Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid ß, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid ß, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid ß, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

Heat adaptation from regular hot water immersion decreases proinflammatory responses, HSP70 expression, and physical heat stress.

Hot-water immersion (HWI) is a type of thermal therapy for treating various diseases. In our study, the physiological responses to occasional and regular HWI have been explored. The rats were divided into a control group, occasional group (1D), and regular group (7D). The 1D and 7D groups received 42°C during 15mins HWI for 1 and 7 days, respectively. The blood samples were collected for proinflammatory cytokines examinations, the heart, liver and kidney were excised for subsequent IHC analysis to measure the level of heat shock protein 70 (HSP70). The results revealed that the body temperature increased significantly during HWI on Day 3 and significantly declined on Days 6 and 7. For the 7D group, body weight, heart rate, hematocrit, platelet, osmolarity, and lactate level were lower than those in the 1D group. Furthermore, the levels of granulocyte counts, tumor necrosis factor-α, and interleukin-6 were lower in the 7D group than in the 1D group. The induction of HSP70 in the 1D group was higher than in the other groups. Physiological responses to occasional HWI are disadvantageous because of heat stress. However, adaptation to heat from regular HWI resulted in decreased proinflammatory responses and physical heat stress.

Parenteral nutrition-associated liver injury and increased GRP94 expression prevented by ω-3 fish oil-based lipid emulsion supplementation.

OBJECTIVE: Parenteral nutrition in infants with gastrointestinal disorders can be lifesaving, but it is also associated with parenteral nutrition-associated liver disease. We investigated the effects of incorporating ω-3 fish oil in a parenteral nutrition mixture on signs of parenteral nutrition-associated liver disease and explored the mechanism involved in this process. METHODS: Seven-day-old New Zealand rabbits were divided into 3 groups of 8, and for 1 week they were infused via the right jugular vein with standard total parenteral nutrition with soybean oil (TPN-soy) or TPN with ω-3 fish oil-based lipid emulsion (TPN-FO), or naturally nursed with rabbit milk (control). Serum and liver tissues were analyzed for serological indicators and pathology, respectively. Reverse-transcriptase polymerase chain reaction was used to evaluate the messenger RNA levels of the endoplasmic reticulum stress chaperone protein glucose-regulated protein 94 (GRP94) in liver tissues and GRP94 protein levels were compared through immunohistochemistry and Western blot assays. RESULTS: TPN-soy animals had significantly higher serum total bilirubin, direct bilirubin, and γ-glutamyl transpeptidase and lower serum albumin than the controls (P < 0.01, each) or the TPN-FO group, which were similar to the controls (P < 0.01 cf. TPN). Damage to liver tissues of the TPN-FO group was much less than that of the TPN-soy group. GRP94 messenger RNA and protein levels in liver tissues of TPN-soy animals were significantly higher than that of the controls or TPN-FO rabbits, which were similar to the controls. CONCLUSIONS: Incorporating ω-3 fish oil in parenteral nutrition emulsion greatly prevented liver dysfunction and liver tissue damage in week-old rabbit kits, possibly by preventing endoplasmic reticulum stress.

Grape seed extract supplementation attenuates the heat stress-induced responses of jejunum epithelial cells in Simmental × Qinchuan steers.

