SIRT1 plays a neuroprotective role in traumatic brain injury in rats via inhibiting the p38 MAPK pathway.

Traumatic brain injury (TBI) is a major cause of disability and death in patients who experience a traumatic injury. Mitochondrial dysfunction is one of the main factors contributing to secondary injury in TBI-associated brain damage. Evidence of compromised mitochondrial function after TBI has been, but the molecular mechanisms underlying the pathogenesis of TBI are not well understood. Silent information regulator family protein 1 (SIRT1), a member of the NAD+-dependent protein deacetylases, has been shown to exhibit neuroprotective activities in animal models of various pathologies, including ischemic brain injury, subarachnoid hemorrhage and several neurodegenerative diseases. In this study, we investigated whether SIRT1 also exert neuroprotective effect post-TBI, and further explored the possible regulatory mechanisms involved in TBI pathogenesis. A lateral fluid-percussion (LFP) brain injury model was established in rats to mimic the insults of TBI. The expression levels of SIRT1, p-p38, cleaved caspase-9 and cleaved caspase-3 were all markedly increased and reached a maximum at 12 h post-TBI. In addition, mitochondrial function was impaired, evidenced by the presence of swollen and irregularly shaped mitochondria with disrupted and poorly defined cristae, a relative increase of the percentage of neurons with low ΔΨm, the opening of mPTP, and a decrease in neuronal ATP content, especially at 12 h post-TBI. Pretreatment with the SIRT1 inhibitor sirtinol (10 mg/kg, ip) induced p-p38 activation, exacerbated mitochondrial damage, and promoted the activation of the mitochondrial apoptosis pathway. In contrast, pretreatment with the p38 inhibitor SB203580 (200 µg/kg, ip) significantly attenuated post-TBI-induced expression of both cleaved caspase-9 and cleaved caspase-3 and mitochondrial damage, whereas it had no effects on SIRT1 expression. Together, these results reveal that the 12 h after TBI may be a crucial time at which secondary damage occurs; the activation of SIRT1 expression and inhibition of the p38 MAPK pathway may play a neuroprotective role in preventing secondary damage post-TBI. For this reason, both SIRT1 and p38 are likely to be important targets to prevent secondary damage post-TBI.

Glovebox-assisted magnetic force microscope for studying air-sensitive samples in a cryogen-free magnet.

Most known two-dimensional magnets exhibit a high sensitivity to air, making direct characterization of their domain textures technically challenging. Herein, we report on the construction and performance of a glovebox-assisted magnetic force microscope (MFM) operating in a cryogen-free magnet, realizing imaging of the intrinsic magnetic structure of water and oxygen-sensitive materials. It features a compact tubular probe for a 50 mm-diameter variable temperature insert installed in a 12 T cryogen-free magnet. A detachable sealing chamber can be electrically connected to the tail of the probe, and its pump port can be opened and closed by a vacuum manipulator located on the top of the probe. This sealing chamber enables sample loading and positioning in the glove box and MFM transfer to the magnet maintained in an inert gas atmosphere (in this case, argon and helium gas). The performance of the MFM is demonstrated by directly imaging the surface (using no buffer layer, such as h-BN) of very air-sensitive van der Waals magnetic material chromium triiodide (CrI3) samples at low temperatures as low as 5 K and high magnetic fields up to 11.9 T. The system's adaptability permits replacing the MFM unit with a scanning tunneling microscope unit, enabling high-resolution atomic imaging of air-sensitive surface samples.

Isolated scan unit and scanning tunneling microscope for stable imaging in ultra-high magnetic fields.

The high resolution of a scanning tunneling microscope (STM) relies on the stability of its scan unit. In this study, we present an isolated scan unit featuring non-magnetic design and ultra-high stability, as well as bidirectional movement capability. Different types of piezoelectric motors can be incorporated into the scan unit to create a highly stable STM. The standalone structure of scan unit ensures a stable atomic imaging process by decreasing noise generated by motor. The non-magnetic design makes the scan unit work stable in high magnetic field conditions. Moreover, we have successfully constructed a novel STM based on the isolated scan unit, in which two inertial piezoelectric motors act as the coarse approach actuators. The exceptional performance of homebuilt STM is proved by the high-resolution atomic images and dI/dV spectrums on NbSe2 surface at varying temperatures, as well as the raw-data images of graphite obtained at ultra-high magnetic fields of 23 T. According to the literature research, no STM has previously reported the atomic image at extreme conditions of 2 K low temperature and 23 T ultra-high magnetic field. Additionally, we present the ultra-low drift rates between the tip and sample at varying temperatures, as well as when raising the magnetic fields from 0 T to 23 T, indicating the ultra-high stability of the STM in high magnetic field conditions. The outstanding performance of our stable STM hold great potential for investigating the materials in ultra-high magnetic fields.

