Effect of Raptor on invasion ability of glioma cells

AIM：To study the influence of Raptor on the invasion ability of glioma cells. METHODS：The technique of RNA interference was used. U87 cells were transfected with Raptor restricted siRNA plasmid, and the expression level of Raptor in the transfected cells was detected by Western blotting. The invasive ability of the cancer cells in vitro was determined. The phosphorylation level of ARK5 and the expression of MMP-2 and MMP-9 were detected by Western blotting. The expression levels of Raptor in the tumor samples of low-grade gliomas (WTO grade I and grade II) and high-grade gliomas (WTO grade III and grade IV) were also analyzed by immunohistochemical staining. RESULTS：Raptor siRNA was transfected into U87 cells and the cells were named siRaptor/U87 cells. The cells transfected with the control plasmid was named Scr/U87 cells. The expression level of Raptor in siRaptor/U87 cells was lower than that in Scr/U87 cells. The results of in vitro invasion assay showed that the number of siRaptor/U87 cells penetrating the Matrivgel matrix membrane was less than that of Scr/U87 cells (P＜0.01). The protein expression of MMP-2 and MMP-9, and phosphorylation of ARK5 protein in the cells in the experimental group were lower than those in control group. The correlation between the expression of Raptor in gliomas and the degree of deterioration was also observed (P＜0.01). CONCLUSION：The expression of Raptor may contribute to the invasion ability of glioma cells by phosphorylation of ARK5 and increase in the levels of MMP-2 and MMP-9.     

L-Carnitine inhibits H2O2-mediated NFATc3 nuclear translocation

AIM:To explore the effect of L-carnitine on nuclear factor of activated T-cells,cytoplasmic 3 (NFATc3) in cardiomyocytes under H2O2 stimulation. METHODS:Primary cultured neonatal rat myocardial cells were stimulated by H2O2 at concentration of 200 μmol/L for 12 h to induce oxidative stress injury. In treatment group, L-carnitine and cyclosporin A (CsA), a specific inhibitor of calcineurin (CaN), were administered 30 min prior to H2O2 stimulation. After treatment, total, cytoplasmic and nuclear NFATc3 protein levels were determined by Western blotting. The method of immunofluoresence was used to evaluate the distribution of NFATc3. RESULTS:H2O2 treatment produced no effect on the expression of total NFATc3, but caused its translocation from the cytosolic to nuclear compartment, which was greatly blunted by L-carnitine pretreatment. CONCLUSION: L-carnitine antagonized oxidative stress injury via alleviating NFATc3 nuclear translocation.     

Aortic expression of HSP22, TNF-α and eNOS in rats with hyperlipidemia and effects of atorvastatin

AIM:To establish a rat hyperlipidemia model for studying the aortic expression of heat shock protein 22 (HSP22), tumor necrosis factor alpha (TNF-α) and endothelial nitric oxide synthase (eNOS) and the effect of atorvastatin intervention. METHODS:Hyperlipidemia model was established in SD rats. Afterwards, the rats were divided into normal control group, high fat group and high fat+atorvastatin intervention group. The expression of HSP22 and TNF-α in the rat aortas was detected by immunohistochemical assay and the expression of eNOS was assessed by Western blotting. RESULTS:No detectable expression of HSP22 and TNF-α in the normal control group was observed. However, the expression of HSP22 and TNF-α was positive in the high fat group and the atorvastatin intervention group. The mean densities of HSP22 and TNF-α positive particles were significant lower in the atorvastatin intervention group as compared with high fat group (both P＜0.05). The expression of eNOS protein in the high fat group and atorvastatin intervention group was significantly lower than that in normal control group (P<0.01). However, no marked difference of eNOS protein expression between high fat group and atorvastatins intervention group was observed. CONCLUSION: The expression of HSP22 and TNF-α in the rat aortas is increased in the hyperlipidemia rat model. This effect can be restored by atorvastatin treatment. The expression of eNOS in the rat aortas is decreased in the hyperlipidemia rat model, but this tendency could not be attenuated by atorvastatin.     