Grape seed extract (GSE), a rich source of polyphenols, is reported to possess antioxidant, anti-inflammatory and immunomodulatory properties. The objective of the present study was to determine whether GSE could attenuate the heat stress-induced responses of jejunum epithelial cells (JEC) in cattle. The JEC of a steer (Simmental × Qinchuan) were exposed to heat stress for 2 h in the absence (0 µg/ml) or presence (10, 20, 40 and 80 µg/ml) of GSE in the culture medium. When cultured at 40°C, JEC supplemented with GSE exhibited increased glutathione peroxidase activity (P= 0·04), viability (P= 0·004), and mRNA expression of epidermal growth factor (EGF; P= 0·03) and EGF receptor (EGFR; P = 0·01). Under the same conditions, the cells exhibited decreased mRNA expression of IL-8 (P= 0·01) and TNF-α (P= 0·03) and decreased protein concentrations of IL-1ß (P= 0·02), Toll-like receptor 4 (TLR4; P= 0·04) and heat shock protein 70 (HSP70; P< 0·001). When cultured at 43°C, JEC supplemented with GSE exhibited increased catalase activity (P= 0·04), viability (P< 0·001), and mRNA expression of EGF (P< 0·001) and EGFR (P< 0·001) and decreased protein concentrations of IL-1ß (P< 0·001), TLR4 (P= 0·03) and HSP70 (P< 0·001), as well as mRNA expression of IL-8 (P< 0·001), TLR4 (P= 0·002) and TNF-α (P< 0·001). Temperature × GSE concentration interactions were also observed for the concentrations of IL-1ß (P< 0·001), IL-8 (P< 0·001), TNF-α (P= 0·01) and HSP70 (P= 0·04) and viability (P< 0·001) of JEC. The results of the present study indicate that GSE can attenuate the responses of JEC induced by heat stress within a certain range of temperatures.

Cytoprotective mechanisms in rats lung parenchyma with zinc deprivation.

Suboptimal intake of Zinc (Zn) is one of the most common worldwide nutritional problems. The aim of this study is to provide new evidence on the relation between moderate Zn restriction, and cytoprotective functions in airway epithelium. We analyzed the effect of moderate Zn deficiency (ZD) on the expression of several pro and anti-apoptotic proteins and cytoprotective factors (Hsp27 and Hsp 70i), as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control group, Zn-deficient group and Zn-refed group. Our previous findings showed an important oxidative and nitrosative stress during ZD, this situation is accompanied by inflammation and alterations in the expression of matrix extracellular proteins. We observed a strong immunopositive area of anti and pro-apoptotics proteins in ZD groups. The mRNA levels of Nrf-2, Bax and Bad were increased in ZD, while in ZD refed group its levels were similar to the control values. The increased expression of Nrf-2 is likely to be critical for protection of lung under inflammatory process triggered during ZD. Hsp27 and Hsp 70i showed an increase of immunostaining area but they were not significant. During the supplementation period, heat-shock proteins increased significantly. In conclusion, our results provide further evidence of the pathways involved in cytoprotection and apoptosis caused by ZD. Additional studies are required in order to investigate whether Hsp27 and Hsp70 are consistently associated with cellular stress and inflammation in lung. There may be a beneficial role for improved Zn nutrition or Zn supplements early in lung pathology.

Effects of an extremely low-frequency electromagnetic field on stress factors: a study in Dictyostelium discoideum cells.

The development of technologies that generate environmental electromagnetic fields (EMFs) has led public opinion and the scientific community to debate upon the existence of possible effects caused by man-made EMFs on the human population and, more generally, on terrestrial ecosystems. Protozoa are known to be excellent bioassay systems in bioelectromagnetic studies because of their features that combine the reliability of in vivo results with the practicality of in vitro ones. For this reason, we examined the possible stressful effects of a 50-Hz, 300-µT extremely low-frequency electromagnetic field (ELF-EMF) on the protozoan Dictyostelium discoideum, which was used as it is included in the eight bioassay alternatives to vertebrate models for the study of human disease by the U.S. National Institutes of Health. Our results show how a 24-h exposure of D. discoideum cells to ELF-EMF can affect the net fission rate, the activity and presence of the pseudocholinesterase as well as the presence of the heat shock protein-70, while no change in the catalase and glutathione peroxidase activities was observed. However, this effect seems to be transient and all the altered parameters returned to their respective control value after a 24-h stay under dummy exposure conditions.

N-acetylglucosamine biofunctionalized CdSeTe quantum dots as fluorescence probe for specific protein recognition.

