Development of a novel and highly efficient method of isolating bacteriophages from water.

Bacteriophages are widely used to the treatment of drug-resistant bacteria and the improvement of food safety through bacterial lysis. However, the limited investigations on bacteriophage restrict their further application. In this study, a novel and highly efficient method was developed for isolating bacteriophage from water based on the electropositive silica gel particles (ESPs) method. To optimize the ESPs method, we evaluated the eluent type, flow rate, pH, temperature, and inoculation concentration of bacteriophage using bacteriophage f2. The quantitative detection reported that the recovery of the ESPs method reached over 90%. The qualitative detection demonstrated that the ESPs method effectively isolated 70% of extremely low-concentration bacteriophage (100 PFU/100L). Based on the host bacteria composed of 33 standard strains and 10 isolated strains, the bacteriophages in 18 water samples collected from the three sites in the Tianjin Haihe River Basin were isolated by the ESPs and traditional methods. Results showed that the ESPs method was significantly superior to the traditional method. The ESPs method isolated 32 strains of bacteriophage, whereas the traditional method isolated 15 strains. The sample isolation efficiency and bacteriophage isolation efficiency of the ESPs method were 3.28 and 2.13 times higher than those of the traditional method. The developed ESPs method was characterized by high isolation efficiency, efficient handling of large water sample size and low requirement on water quality.

Diazoxide protects rat vascular endothelial cells against hypoxia and cold-induced damage.

The present study aimed to examine the effects of hypoxia and cold on vascular endothelial cells (VECs), as well as the protective ability of novel VECs-protective drugs against these injuries. A rat model simulating exposure to hypoxia and cold at high altitude environments was established. Based on these animal experiments, rat aortic VECs were established as injury models and exposed to hypoxia and/or adrenaline (ADR) in vitro. The results revealed that hypoxia significantly altered the levels of nitric oxide and vascular endothelial growth factor, while the cold temperature significantly increased the release of ADR and noradrenaline. Exposure to hypoxia combined with cold temperature significantly affected all these indices. In vitro experiments demonstrated that hypoxia, ADR (which was used to simulate cold in the animal experiments) and the combination of the two factors resulted in damage to the VECs and endothelial dysfunction. In addition, the results also showed that diazoxide, a highly selective mitoKATP opener, protected VECs against these injuries. In conclusion, hypoxia and cold temperature induced endothelial cell dysfunction and endocrine disorders, respectively. Improving endothelial function using diazoxide may be an effective therapeutic strategy in patients with altitude-associated disorders. However, the potential for clinical application requires further study.

[Effects of acute cold exposure on pulmonary proinflammatory cytokine of rat].

OBJECTIVE: To study the effects of acute cold exposure on the inflammation and pathologic injuries in pulmonary of rats, and explore the mechanism induced by cold stress. METHODS: Forty male Wistar rats were randomly divided into five groups(n = 8): control group (23 ± 2) °C 2.5 h, -25°C 0.5 h group, -25°C 1 h group, -25°C 2 h group and -25°C 2.5 h group. Rats were exposed to cold at -25°C and no wind by keeping them in a low temperature chamber except control group. Rectal temperatures of the rats were measured before and after cold exposure. The morphological changes of pulmonary were observed by the optics microscope. The levels of tumer necrosis factor-α(TNF- α), interleukin-6 (IL-6) and interleukin-ß (IL-1ß) in lung tissue homogenate were measured by ELISA. RESULTS: Compared to the control group, body core temperatures of the -25°C 1 h group, -25°C2 h group and -25°C 2.5 h group were decreased significantly, and the D-values of rectal temperature were increased before and after cold exposure (P < 0.05). The infiltration of inflammatory cells and alveolar edema fluid appeared in the lung tissue of the -25°C 2.5 h group. The concentrations of tumor necrosis factor-α (TNF α), interleukin-6 (IL-6) and inter- leukin-1ß (IL-1ß) in lung tissue homogenate were increased significantly in -25°C l h group, -25°C 2 h group and -25C° 2.5 h group (P < 0. 05). CONCLUSION: The infiltration of inflammatory cells and the increase in proinflammatory cytokine from pulmonary may lead to the lung tissue injury after acute cold exposure.

[Regulative effects of vessel active drugs on extremital skin temperature of experimental animals exposed to cold].

OBJECTIVE: Using an experimental model of animals exposed to cold to evaluate the regulative effects of prazosin hydrochloride (Pra) and racanisodamine (Ani) on extremital skin temperature of rats and mice. METHODS: Eighty animals were randomly divided into eight groups according to the drug dosage. After been administered with drugs by intragastric at room temperature for 60 min, the animals were moved into specified temperature (5 degrees C,18 degrees C) environment and the skin temperatures at the 1/3 site at the proximal end of tail were measured by infrared camera on 180 min and 300 min. Effects of drug were evaluated by changes in tail skin temperatures. RESULTS: Pra and Ani combination raised the extremital skin temperature of experimental animals significantly in a dose-dependent manner, while single use of Pra was not potent to rats and less potent to mice, and single use of Ani could not raise extremital skin temperature of both rats and mice. Change of rectal temperature in mice showed that Pra and Ani combination did not affect core temperature. CONCLUSION: Pra and Ani combination could significantly raise extremital skin temperature of rats and mice exposed to cold, and would not affect their core (rectal) temperature.

