Normal Rat Serum Research Articles

Depletion of GR-1-Positive Cells Is Associated with Reduced Neutrophil Inflammation and Astrocyte Reactivity after Experimental Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity with 25% of patients dying within the first 48h and a high incidence of poor outcomes. Because of high early mortality rates, an understanding of acute brain injury mechanisms is essential. In this study, we have investigated the putative role of acute inflammation in brain injury after experimental ICH. We depleted GR-1(+) cells in mice by intraperitoneal administration of anti-GR-1 antibody or normal rat serum (control). We then induced ICH by infusion of autologous whole blood into the striatum and compared functional outcome and brain injury markers between the two groups. We found that administration of anti-GR-1 antibody led to a profound decrease in circulating GR-1(+) cells (1.5 ± 0.34% vs. 50.3 ± 8.3% of CD45(+) cells, p ≤ 0.01) and that brain neutrophils decreased by approximately 50% (p ≤ 0.05). We observed a reduction in astrocyte immunoreactivity in the GR-1(+) cell-depleted group (p ≤ 0.05). Conversely, we did not find attenuation of brain edema or differences in behavioral deficits between the two groups. In summary, our results are promising and suggest that larger studies or different neutrophil manipulations may produce greater attenuation of injury after ICH.

Rat Kidney biomarkers: 613‐APS (renal)

Multiplex measurement of rat kidney cytokines in serum and urine using the Fluorokine® MAP assayJane Schmidt Ph.D, Michael Rynning, Richard Fuerstenberg R&D Systems, Inc., Minneapolis, MNDrug toxicity can result in major organ damage to the kidneys. The traditional methods for monitoring drug‐induced nephrotoxicity in toxicology studies include measurement of serum creatinine and blood urea nitrogen levels, but in recent years more sensitive kidney biomarkers have emerged. The PCK rat is a recently identified rodent model of polycystic kidney disease that can be used as a model for general kidney disease or damage. We measured TIM‐1/KIM‐1, FABP‐1, Lipocalin‐2, Cystatin C, and Osteopontin in serum and urine from normal (Sprague Dawley) and PCK rats using the Fluorokine MAP Rat Kidney Toxicity Panel and observed a significant increase of TIM‐1/KIM‐1 and Lipocalin‐2 in the PCK rat serum compared to normal rat serum. We also observed statistically significant differences for Lipocalin‐2 and Cystatin C in PCK urine when compared to normal urine. Fluorokine Multianalyte Profiling (MAP) kits are bead‐based multiplex immunoassays designed for use on Luminex® 100/200™, Bio‐Rad® Bio‐Plex® analyzers and offer the ability to quantify multiple analytes simultaneously with less user time, less sample volume, and lower cost per result compared to conventional ELISAs. Five biomarkers can be measured in a user‐defined multiplex using the Rat Kidney Toxicity Panel. The assay is fully validated for accurate, precise, and reproducible results in rat serum, plasma, and urine.

Increased level of soluble syndecan-1 in serum correlates with myocardial expression in a rat model of myocardial infarction

A candidate marker for ventricular remodeling after myocardial infarction (MI), syndecan-1 (Sdc1), has been shown to be upregulated in myocardial tissues. However, the clinical potential of this marker depends on the ability to obtain samples safely and noninvasively. Therefore, we investigated the expression of soluble Sdc1 in the serum of rats after MI. Anterior descending coronary arteries of Sprague-Dawley rats were ligated, and MI was confirmed by morphologic and physiologic methods. Rats that underwent surgery without ligation served as the control group. We analyzed the expression of Sdc1 mRNA by quantitative reverse-transcription polymerase chain reaction and that of Sdc1 protein by western blot in heart tissue from the MI border and compared it to the expression of soluble Sdc1 in serum. The myocardial levels of expression of Sdc1 mRNA and protein were very low in the sham group but increased significantly in the MI group (P<0.01). The expression of myocardial Sdc1 reached a peak at day 3 and declined gradually thereafter, although the levels at 14 days remained significantly higher than those in the sham group. The expression of soluble Sdc1 in the sera of the rats in the MI group followed a similar pattern and was linearly correlated with the expression of Sdc1 protein in the MI border zone (r=0.952, P<0.01). Soluble Sdc1 was also detected at low levels in normal rat serum. These results may facilitate additional exploration of the utility of serum Sdc1 as a biomarker for MI in humans.