Atomically resolved low-temperature scanning tunneling microscope operating in a 22 T water-cooled magnet.

We present the design and construction of a nonmetallic tip-sample mechanical loop featured Scanning Tunneling Microscope (STM) that operates in a 22 T water-cooled magnet at a low temperature of l.8 K. The STM head mainly consists of a spider-drive motor, stand-alone scanner, moveable sapphire sample holder, and sapphire frame. All parts exist in the tip-sample mechanical loop are made of sapphire to reduce the interference from high magnetic fields. Except for the necessary movement of the tip and scanner, all STM parts are stationary. More importantly, the tip-sample mechanical loop is separate from the motor after detecting the tunneling current, which helps prevent the high voltage signal interference from entering the tip-sample junction, leading to a high stable imaging. A Janis liquid helium cryostat is used to obtain a variable temperature range from 1.8 K to 300 K, and the STM head is cooled down via helium exchange gas. The STM head hangs at the bottom of a probe with a two-stage spring suspension to prevent the huge vibration generated by the water-cooled magnet from entering the tip-sample junction. The performance is demonstrated by atomically resolved STM images of graphite surface at 0 T and 22.8 T under room temperature. Furthermore, the obtained atomic-resolution images of NbSe2 at 1.8 K and 22 T, as well as high-resolution dI/dV spectrums at temperatures from 1.8 K to 8.5 K and magnetic fields from 0 T to 22 T are displayed. This is the first STM capable of atomic-resolution imaging and dI/dV measurement at 1.8 K in a 22 T water-cooled magnet. The high immunity to the magnetic field makes the nonmetallic tip-sample mechanical loop widely useable for atomic-resolution STM imaging in ultra-high magnetic field conditions.

Cryogenic spectroscopic imaging scanning tunnelling microscope in a water-cooled magnet down to 1.7 K.

Spectroscopic-imaging scanning tunnelling microscope (SI-STM) in a water-cooled magnet (WM) at low temperature has long been desirable in the condensed matter physics area since it is crucial for addressing various scientific problems, such as the behaviour of Cooper electrons crossing Hc2 in a high-temperature superconductor. Here we report on the construction and performance of the first atomically resolved cryogenic SI-STM in a WM. It operates at low temperatures of down to 1.7 K and in magnetic fields of up to 22 T (the WM's upper safety limit). The WM-SI-STM unit features a high-stiffness sapphire-based frame with the lowest eigenfrequency being 16 kHz. A slender piezoelectric scan tube (PST) is coaxially embedded in and glued to the frame. A well-polished zirconia shaft is spring-clamped onto the gold-coated inner wall of the PST to serve both the stepper and the scanner. The microscope unit as a whole is elastically suspended in a tubular sample space inside a 1K-cryostat by a two-stage internal passive vibrational reduction system, achieving a base temperature below 2 K in a static exchange gas. We demonstrate the SI-STM by imaging TaS2 at 50 K and FeSe at 1.7 K. Detecting the well-defined superconducting gap of FeSe, an iron-based superconductor, at variable magnetic fields demonstrates the device's spectroscopic imaging capability. The maximum noise intensity at the typical frequency is 3 pA per square root Hz at 22 T, which is only slightly worse than at 0 T, indicating the insensitivity of the STM to harsh conditions. In addition, our work shows the potential of SI-STMs for use in a WM and hybrid magnet with a 50 mm-bore size where high fields can be generated.

A hybrid magnet based scanning tunneling microscope.