Hsp70 proteins are implicated in resistance to chemotherapy in cancers, the detection of which is important for cancer treatment and prognosis. In this work, we report the study on the detection of specific intracellular target protein in fixed cells using GlcNAc-conjugated CdSeTe QDs. The QDs were coupled with Con A via a carbodiimide reaction and then were further assembled with GlcNAc by lectin-carbohydrate interaction between Con A and GlcNAc. The obtained QDs-Con A-GlcNAc conjugates have an emission wavelength at 650 nm that is close to the near-infrared (NIR) regions and a specific recognition for Hsp70. These results show that the QDs-Con A-GlcNAc probe can be a promising tool for direct localization of the Hsp70 protein.

Supplementation with essential amino acids in middle age maintains the health of rat kidney.

Chronic kidney diseases are a social and economic problem, and diet has long been recognized as a fundamental modulator of kidney health in human and experimental models. Age-dependent alterations in mitochondrial function play a crucial role in the development of diseases of aging, and mitochondrial disorders have been observed in experimental models of kidney failure. Recently, the beneficial dietary effect of a specific mixture of essential amino acids (EAA) has been studied in elderly subjects, but no data were collected from the kidney. The aim of this study was to assess whether daily supplementation of the diet with EAA at the beginning of senescence could preserve renal health. We used middle-aged (18-month-old) male Wistar rats fed a standard diet and water ad libitum (M-aged group) or a diet with added EAA (1.5 g/kg per day) dissolved in drinking water for 3 months (M-aged+EAA group). Young (2-month-old) rats fed a standard diet for 3 months were used as controls. Mitochondrial morphology and markers for collagen, cyt-c-oxidase, HSP60, GRP75, eNOS, iNOS, Bax, Bcl2 and VEGF were analyzed in glomeruli and tubules. EAA supplementation limited fibrosis and increased the capillary tuft area in the glomeruli of M-aged rats. VEGF and eNOS were enhanced in glomeruli and the peritubular space with the EAA-supplemented diet. Mitochondrial cyt-c oxidase, Bcl2, and chaperones increased in the distal tubules of the EAA group to levels similar to those observed in the young group. Mitochondrial area and density after EAA intake did not differ from young groups. The results suggest that prolonged EAA intake could represent a strategy for maintaining the healthy status of the kidney in M-aged animals.

Expression of heat shock proteins, Hsp70 and Hsp25, in the rat gingiva after irradiation with a CO2 laser in coagulation mode.

BACKGROUND AND OBJECTIVE: The therapeutic rationale of low-energy pulsed CO(2) laser coagulation mode has not been clarified yet. We conducted this study to characterize the effect of low-energy pulsed CO(2) laser coagulation mode irradiation of the rat gingiva in terms of the expression of heat shock proteins. MATERIAL AND METHODS: Laser irradiation was achieved with the parameters of 5 W, 600 mus pulse duration, and fluence of 326 J/cm(2). The gingiva dissected at different times after irradiation was processed for immunohistochemical examination of the expression of the heat shock proteins, Hsp70 and Hsp25. RESULTS: One hour after irradiation, the epithelial keratinocytes facing the laser wound exhibited an overexpression of Hsp70 in their nucleus. The connective tissue cells facing the laser wound, which included fibroblasts and capillary endothelial cells, showed de novo expression of Hsp70 at 3 h post-irradiation, the level of which peaked at 1 d and thereafter decreased. An enhanced and/or de novo expression of Hsp25 in the connective tissue cells facing the laser wound became evident at 3 h after irradiation, and after 1 d the Hsp25-expressing cells increased in number and spread over the wound as wound repair progressed. There was a temporospatial difference in the expression pattern between Hsp70 and Hsp25, with only a few cells appearing to co-express both heat shock proteins. CONCLUSION: The CO(2) laser treatment in coagulation mode produced the expression of heat shock proteins, and the findings suggest that while Hsp70 mainly conferred cell protection, Hsp25 was involved in the progress of wound repair as well as cell protection.

