jurkat E6.1 T-lymphocyte leukemia [JELT] cell line was investigated in vitro. JETL cells were irradiated with LPLI at 635nm and 780m wavelengths [energy density 9.174 J/cm[2], and assessed for the expression of HSP70 and HSP90 by flow cytometry after 24, 48 and 72 incubation time periods [ITPs]. At 24 hours ITP post-irradiation, control cultures showed that 10.7% of cells expressed HSP70, while LPLI cultures at 635nm and 780nm manifested a higher expression [32.1and 21.3%, respectively], and the difference was significant [P