In this paper, a scanning tunneling microscope (STM) is presented that operates in a 27.5 T magnetic field within a hybrid magnet. The coarse approach of the STM is realized by using an inertial piezoelectric motor, and the scanning is realized by using a miniature scanner, which stands alone on a sapphire base. A combined vibration isolation system consisting of a brick-rubber-brick stack and two springs is used to isolate the vibration generated from the magnet. An enclosed copper shield is used to prevent sound from entering the tip-sample junction. The sound and vibration isolation measures highly improve the stability of the STM imaging. All the materials selected to construct the STM head are nonmagnetic. The drift rates of the STM in the X-Y plane and Z direction are as low as 26.2 pm/min and 34.6 pm/min, respectively, under ambient conditions. The high performance of the homebuilt STM was demonstrated by graphite hexagonal lattice images obtained in magnet fields ranging from 0 T to 27.5 T even without the protection of a vacuum and low temperatures. As far as known, this is the first STM that operates in a hybrid magnet. It is also the first STM that can obtain graphite hexagonal lattice images in magnetic fields up to 27.5 T. Our results greatly contribute to the further STM studies under ambient conditions and ultrahigh magnetic fields.

30 T scanning tunnelling microscope in a hybrid magnet with essentially non-metallic design.

We report on the construction and performance of the first hybrid resistive-superconducting magnet (HM) based scanning tunnelling microscope (STM) above 30 T. This custom-design HM-STM features a novel design of the STM head unit, whose tip-sample approach is implemented using a slender piezoelectric tube (PZT). The scanner shares part of PZT by fixing a sapphire frame onto the front quarter of PZT to construct a compact tip-sample loop, realising an outer diameter of 8.8 mm, which makes it compatible with a narrow sample space. Its main components are made of non-metallic materials of sapphire, which allows it to be immune from eddy currents and to operate under the condition of strong magnetic field fluctuation from a hybrid magnet, as well as cryogen-free cryocooler magnet systems. To analyse the stiffness of the STM head unit, the eigenfrequencies with 11 kHz and 12 kHz in bending modes, 25 kHz in a torsional mode, and 67 kHz in a longitudinal mode were simulated by finite element analysis; also, the drifting rates of the STM in ambient conditions in the X-Y plane and Z direction were measured at 25.5 and 38.2 pm/min, respectively. We present the first atomic images in magnetic fields up to 30.1 T in an HM. The raw data show the stable and distinguished performance while ramping up to maximum fields, indicating the new device's potential capability of operating in the future 45T-hybrid magnet and hundred-field pulsed magnet. Meanwhile, our compact and concentric cylindrical STM insert can operate in the low-temperature tubular sample space housed by the HM bore to develop low-temperature and extreme high-magnetic field STM.

Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway.

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

Lactosyl derivatives function in a rat model of severe burn shock by acting as antagonists against CD11b of integrin on leukocytes.

Severe burn shock remains an unsolved clinical problem with urgent needs to explore novel therapeutic approaches. In this study, the in vivo bioactivity of a series of synthetic lactosyl derivatives (oligosaccharides) was assessed on rats with burn shock to elucidate the underlying mechanisms. Administration of An-2 and Gu-4, two lactosyl derivatives with di- and tetravalent beta-D: -galactopyranosyl-(1-4)-beta-D: -glucopyranosyl ligands, significantly prolonged the survival time (P < 0.05 vs. saline), stabilized blood pressure and ameliorated the injuries to vital organs after burn. Flow chamber assay displayed that An-2 and Gu-4 markedly decreased the adhesion of leukocytes to microvessel endothelial cells. Competitive binding assay showed that a CD11b antibody significantly interrupted the interaction of An-2 and Gu-4 with leukocytes from rats with burn shock. With fluorescent microscopy, we further found that the oligosaccharides were selectively bound to leukocytes and with a colocalization of CD11b on the cell membrane. Interestingly, the lectin domain-deficient form of CD11b failed to bind with An-2 and Gu-4. The results suggest that both An-2 and Gu-4 significantly inhibit the adhesion of leukocytes to endothelial cells by binding to CD11b and thereby exert protective effects on severe burn shock.

[Role of deacetylase sirtuins in sepsis: beneficial or harmful?]

OBJECTIVE: Sepsis, life-threatening organ dysfunction caused by a dysregulated host response to infection, is a major public health concern. To date, the mechanism of sepsis is not completely understood, which is still a huge task ahead of numerous clinical and laboratory researchers. Recently, increasing evidences show that deacetylase sirtuins play an important role in sepsis and the function of sirtuins are varied in different stages of sepsis. More importantly, the mechanism of sirutins is not fully understood. The sirtuins family is composed by sirtuin 1-7 members. Among them, sirtuin 1 is widely reported. In addition to sirtuin 1, other members of sirtuins are also involved in the regulation of inflammation or metabolism signaling following sepsis. Of note, the sirtuins may interact with each other and form a precious control mechanism. Herein, we tried to summarize the recent paper from PubMed, to explain the possible mechanism of distinct role of sirtuin 1/2, to generalize the downstream effects of sirtuin 3 action, and to describe the interactions among sirtuins members on sepsis, which might be helpful for our future research and potential clinical applications.