[Effect of iptkalim on myocardial enzymes and free radicals metabolism with hypoxic pulmonary hypertension].

OBJECTIVE: To explore the effects of iptkalim on myocardial enzymes and free radicals metabolism with hypoxic pulmonary hypertension (HPH), in order to provide evidence for the mechanism of iptkalim on clinical treat. METHODS: 110 young men stayed at high altitude above 5 000 m were divided into iptkalim group (n = 74) and placebo group (n = 36), aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (gamma-GT), creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), malonaldehyde (MDA), nitric oxide(NO) and nitric oxide synthase(NOS) were detected before and after took medicines for 6 mouths. RESULTS: After took medication for 6 mouths, ALT, AST, gamma-GT, CK and LDH were reduced, SOD, NO, and NOS were increased, MDA were reduced, there were very significant difference (P < 0.05). CONCLUSION: Oxygen free radicals have taken part in the process of HPH, iptkalim have the effect of anti-peroxidation of lipid and protect myocardial cells stress injured by hypoxia which related with mitochondrial membrane and cell membrane's K(ATP) channel activation.

[Effect of neotype carbonic anhydrase target-based inhibitors(P-8) on the hypoxic tolerance in mice].

OBJECTIVE: To explore the effects of different doses of P-8 in increasing the Hypoxia tolerance of mice and the mechanisms involved. METHODS: The health mice were placed into the oxygen deficit bottles and measured the survival time in the condition of hypoxia. The male mice were put into the ladder cage, then placed them into the hypobaric champer to determine the survival time of mice with decompression hypoxia (min). We observed the activity changes of the mice's organization carbonic anhydrase II (CAII). By using the drug in prophylaxis, we investigated the effects of carbonic anhydrase target-based inhibitors P-8 for improving the hypoxia tolerance. RESULTS: (1) In improving the endurance of mice in the condition of hypoxia, the survival time of 6.25 mg/(kg x d) and more doses of P-8 groups were (27.38 +/- 4.63, 29.53 +/- 4.43, 29.67 +/- 7.28, 31.55 +/- 6.34, 32.45 +/- 6.65, 36.81 +/- 7.24 and 35.41 +/- 4.20) min, compared with the control group (22.90 +/- 3.19) min , the survival time significantly prolonged (P < 0.05, P < 0.01); compared to the same dose of acetazolamide groups (24.54 +/- 3.17, 22.70 +/- 3.04, 22.67 +/- 2.99, 23.93 +/- 0.96, 27.87 +/- 5.06, 30.79 +/- 5.12 and 35.14 +/- 6.46) min, the survival time significantly prolonged; P-8 groups and Acetazolamide's minimum effective dose were 6.25 and 100 mg/(kg x d), the potency of P-8 is 16 times Acetazolamide. (2) In improving the endurance of mice in the condition of hypoxia, the survival time of middle and high doses of P-8 groups [(24.82 +/- -3.92, 28.27 +/- 5.89) min] were significantly longer than those in control group [(21.96 2.51) min, P < 0.05]; compared with the acetazolamide (23.11 +/- 3.71) min, the survival time of high dose of P-8 group was significantly prolonged. (3) Compared with the normal control group, P-8 [(25 mg/(kg x d), 50 mg/(kg x d), 100 mg/(kg x d), 200 mg/(kg x d)] dose groups inhibited the activity of carbonic anhydrase II (CAII) in the renal (P < 0.05, P < 0.01); P-8 [100 mg/(kg x d) and 200 mg/(kg x d)] dose group significantly inhibited the activity of carbonic anhydrase II (CA II) in the brain (P < 0.05). CONCLUSION: P-8 treatment improved the endurance of mice in the condition of hypoxia and worked better than Acetazolamide. The mechanism may be related to the inhibition of carbonic anhydrase organization.

[Changes of VEGF, TNF-alpha, IL-6 and NO in serum of patients with HAPE].