Treatment of premenstrual syndrome with Chinese medicine formula, Baixiangdan capsule: implications for neuron viability and GABAA receptor modulation in rat cortex

To explore the effects of Baixiangdan capsule (BXD), a Chinese medicinal formula, on the premenstrual syndrome (PMS) rats with Liver-qi invasion and the possible underlying micro-mechanisms. Wistar rats were randomly assigned to two groups, nomal group and BXD group After evaluated by macro-behavior observation and open-field test, MTT assay and the whole-cell patch clamp recording were performed respectively to evaluate the effects of serum from BXD capsule-treated rats on the viability and GABA(A) R-induced currents of cortical neurons in vitro. In the open-field test, the crossing score, rearing score and total score of BXD rats decreased significantly (P < 0.05), compared by the normal rats. Compared with cells exposed to serum of normal rats, the viability values of those incubated with serum of BXD group for 24 h and 48 h significantly increased (P < 0.05), measured by MTT assay. The results of whole-cell patch clamp recording showed that concentration-response relationship curves revealed an EC50 value of (29.0 +/- 4.4) micromol x L(-1) and a Hill coefficient of 1.07 for normal-exposed cultures, (63.5 +/- 8.2) micromol x L(-1), 1.04 for BXD-exposed cultures after incubation for 24 h. Furthermore, the difference in EC50 values was statistically significant (P < 0.01), that in the Hill coefficient was not obvious. BXD capsule could significantly decrease crossing score and total score of open-field test and effectively enhance the neuron viability and GABA(A) receptor activity in rat cortex. Paeonimetabolins I and paeonol may play a significant role in treating PMS model rats with Liver-qi invasion by BXD capsule, and paeonol may target at GABA(A) receptor, especially.

Effects of Chinese herbal medicine Xuefu Zhuyu Decoction on angiotensin Ⅱ-induced proliferation and extracellular matrix synthesis of rat cardiac fibroblasts

To investigate the effects of Xuefu Zhuyu Decoction, a compound Chinese herbal medicine, on the proliferation and extracellular matrix synthesis of cardiac fibroblasts (CFs) from neonatal rats induced by angiotensin II, and to explore the mechanism of Xuefu Zhuyu Decoction in improving myocardial fibrosis. CFs derived from neonatal rats were cultured with enzymatic dissociation to establish a cell model of cardiac fibrosis induced by angiotensin II in vitro. CFs were divided into 6 groups, including blank control group, angiotensin II group, valsartan-medicated serum group, and 5%, 10% and 15% Xuefu Zhuyu decoction-medicated serum group. CFs in the blank control group were incubated with normal rat serum; CFs in the other groups were stimulated with angiotensin II at a dose of 10-6 mol/L, and then incubated with normal rat serum and 5%, 10% and 15% Xuefu Zhuyu decoction-medicated serum for 24 h, respectively. Methyl thiazolyl tetrazolium assay was employed to measure CF proliferation, and collagen synthesis was detected by analysis of the hydroxyproline in CFs. Radioimmunoassay was used to measure concentrations of procollagen III and hyaluronic acid. Enzyme-linked immunosorbent assay was used to measure fibronectin expression. Compared with the blank control group, angiotensin II induced cardiac fibroblast proliferation, stimulated DNA and collagen syntheses (P<0.01), and increased fibronectin, hyaluronic acid and procollagen III concentrations (P<0.01). Xuefu Zhuyu decoction-medicated serum inhibited CF proliferation and DNA and collagen syntheses (P<0.05), and decreased the concentrations of fibroectin, hyaluronic acid and procollagen III (P<0.01). Angiotensin II can induce cardiac fibrosis, stimulate cardiac fibroblast proliferation and extracellular matrix synthesis, while Xuefu Zhuyu Decoction can prevent cardiac fibrosis, the mechanism of which may be related to inhibiting CF proliferation and extracellular matrix synthesis.