Effects of chromium histidinate on renal function, oxidative stress, and heat-shock proteins in fat-fed and streptozotocin-treated rats.

OBJECTIVE: Chromium is an essential element for carbohydrate, fat, and protein metabolism. The therapeutic potential of chromium histidinate (CrHis) in the treatment of diabetes has been elucidated. The present study investigated the effects of CrHis on serum parameters of renal function, on oxidative stress markers (malondialdehyde [MDA] and 8-isoprostane), and on the expression of heat-shock proteins (HSPs) in rats. METHODS: Male Wistar rats (n=60, 8 weeks old) were divided into four groups. Group 1 received a standard diet (12% of calories as fat). Group 2 received a standard diet, plus CrHis. Group 3 received a high-fat diet (40% of calories as fat) for 2 weeks, and was then injected with streptozotocin (STZ) on day 14 (STZ, 40 mg/kg intraperitoneally). Group 4 was treated in the same way as group 3 (HFD/STZ), but was supplemented with 110 microg CrHis/kg/body weight/day. Oxidative stress in the kidneys of diabetic rats was evidenced by an elevation in levels of MDA and 8-isoprostane. Protein concentrations of HSP60 and HSP70 in renal tissue were determined by Western blot analyses. RESULTS: Chromium histidinate supplementation lowered kidney concentrations of MDA, 8-isoprostane levels, serum urea-N, and creatinine, and reduced the severity of renal damage in the STZ-treated group (i.e., the diabetes-induced group). The expression of HSP60 and HSP70 was lower in the STZ group that received CrHis than in the group that did not. No significant effect of CrHis supplementation was detected in regard to the overall measured parameters in the control group. CONCLUSIONS: Chromium histidinate significantly decreased lipid peroxidation levels and HSP expression in the kidneys of experimentally induced diabetic rats. This study supported the efficacy of CrHis in reducing renal risk factors and impairment because of diabetes.

[Effect of preventive acupuncture and moxibustion at "Guanyuan" (CV 4) on the expression of HSP 70 and HSP 70 mRNA in spleen and the contents of serum IL-2, TNF-alpha in menopausal rats].

OBJECTIVE: To observe the influence of preventive acupuncture (PA) and preventive moxibustion (PM) at "Guanyuan" (CV 4) on the immune function in natural climacteric rats. METHODS: A total of 160 female SD rats were randomized into control, PA and PM groups, the former one group was further divided into 10 month (mon), 12 mon, 14 mon and 16 mon subgroups, and the later two groups were further divided into 12 mon, 14 mon and 16 mon subgroups, with 16 rats in each. In addition, other 16 female SD rats aged 3.5 mon were used as the young control (YC) group. "Guanyuan" (CV 4) was punctured with an acupuncture needle and the needle was retained for 20 min, or given with one ignited moxa-cone from the age of 10 mon on. The treatment was conducted twice every week, 8 weeks altogether. The expression of HSP 70 and HSP 70 mRNA of the spleen tissue was detected by using immunohistochemistry and in situ hybridization respectively, and serum IL-2 and TNF-alpha contents were assayed by using radio-immunoassay. RESULTS: In comparison with YC group, 1) the expression of spleen HSP 70 and HSP 70 mRNA increased significantly in 10 mon control (mon-C), 12 mon-PM and 12 mon-PA groups, and 14 mon-PA group (only HSP 70 mRNA) (P < 0.05, P < 0.01); 2) HSP 70 expression decreased remarkably in 14 mon-C, 16 mon-C and 16 mon-PA groups (P < 0.05, P < 0.01); 3) IL-2 contents decreased evidently in 12 mon-C and 14 mon-C groups, and TNF-alpha contents increased obviously in 12 mon-PM, 12 mon-PA and 16 mon-C groups (P < 0.05). In comparison with the corresponding same age control groups, HSP 70 and HSP 70 mRNA expression increased significantly in 12 mon-PM and 12 mon-PA groups, 14 mon-PM and 16 mon-PM (HSP 70 only), 14 mon-PA (HSP 70 mRNA only) groups (P < 0.05, P < 0.01); IL-2 level of 12 mon-PM group, and TNF-alpha contents of 12 mon-PM and 12 mon-PA groups increased markedly (P < 0.05, P < 0.01). No significant differences were found between PM and PA groups in most different age groups (P > 0.05). CONCLUSION: Both preventive acupuncture and preventive moxibustion can upregulate the expression of spleen HSP 70 and HSP 70 mRNA and serum IL-2 and TNF-alpha levels, which may contribute to their effects in enhancing the immune function in menopausal rats.