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

Effects of Kv1.5 on proliferation and apoptosis of rat PASMCs under hypoxia+hypercapnia condition and relationship with MAPK pathway

AIM：To investigate the effects of voltage-dependent K＋ channel 1.5 (Kv1.5) on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia+hypercapnia condition and the relationship with mitogen-activated protein kinase(MAPK) signal pathway. METHODS：The PASMCs isolated from the male SD rat were cultured under hypoxia+hypercapnia condition, and randomly divided into normal group (N group), hypoxia+hypercapnia group (HH group), hypoxia+hypercapnia+DMSO incubation group (HD group), hypoxia+hypercapnia+U0126 (an extracellular signal-regulated kinase 1/2 inhibitor) incubation group (HU group), hypoxia+hypercapnia+SB203580 (a p38 mitogen-activated protein kinase inhibitor) incubation group (HS group), and hypoxia+hypercapnia+anisomycin (an agonist of MAPK) incubation group (HA group). Cell Counting Kit-8 was used to detect the cell viability. The protein expression of Kv1.5, PCNA and Bax was detected by Western blotting. RESULTS：Compared with N group, the cell viability and PCNA protein expression in HH group and HD group were significantly raised (P<001), but Kv1.5 and Bax proteins were significantly decreased (P<0.01). No difference between HH group and HD group was observed (P>005). Compared with HD group, the cell viability and PCNA protein expression in HU group, HS group and HA group were decreased (P<0.05 or P<0.01), but Kv1.5 protein and Bax protein were raised (P<0.01), with the most significant changes in HA group. ^{CONCLUSION：}The regulation of Kv1.5 to the proliferation and apoptosis of PASMCs under hypoxia+hypercapnia condition might have a relationship with the activation of MAPK signal pathway.     

大棚种植菜用甘薯茎尖矿物质和氨基酸含量分析

采用感应耦合等离子质谱和氨基酸自动分析仪,对种植于大棚内的7种菜用甘薯茎尖的12种矿质元素和17种氨基酸含量进行分析。结果表明,甘薯茎尖含有丰富的矿质元素和氨基酸,不同甘薯茎尖的矿质元素和氨基酸的含量略有区别。常量元素方面,镁的含量波动较小,为2 904.53~3 903.17 mg/kg,钾、钙、钠的含量波动较大,特别是钠,为544.24~11 086.49 mg/kg;微量元素方面,7种甘薯茎尖含量较大的分别为铁、锌、锰、硼,在18.64~115.88 mg/kg,含量较小的分别为铜、铬、钼、钴,在0.02~21.65 mg/kg。7种甘薯茎尖的氨基酸含量丰富,氨基酸总量在26.61%~30.03%,人体必需氨基酸含量比例E/T值在33.20%~37.66%,人体必需氨基酸含量与非必需氨基酸含量之比E/N值在0.50~0.60,基本符合FAO/WHO关于理想蛋白质的要求。     

Evaluation of feeding spray-dried bovine plasma protein on production performance of laying hens exposed to high ambient temperatures