Insulin suppresses the expression and function of breast cancer resistance protein in primary cultures of rat brain microvessel endothelial cells

The aim of this study was to investigate the role of insulin in the regulation of breast cancer resistance protein (BCRP) function and expression using primary cultured rat brain microvessel endothelial cells (rBMECs) as an in vitro model of the blood brain barrier (BBB). The prazosin uptake assay and western blot analysis were used to assess the function and expression of BCRP, respectively. It was noted that the uptake of prazosin by rBMECs was time-, concentration- and temperature-dependent. The BCRP inhibitors novobiocin and imatinib mesylate significantly increased the uptake of prazosin by the cells in a concentration-dependent manner. The cells were also incubated with sera from diabetic rats for 72h, serving as a diabetic in vitro model. We found that the uptake of prazosin by rBMECs incubated in the diabetic rat sera was 39.8% of that in normal rat sera, and insulin treatment reversed this decrease. Further results showed that insulin down-regulated the function and expression of BCRP in rBMECs in a concentration-dependent manner. Treatment with an antibody against the insulin receptor abolished the down-regulation of BCRP function and expression that was induced by insulin. These results indicate that insulin suppressed the function and expression of BCRPs in rBMEC primary cultures.

Determination of monoamine neurotransmitters in serum of pain model rats

Objective: To investigate the effect of pain on contents of monoamine neurotransmitters in serum of pain model rats.Methods: The model was established by subcutaneous injection of 5% formaldehyde at the sole of left hind limb and the animal behavior was observed and recorded.Contents of norepinephrine(NE) and dopamine in serum of normal and model rats were determined by fluorescence spectrophotometry.The excitation emission wavelength of NE was 384 nm/483 nm,and that of dopamine was 324/373 nm.Results: The linear response of NE ranged from 0.01-0.25 μg/ml(r=0.996 5,n=7),that of dopamine ranged from 0.01-0.20 μg/ml(r=0.993 5,n=7).Intra-day and inter-day RSD of both NE and dopamine were less than 20%.The relative recovery rates of NE and dopamine were 99.8%-113.5% and 99.4%-106.0%,respectively.The contents of NE and dopamine in serum of rats in control group were(0.088±0.030),(0.047±0.005) μg/ml,and those of the rats in model group were(0.122±0.010),(0.064±0.010) μg/ml,respectively.Conclusion: Fluorescence spectrophotometry is accurate and sensitive for the determination of NE and dopamine in serum of rats.Subcutaneous injection of formaldehyde engenders symptoms of pain and probably raises the contents of NE and dopamine in serum of pain model rats.

Determination of time-dependent accumulation of d-lactate in the streptozotocin-induced diabetic rat kidney by column-switching HPLC with fluorescence detection

For better understanding the complete metabolism and the physiological role of d-lactate, the concentrations of d-lactate in the serum, liver and kidney of normal and diabetic rats were determined by our established column-switching HPLC method with pre-column fluorescence derivatization. Eight-week-old male Sprague–Dawley rats were administered with streptozotocin (STZ) (80 mg/kg) or citrate buffer intraperitoneally. The tissues were then removed and homogenized after 4, 8, 12 and 16 weeks of drug administration, respectively. The homogenates were centrifuged at 1200 × g for 10 min, then the supernatants were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on an ODS column followed by a Chiralpak AD-RH chiral column for enantioseparation. The results showed that the d-lactate content elevated in all the 3 examined tissues under diabetic stages. In addition, d-lactate concentrations in rat kidney were accumulated significantly and time-dependently in diabetic groups after receiving STZ for 4, 8, 12 and 16 weeks (2.99, 13.11, 18.19, 23.23 vs. 0.79 μmol/mg protein as control group). Moreover, the kidney of induced 12-week diabetic rat renal showed some histological changes of progressive diabetic nephropathy. The results suggest that d-lactate may be used as a marker of diabetic nephropathy.