The effects of vitamin C and E supplementation on heat shock protein 70 response of ovary and brain in heat-stressed quail.

1. The effects of vitamin C (L-ascorbic acid) and vitamin E (alpha-tocopherol acetate) supplementation on egg production and heat shock protein 70 (Hsp70) response of ovary and brain in the Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature were evaluated. 2. The birds (n = 540; 55 d old) were randomly assigned to 18 groups consisting of 3 replicates of 10 birds each in a 2 x 3 x 3 factorial arrangement of treatments. Birds were kept in wire cages in a temperature-controlled room at either 22 degrees C (thermo-neutral, TN) or 34 degrees C (heat stress, HS) for 8 h/d (09:00 to 17:00 h; until the end of the study) and fed on a basal diet or the basal diet supplemented with either three levels of vitamin C (0, 250 and 500 mg of L-ascorbic acid/kg of diet) or three levels of vitamin E (0, 250 and 500 mg of dl-alpha-tocopheryl acetate/kg of diet). 3. Feed intake and egg production were not affected by vitamin C and E supplementation under thermo-neutral conditions. However, feed intake and egg production were increased with the vitamin C or E supplementation either singly or in combination in heat-stressed quail. When vitamin was added, feed intake and egg production of quails under TN were different from those raised under HS. However, in the absence of supplemental vitamins, feed intake and egg production at TN and HS were no different. 4. Heat exposure increased serum corticosterone levels and Hsp70 expression. Serum corticosterone level was significantly decreased by vitamin C or E supplementation in HS groups for quail. Ovary and brain Hsp70 expression linearly decreased as dietary vitamin C or vitamin E supplementation increased in heat-stressed groups. However, Hsp70 expression of ovary and brain was not affected by vitamin C or E supplementation under thermo-neutral conditions. 5. The present study showed that a combination of dietary vitamin C (500 mg) and E supplementation (500 mg) may alleviate some heat stress effects of heat shock proteins of ovary and brain and egg production of Japanese quail.

A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants.

To investigate the function of nuclear-localized plant HSP70, we used NtHSP70-1 isolated from Nicotiana tabacum. The subcellular localization of NtHSP70-1 was identified by fluorescence microscopy for NtHSP70-1/GFP or smGFP fusion proteins in onion epidermal cells, obtained using particle gun bombardment. To analyze the drought-stress tolerance and thermoprotective role of NtHSP70-1, we obtained transgenic tobacco plants that constitutively expressed elevated levels of NtHSP70-1 as well as transgenic plants containing either the vector alone or else having NtHSP70-1 in the antisense orientation. From analysis for genomic DNA in transgenic seedlings after heat stress, NtHSP70-1 helps to prevent the fragmentation and degradation of nuclear DNA during heat stress. In addition, seedlings constitutively overexpressing NtHSP70-1 grew to be healthy plants, whereas transgenic vector or antisense seedlings resulted in death after heat-/drought-stress.

Dehydroepiandrosterone: a modulator of cellular immunity and heat shock protein 70 production during polymicrobial sepsis.