The purpose of this study was to evaluate feeding 2 levels of spray-dried bovine plasma protein (SDP) on production performance of laying hens subjected to high ambient temperatures. Two groups of 96 Hy-Line W-98 hens (38 wk of age) were housed in each of 2 environmentally controlled chambers. At 40 wk of age, all hens were fed 3 diet treatments consisting of (1) a control diet (0% SDP); (2) the control diet supplemented with 0.75% SDP; and (3) the control diet supplemented with 1.50% SDP. Hens in each chamber (8 cages of 4 hens per cage) were ad libitum fed 1 of each diet for 5 wk. The heat stress (HS) chamber was maintained at 21°C (wk 1), 29°C (wk 2), and 35°C (wk 3 to 5). The thermoneutral chamber was maintained at 21°C during wk 1 to 5. A significant main effect of week was observed for hens maintained in the HS chamber for egg production, egg weight, egg mass, and feed consumption, which resulted in acute heat stress causing a reduction in these parameters. Hens fed the 1.50% SDP diet in the HS chamber produced greater (P < 0.05) egg mass on average than hens fed the control or 0.75% SDP diet (wk 1 to 5). During the second week of acute HS (wk 4), hens fed the control and 1.50% SDP diets had greater (P < 0.05) egg production than those fed the 0.75% SDP diet. During wk 5, hens in the HS chamber that were fed the 1.50% SDP diet produced more (P < 0.05) eggs than those fed the control diet. Therefore, based on the results of this study, acute HS negatively affected short-term production performance. In addition, feeding hens an SDP-supplemented diet may have a slight positive effect on production performance when maintained in acute HS conditions.     

PREVALENCE OF COMPUTED TOMOGRAPHIC SUBCHONDRAL BONE LESIONS IN THE SCAPULOHUMERAL JOINT OF 32 IMMATURE DOGS WITH THORACIC LIMB LAMENESS

Osteochondrosis is a common developmental abnormality affecting the subchondral bone of immature, large breed dogs. The purpose of this retrospective study was to describe CT lesions detected in scapulohumeral joints of 32 immature dogs undergoing CT for thoracic limb lameness. Eight dogs (14 scapulohumeral joints) had arthroscopy following imaging. Thirteen dogs (19 scapulohumeral joints) were found to have CT lesions, including 10 dogs (16 scapulohumeral joints) with subchondral bone lesions and 3 dogs with enthesopathy of the supraspinatus tendon. In one dog, subchondral bone lesions appeared as large oval defects within the mid‐aspect of the glenoid cavities, bilaterally. These lesions resembled osseous cyst‐like lesions commonly identified in the horse. This is the first report of such a presentation of a subchondral bone lesion in the glenoid cavity of a dog. In all dogs, small, focal, round or linear lucent defects were visible within the cortical bone at the junction of the greater tubercle and intertubercular groove. These structures were thought to represent vascular channels. Findings from this study support the use of CT as an adjunct modality for the identification and characterization of scapulohumeral subchondral bone lesions in immature dogs with thoracic limb lameness.     

Paraoxonase activity as a tool for clinical monitoring of dogs treated for canine leishmaniasis

This study was designed to determine if the activity of paraoxonase (PON1), an antioxidant enzyme that works as a negative acute phase reactant, is a better predictor for the clinical recovery of leishmaniotic dogs receiving standard treatments compared with inflammatory markers such as C reactive protein (CRP) and electrophoretic fractions. For this purpose we tested 20 healthy dogs (controls) and 39 leishmaniotic dogs classified as sick (group A, n = 23) or severely sick (group B, n = 16) and tested at admission and after 3, 7, 14, 21, 28, 35 and 42 days.At admission, CRP and electrophoresis were altered in both groups, while PON1 activity was abnormal only in group B. There were no differences related to the outcome (mortality, complications or time of recovery). PON1 activity normalized in about 2 weeks in dogs that had abnormal values at admission and a final positive outcome; CRP normalized in 4–6 weeks and electrophoretic fractions were still altered after 6 weeks. The results show that, at admission, inflammatory markers did not predict the outcome of leishmaniasis. PON1 activity decreased only in some dogs with systemic inflammation but not in those with mild leishmaniasis: when decreased, PON1 normalized earlier than other markers in dogs that responded to treatment. This finding most likely depends on the rapid decrease in oxidative phenomena. PON1 activity should therefore be tested on admission: if low values are recorded, severe inflammation may be suspected and PON1 measurement may be repeated during treatment to early identify responsive dogs.     

Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro

Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers.Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P < 0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P < 0.001). Activation with PMA induced a loss of viable neutrophils after 3 h, which was greater with AOPP-BSA incubation (P < 0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.