Effects of blueberry on the proliferation and activation of hepatic stellate cell and its mechanism

To investigate the effects of blueberry on the proliferation and activation of hepatic stellate cell (HSC) and its mechanism. Rat HSC were isolated by type IV collagenase digestion and Nycodenz density gradient centrifugation. The cultured HSC was incubated at different concentrations of 10% blueberry serum. The 10% DSHX serum was used as positive control and 10% normal rat serum group as control. MTT colorimetric assay was used to detect the HSC proliferation. ColI of culture supernatant was detected by ELISA. The expression of α-SMA in HSC was measured by immunocytochemistry staining while the expressions of Nrf2 and HO-1 were determined by Western blot. Compared with controls, the low and high-dose blueberry serum groups significantly inhibited the HSC proliferation (P < 0.05). It had the same inhibitory effects as the positive control serum group (P > 0.05). ColI of culture supernatant obviously decreased (P < 0.05). And the expression levels of α-SMA in low and high-dose blueberry serum groups decreased significantly (P < 0.05). And there were similar effects in low & high-dose blueberry serum and positive control serum groups. Western blot showed that the expressions of Nrf2 and HO-1 in blueberry and positive control serum groups were higher than that in control group. And the increment was more significant in the low and high-dose blueberry serum groups (P < 0.05). Blueberry can significantly inhibit the proliferation and activation of HSC and reduce the synthesis of extra-cellular matrix. It may have potential preventive and protective effects on hepatic fibrosis. The mechanism may be related to an elevated expression of HO-1 through the Nrf2 pathway.

Effect of qinghuobaiduyin on the expression of high mobility group box chromosomal protein 1 in macrophage

To observe the expression of high mobility group box chromosomal protein 1(HMGB1) in RAW264.7 macrophages after interfering with burning serum and qinghuobaidu-yin (QHBDY), and to find out the endogenous protection mechanism of QHBDY resisting inflammation reaction. RT-PCR was used to detect the expression of HMGB1 in RAW264.7 macrophages after interfering RAW264.7 macrophages with normal SD rat serum, burning SD rat serum, and QHBDY feeding SD rat serum. Small quantity of HMGB1 mRNA was expressed in RAW264.7. The expression of HMGB1 mRNA fluctuated around the standard level after interfering with normal serum of SD rats. The expression of HMGB1 mRNA rose at 3 h, and then decreased to the standard level; at 18 h, it rose rapidly; at 36 h, it reached the peak; and at 48 h, it remained at the high level after interfering with burning serum. The expression of HMGB1 mRNA increased at 3 h, and then decreased to the standard level. At 24 h, it started to rise after interfering with herb serum, and was lower than that of; the burning serum group (P<0.05). Burning serum can increase the expression of HMGB1 mRNA in RAW264.7. QHBDY can decrease the high expression of HMGB1 mRNA in RAW264.7 caused by burning serum.

Anti-inflammatory effect of the polysaccharides of Golden needle mushroom in burned rats

The present study was to investigate chemical components of polysaccharides isolated from Golden needle (GNP) mushroom using high performance liquid chromatography (HPLC) and nuclear magnetic resonance spectroscopy (NMR). Then, anti-inflammatory effect of the polysaccharides was also evaluated. Results indicated that GNP was composed of the three monosaccharides (glucose, mannose and xylose) with a molar ratio of 3.5:0.8:1.4. GNP could significantly decreased CD4 + CD8 +, ICAM-1, and MPO in serum and colon of normal and burned rats. It could be concluded that GNP possessed strong anti-inflammatory activity in burned rats.

Inducing Effects of Zhichan Soup on Neuron Stem Cell Differentiation

Abstract This is an experiment to observe the inducing effects of Zhichan Soup on neuron stem cell (NSC) differentiation. NSCs were divided into three groups. The normal group was treated with the normal medium. The herb group was treated with the Zhichan Soup-medicated sera. The control group was treated with the normal rat sera. After the 21-day treatment, TH and DAT proteins were checked by immunocytochemistry, and Shh, Ptx3 and Nurrl mRNA were checked by RT-PCR. Results of this experiment indicated that neither TH nor DAT proteins were found in any groups. Compared with other groups, the Zhichan Soup group had registered a significantly enhanced expression of Shh, Ptx3 and Nurrl mRNA. It is believed that the Zhichan Soup-medicated serum may be able to improve the expression of Shh, Ptx3 and Nurrl in a micro-environment to facilitate NSCs to differentiate into dopamine neurons.