OBJECTIVE: To explore the possible pathophysiological process and mechanisms underlying the development and formation of high altitude pulmonary edema(HAPE) by observing the changes in contents of VEGF, TNF-alpha, IL-6 and NO in serum from the initiated and recovery of HAPE patients. METHODS: We studied 10 HAPE patients in a Chinese population. The patients were divided into two groups including HAPE initiate group and the recovery group. Contents of VEGF, TNF-alpha, IL-6 and NO in serum of the two groups were determined to study the process of HAPE. RESULTS: VEGF levels in the HAPE initiate one and the recovery groups were (167.9 +/- 26.5 and 53.1 +/- 17.0 pg/ ml), respectively. There was a significant decrease of VEGF content in recovery one compared to the HAPE group. The same results for TNF-alpha were gained. The levels of TNF-alpha in recovery group was much lower than that in the HAPE initiate one. They were (29.2 +/- 6.8) pg/ml and (86.2 +/- 24.1) pg/ml, respectively. The contents of IL-6 in HAPE initiate group and the recovery group were (32.3 +/- 16.5) pg/ml and (12. 5 +/- 8.0) pg/ml, respectively. But no significance existed. The level of NO in HAPE initiate group was (33.8 +/- 3.3) micromol/L, and it remarkably increased to (74.1 +/- 6.2) micromol/L in recovery one. CONCLUSION: VEGF, TNF-alpha, IL-6 and NO participated in the different aspects of the pathophysiological process and might have influence on HAPE.

[Protective effects of new compound codonopsis tablets against acute mountain sickness].

OBJECTIVE: To study on the protective effects of new compound codonopsis tablets against acute mountain sickness (AMS). METHODS: Forty-five male plain resident soldiers stayed at 1400 m altitude for 3 months were randomly divided into two groups, control (15 men) and treatment group (30 men). Single blind trial was used in this study. The subjects in the two groups took placebo and new compound codonopsis tablets respectively for 5 days before climbing to high mountain, and continued to take for another 10 days until the 3rd day after arriving at 5200 m altitude. On the 1st , 3rd, and 5th day after they arrived at 5200 m altitude, the score and the degree of AMS symptoms of soldiers were followed up and recorded according to State Military Standard GJB1098-91--"Principles of diagnosis and treatment of benign form of acute mountain sickness", heart rate (beats/min) and arterial oxygen saturation (%) were detenrmined. On the 6th day after they arrived at high altitude, forced vital capacity(FVC), forced expired volume in one second(FEV1.0), FEV1% (FEV1.0/FVC), FEF25%-75%, peak expiratory flow (PEF) and maximal voluntary ventilation (MVV) were detected, total frequency of hands cross movement and memory of order numbers test were measured. RESULTS: Comparison with control, AMS symptoms of treatment group reduced on the 1st, 3rd, and 5th day after arriving at 5200 m high altitude (P < 0.01). The degree of AMS symptoms of treatment group was significantly different from that of control. The proportion of slight symptoms in treatment group was high, and that of relative serious symptoms in control was high. Compared with control, FVC, FEV1.0, FEF25%-75%, PEF and MVV of treatment group increased (P < 0.05, P < 0.01), and Ttis, Ctis of treatment group increased (P < 0.05, P < 0.01), Atime decreased markedly (P < 0.05), there was no statistically significant difference in Etis and Sum between the two groups. CONCLUSION: New compound codonopsis tablets could decrease the incidence of AMS, mitigate the symptoms of AMS, and improve breathing function and fingers movement function. New compound codonopsis tablets have an obvious effect on prevention and treatment of acute mountain sickness.

[Cloning and sequencing of alpha, beta globin coding genes in Tibetans living at high altitude].

AIM: To explore the molecular biological mechanism of hemoglobin with high oxygen affinity in Tibetans by determining the sequence of globin cDNA in Tibetans living at high altitude. METHODS: Total RNA was isolated from human bone marrow samples of three Tibetans who live in Qinghai-Tibet plateau. cDNA fragments coding for alpha and beta genes of human hemoglobin were obtained through RT-PCR and were ligated to plasmid pGEM-T easy vectors, and then the ligation liquid were transformed to Escherichia coli and cloned and sequenced. Nucleotide sequences were compared with GenBank data by BLAST method. RESULTS: sequence of a globin cDNA in Tibetans were the same with the registering globin genes in the GenBank, and Hb Abruzzo (beta143 (H21), His- > Arg) gene mutation, a high oxygen affinity beta globin mutation, was found in one Tibetan' beta goblin coding gene (CAC- > CGC). CONCLUSION: This hemoglobin gene mutation may be associated with high altitude adaptation of Tibetans living at high altitude.

[Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation].

The effects of hypoxia on the level of reactive oxygen species (ROS), IkappaBalpha tyrosine phosphorylation, transcription of P65 mRNA and NF-kappaB activation in isolated rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry, Western blotting and RT-PCR. The results obtained are as follows. (1) During hypoxia, the levels of intracellular ROS began to increase at 1 h, then reached a peak at 2 h, and began to decrease after 3 h. IkappaBalpha tyrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. NF-kappaB activation began to increase after 3 h hypoxia, and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 mmol/L) was added into the medium, the level of IkappaBalpha phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 micromol/L), NF-kappaB activation induced by hypoxia was blocked significantly. (3) The expression of p65 mRNA was also elevated markedly during hypoxia. These results suggest that hypoxia may lead to IkappaBalpha phosphorylation and NF-kappaB activation through intracellular ROS, and that the regulation of NF-kappaB activity may involve IkappaBalpha phosphorylation and the expressions of each subunit gene of NF-kappaB.