OBJECTIVE: DHEA is an immunomodulatory steroid hormone that improves survival during systemic inflammation. A DHEA-induced modulation of heat shock protein response may be an alternative mechanism contributing to the beneficial effects of this hormone. We investigated the effect of DHEA administration on survival, cellular immune functions, and HSP-70 production in septic mice. DESIGN AND SETTING: Randomized animal study, level I trauma center, university research laboratory. SUBJECTS: Male NMRI mice. INTERVENTIONS: Mice were subjected to sham operation (laparotomy, LAP) or sepsis (cecal ligation and puncture, CLP) with or without administration of either saline 0.9% (LAP, CLP) or 20 mg/kg DHEA subcutaneously (LAP/DHEA, CLP/DHEA). Survival was monitored over a 48-h period. Splenocyte apoptosis rate (AnnexinV binding), splenocyte proliferation ([3H]thymidine incorporation), TNF-alpha plasma concentration (ELISA), and HSP-70 concentration (ELISA) in tissue extracts from liver, lung, and spleen were monitored 48 h after onset of sepsis. RESULTS: DHEA administration improved the survival of septic mice (78% vs. 50%). This effect was paralleled by increased splenocyte proliferation, decreased cellular apoptosis rate of splenocytes, and attenuation of TNF-alpha release. Furthermore, an increased HSP-70 concentration was observed in lungs and spleens of DHEA-treated septic animals. CONCLUSIONS: DHEA-treatment decreased the mortality rate of septic mice. This was accompanied by improved cellular immune functions and an augmented heat shock response (HSP-70) of lungs and spleens. Further studies are required to demonstrate a direct relationship between the improved survival and the observed alterations in the immune system in DHEA-treated animals.

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion injury in rats.

Hyperbaric oxygen (HBO) therapy is an effective adjunct in treating ischemia-reperfusion (I/R) injury of brain, small intestine, testis, and crushing extremities. This study was designed to test the hypotheses that preconditioning the rats with HBO could protect the liver against subsequent I/R injury. Daily treatment with one-dose HBO (90 min, 2.5 ATA) was brought about for male Sprague Dawley rats for 1 to 3 days before an I/R injury of liver. Hepatic expression of heat-shock protein 70 (Hsp70), total concentration of glutathione (GSH), activity of catalase, superoxide dismutase (SOD), and serum AST and ALT were estimated before and after HBO, as well as after I/R injury. The results showed that activity of hepatic catalase was decreased by one dose, but not three doses, of HBO as compared with baseline data. However, hepatic Hsp70 expression fluctuated insignificantly. AST and ALT increase less in rats preconditioned with one-dose HBO as compared with those without HBO or with three-dose HBO. Our results showed preconditioning by one-dose HBO protects rat liver against subsequent ischemia-reperfusion injury.

[Effects of heating on the epithelia of benign prostate hyperplasia in vitro].

OBJECTIVE: To investigate the biological changes of the epithelia of benign prostate hyperplasia under heating at different temperatures and at different points of time after heating. METHODS: Cell morphology, MTT, flow cytometer, and the immunocytochemical method for detecting heat shock protein 70 and prostate specific antigen were used to observe the effect of heating on the primarily cultured epithelia of benign prostate hyperplasia at 45 degrees C and 60 degrees C and at 1 hour and 12 hours after heating. RESULTS: Heating at 45 degrees C for 15 min resulted in apoptosis, and at 60 degrees C, necrosis in most of the cells. The inhibiting effect of heating on the growth of cells was observed, more significant in the 60 degrees C group than in the 45 degrees C group. The cell phase arrest induced by heat, mainly G0/G1 arrest, was more significant in the 12 h group than in the 1 h group. Heating up-regulated the expressions of heat shock protein 70 and prostate specific antigen in cells. CONCLUSION: Heating can induce apoptosis, necrosis and growth suppression of the epithelia of benign prostate hyperplasia. Its process and mechanism are correlated with the cell phase arrest and the up-regulation of the expressions of heat shock protein 70 and prostate specific antigen.

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.

A field methodology to study effects of UV radiation on fish larvae.