Fully phosphorylated fetuin-A forms a mineral complex in the serum of rats with adenine-induced renal failure

The serum glycoprotein fetuin-A is an important inhibitor of extra-osseous calcification, but correlations between serum fetuin-A levels and the extent of vascular calcification are controversial. In this study, we used a rat model of adenine-induced renal failure with secondary hyperparathyroidism that exhibits all characteristic features of patients with chronic kidney disease. These rats had medial vascular calcification along with reduced levels of both serum and hepatic fetuin-A. Treatment with an inhibitor of ectopic calcification, alendronate, decreased bone turnover and eliminated completely the vascular calcification in this rat model, but there was no change in the levels of hepatic and serum fetuin-A. Centrifugation of the serum of untreated rats with renal failure gave a small precipitate composed of fetuin-A, calcium, magnesium, and phosphate; this complex, absent from normal rat serum, was not found in the serum of alendronate-treated rats with renal failure. Rat serum contained three types of phosphorylated fetuin-A, as well as unphosphorylated forms, but only the fully phosphorylated fetuin-A was present in the mineral complex. The amount of this complex reflected the risk of mineral precipitation. Our results suggest that the measurement of serum fetuin-mineral complex rather than fetuin-A alone might provide a better indication of extra-osseous calcification propensity.

Role of FSH in regulating testicular germ cell transformations in the rat: a study using DNA flow cytometry.

The role of FSH in regulating testicular germ cell transformations during initiation and maintenance of spermatogenesis in the pubertal and adult rat has been studied using DNA flow cytometry (FCM). The cell types were quantified on the basis of their DNA content using DNA specific fluorochrome DAPI (4,6-diamidino phenylindole). Pubertal (30-d old) and adult (100-d old) rats were deprived of endogenous FSH support for 10 d by daily injection (200 microliters d-1) of a characterized FSH antiserum; the control group received an equivalent volume of normal rat serum. FSH deprivation did not lead to any change in serum testosterone levels. The relative proportion of testicular germ cells in the FSH deprived pubertal rat showed a 90% reduction in 1C (round spermatids) and 260% and 90% increase in 2C (spermatogonia) and 4C (spermatocytes) cells respectively. While the overall conversion of 2C to 1C (1C:2C ratio) was reduced by 98%, the transformation of 2C to 4C (4C:2C ratio) and 4C to 1C (meiotic division 1C:4C ratio) was inhibited by 43% and 93% respectively. In the FSH-deprived adult rat the overall conversion of 2C to 1C was reduced by 26% (P < 0.05) only. The 2C and 4C population of cells increased by 47% and 97% respectively (P < 0.025) and the 4C:2C ratio by 47% (P < 0.05). While the meiotic division (1C:4C ratio) was reduced by 54% (P < 0.001), the post-meiotic differentiation of round spermatids to elongate-spermatids (HC:1C) was inhibited by 68% (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Activity of the First Epidermal Growth Factor-Like Domain of Rat Coagulation Factor VII

Abstract: The first epidermal growth factor-like (EGF1) domain of coagulation factorVII (FVII) is essential for binding with tissue factor (TF) in human. We hypothesized that the function of rat FVII (rFVII) EGF1 domain might be the same as the human being's. The clone and expression of rFVII EGF1 domain would be helpful in studying the anticoagulant drugs based on FVII/TF interaction. Accordingly, the model of rat TF expression in vitro was established by lipopolysaccharide(LPS) induction. We amplified the EGF1 domain from a rat liver by reverse transcriptase PCR (RT-PCR), and then a fusion expression vector named pET28a-EGFP-EGF1 was constructed. After expression in Ecoli BL21 cells, the EGFP-EGF1 fusion proteins were analyzed for binding affinity to rat TF by confocal microscope and flow cytometry. Prothrombin time assay was employed to test biological activity of the fusion protein by using FVII depleted human plasma. The resulting plasmid expressed fusion protein EGFP-EGF1 in the soluble form in E.coli BL21, and the recombinant protein was purified successfully with a major band at 36KD in SDS-PAGE(Figure 1). The purified EGFP-EGF1 fusion proteins could definitely depress recombinant mouse FVII((rmFVII) binding with rat TF(rTF)(70.06%VS 53.80%, P< 0.01)(Figure 2 &3). However, the proteins lost the activity of coagulation when compared to that of blood serum of normal SD rats (56.8sVS 17.8s, P< 0.01). (Table 1)We conclude that the rat EGF1 region could specifically bind to TF without the ability of coagulation, and it might facilitate the development of molecular target study in anti-thrombosis treatment.Table 1. Prothrombin Time test of Fusion proteins [Display omitted] [Display omitted] [Display omitted] [Display omitted] [Display omitted]

Glycine decreases developmental damage induced by hyperglycaemia in mouse embryos

Hyperglycaemia induces neural tube defects and growth retardation in cultured mouse and rat embryos. In this study the possibility that glycine could prevent hyperglycaemia-induced embryopathy was researched. Early somite mouse embryos were cultured in normal medium, hyperglycaemic medium (50 mmol L(-1) glucose), or with glycine (1 mmol L(-1)) supplementation of normal and hyperglycaemic rat serum for 48 h. The embryo growth and differentiation were determined to estimate developmental and congenital malformations as well as lipid peroxidation levels. Adding glycine to the control culture medium did not affect embryonic development. Whereas the amino acid protected against telencephalon dysmorphogenesis, the decreased DNA content and number of somites, and the morphological score affectation induced by the hyperglycaemic medium, it had no preventive effect on the retarded differentiation of the otic system. Moreover, it prevented the high hyperglycaemia-induced lipoperoxidation levels of embryonic tissues. Embryos were partially protected from the hyperglycaemia-induced teratogenesis due to the antioxidative effect of glycine. As no other mechanisms related to the antiglycation or other protective effects of glycine were examined, the mechanism whereby it acted as an antiteratogenic agent needs further study.

Circulatory antigen of Heymann nephritis. III. Presence of the 70-kD circulatory protein in the immune deposits of Heymann nephritis

An antigen of 70 kD size has been isolated previously from normal rat serum which has immunological cross-reactivity to the Heymann nephritis antigen, F x 1A. Its role in the pathogenesis of Heymann's nephritis was unknown. In this investigation we tested for the presence of 70-kD circulatory antigen in the glomerular immune deposits of Heymann's nephritis. Further, its presence was correlated with severity of disease. It was observed that the presence of the 70-kD antigen strongly correlated with the existence of electron-dense deposits in the lamina rara externa (LRE) of the glomerular capillary wall and with pathologic proteinuria. Temporally, the presence of the 70-kD antigen in the immune deposits was followed by large electron-dense deposits, enhanced complement activity and proteinuria. The data suggest that in the growing immune complex lattice in the LRE, the 70-kD circulatory antigen by virtue of its small size, mobility and antigen cross-reactivity facilitates cross linking and coalescence of immune complexes, resulting in electron-dense immune deposits (EDD) formation which initiates complement activation and consequent proteinuria.

Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect.

HSP70 Inhibits Burn Serum-Induced Apoptosis of Cardiomyocytes via Mitochondrial and Membrane Death Receptor Pathways

The objective is to determine if Hsp70 is involved in the mechanism by which serum from burn rats induces apoptosis of cardiomyocytes. Cardiomyocytes in primary culture were assigned to the following groups: normal controls, normal rat serum, burn serum, burn serum plus empty vector control, and burn serum plus Hsp70 transfection. Each group, except normal controls, was cultured with the corresponding serum for 2 hours. The empty vector controls and the Hsp70 groups were transfected, respectively, with the empty vector or pcDNA3.1-Hsp70 recombinant plasmid 36 hours previously. Hsp70 protein levels were assessed by Western blot. Apoptotic cells were counted after Hoechst 33258 staining. Apoptosis of cardiomyocytes was also detected by the presence of "ladder" bands in agarose gels following electrophoresis of extracted DNA. Activation of caspase-3, -8, and -9 and Bid cleavage were assessed by Western blot. Hsp70 overexpression inhibited burn serum-induced apoptosis of cardiomyocytes; and burn serum-induced activation of caspases-3, -8, and -9; and Bid cleavage into tBid. Hsp70 inhibits burn serum-induced apoptosis by interfering with death receptor and mitochondrial pathways.

An association betweenSchistosoma mansoniworms and an enzymatically-active protease/peptidase in mouse blood

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.