There is a considerable lack of in situ specific information about the effects of UV-B radiation on limnic animals studied in the field. We exposed larval pike (Esox lucius L.) in two types of cuvettes (glass and quartz) placed at different depths (5 or 15 cm) to natural solar UV or to artificially enhanced UV-B (lamps on 3 h per day), simulating the scenarios for coming decades. Dose realism and comparability with earlier laboratory experiments was the main purpose, and therefore UV-B irradiances to the surface as well as underwater irradiances were directly measured. Result showed that UV-B dose rates in natural waters are low even though DOC concentration was low (4.8 mg/l) in our study lake. A slight increase in ambient UV-B dose rates was enough to cause neurobehavioral symptoms in pike larvae. However, the dose rates applied were inadequate to affect superoxide dismutase (SOD) or HSP70. While assessing the suggested risks due to increased UV, conclusions emphasize the importance of conducting field UV studies as supplements to laboratory experiments. We also recommend direct measurements of UV-radiation at sites where the target organisms are actually exposed.

Tumor regression by combined immunotherapy and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma.

Immunotherapy (IT) has become an accepted therapeutic modality. We previously reported that intracellular hyperthermia (IH) using magnetic nanoparticles induces antitumor immunity. We undertook these studies in order to study the combined effects of IT and IH on melanoma. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs were injected into a B16 melanoma nodule in C57BL/6 mice, which were subjected to AMF for 30 min. The temperature at the tumor reached 43 degrees C and was maintained by controlling the magnetic field intensity. At 24 h after IH, interleukin-2 (IL-2) or granulocyte macrophage-colony stimulating factor (GM-CSF) was injected directly into the melanoma. Mice were divided into six groups: group I (control), group II (IH), group III (IL-2), group IV (GM-CSF), group V (IH + IL-2), and group VI (IH + GM-CSF). Complete regression of tumors was observed in mice of groups V and VI (75% (6/8) and 40% (4/10) of the mice, respectively), while no tumor regression was observed in mice of the other groups. This study supports the combined use of IT and IH using MCLs in patients with advanced malignancies.

Hyperthermic pre-conditioning protects retinal neurons from N-methyl-D-aspartate (NMDA)-induced apoptosis in rat.

Glutamate-induced excitotoxicity is associated with a selective loss of retinal neurons after retinal ischemia and possibly in glaucoma. Since heat shock protein (HSP) 70 is known to play a protective role against ischemic neuronal injury, which is also linked to excitotoxicity, we studied the expression of inducible (HSP72) and constitutive (HSC70) forms of HSP70 in apoptosis of retinal ganglion cells (RGCs) after intravitreal injection of 8 nmoles N-methyl-D-aspartate (NMDA), a glutamate receptor agonist. Approximately 18 h after NMDA injection, there were increased numbers of TUNEL-positive cells and cells with elevated HSP72 immunoreactivity in the retinal ganglion cell layer (RGCL), but there were no noticeable changes in HSC70 immunoreactivity. These HSPs positive cells were also Thy-1 positive, a marker for RGCs. Hyperthermic pre-conditioning, which is known to induce HSPs, given 6 or 12 h prior to NMDA injection ameliorated neuronal loss in the RGCL as counted 7 days after NMDA injection but pre-conditioning at 18 h prior to NMDA injection did not have any ameliorative effect. Quercetin, an inhibitor of HSP synthesis, abolished the ameliorative effect of hyperthermic pre-conditioning. Pre-conditioning elevated HSP72 but not HSC70 immunoreactivity and reduced the number of TUNEL-positive cells in the RGCL at 18 h. Our results suggest that intravitreal injection of NMDA induces an up-regulation of HSP72 in a time-dependent manner but not HSC70 in RGCs, indicating a stress response of HSP72 in RGCs and other inner retinal neurons after exposure to NMDA. Hyperthermic pre-conditioning given within a therapeutic window is neuroprotective to the retina against NMDA-induced excitotoxicity, likely by inhibiting apoptosis through the modulation of HSP72 expression.