Polydatin prevents the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria.

Polydatin is thought to protect mitochondria in different cell types in various diseases. Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury. To investigate the protective effect of polydatin after traumatic brain injury, a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults. Rat models were intraperitoneally injected with polydatin (30 mg/kg) or the SIRT1 activator SRT1720 (20 mg/kg, as a positive control to polydatin). At 6 hours post-traumatic brain injury insults, western blot assay was used to detect the expression of SIRT1, endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side. Flow cytometry was used to analyze neuronal mitochondrial superoxide, mitochondrial membrane potential and mitochondrial permeability transition pore opened. Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy. Our results showed that after treatment with polydatin, release of reactive oxygen species in neuronal mitochondria was markedly reduced; swelling of mitochondria was alleviated; mitochondrial membrane potential was maintained; mitochondrial permeability transition pore opened. Also endoplasmic reticulum stress related proteins were inhibited, including the activation of p-PERK, spliced XBP-1 and cleaved ATF6. SIRT1 expression and activity were increased; p38 phosphorylation and cleaved caspase-9/3 activation were inhibited. Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury. These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria. The mechanisms may be linked to increased SIRT1 expression and activity, which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway. This study was approved by the Animal Care and Use Committee of the Southern Medical University, China (approval number: L2016113) on January 1, 2016.

[Effect of limited volume resuscitation on hemodynamic changes in pregnant rabbit with hemorrhagic shock].

OBJECTIVE: To determine the effects of two fluid resuscitation strategies on the changes of hemodynamic variables, serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in a clinically relevant model of uncontrolled hemorrhagic shock in pregnant rabbits. METHODS: Hemorrhagic shock was induced by bleeding via carotied artery, followed by transection of a medium vessel in gestational sac. Experimental design consisted of three phases, shock phase (0-30 min), prehospital phase (30-90 min) and hospital phase (90-180 min). Twenty pregnant rabbits were randomly divided into two groups (n=10 /group), aggressive fluid resuscitation group (PNL group) and limited volume resuscitation group (PLH group). In the shock phase, animals were hemorrhaged by blood withdrawal to mean arterial pressure (MAP) of 40-45 mm Hg (1 mm Hg=0.133 kPa) via carotid artery. In the prehospital phase, a medium vessel in the gestational sac was transected, then the animals in the PNL group and PLH group were resuscitated with 0.9% normal saline (NS) and shed blood to MAP of 80, 60 mm Hg respectively. In the hospital phase, bleeding was controlled by surgical intervention and all the animals were reinfused with shed blood and NS to MAP 80 mm Hg. Hemodynamic variables and respiration rate were monitored and blood samples were collected for TNF-alpha and IL-6 measurement, and finally subsequent volume resuscitation and survival rate were recorded. RESULTS: (1) At 120 min, the respiration rate and heart rate in the animals assigned to PLH group was (66+/-16) bpm, (235+/-41) bpm respectively, which were significantly lower than those in PNL group (P<0.01), while MAP and central venous pressure in the PLH group was (80.4+/-7.2) mm Hg, (8.0+/-4.4) cm H2O, respectively, which were significantly higher than those in PNL group (P<0.01); (2) The serum concentration of TNF-alpha, IL-6 of all the animals were markedly increased after hemorrhagic shock, and peak at 24 min. The serum concentration of TNF-alpha, IL-6 in animals assigned to PLH group were (105+/-67) ng/L, (118+/-51) ng/L respectively, which were significantly lower than those in PNL group (P<0.01). The serum concentration of TNF-alpha, IL-6 in the animals assigned to PLH group were decreased to normal at 480 min; (3) The subsequent blood transfusion volume and NS resuscitation volume in PLH group in prehospital phase were (16.0+/-2.2) ml, (39.0+/-5.5) ml respectively, while those in hospital phase were (28.0+/-6.7) ml, (90.0+/-7.1) ml respectively, which were significantly lower than those in PNL group (P<0.05); (4) The 24 and 72 hours survival rate in the animals assigned to PLH group were 100%, 90% respectively; which were significantly higher than those in PNL group (P<0.01). CONCLUSION: Limited volume resuscitation improves thermodynamic changes of pregnant rabbit, attenuates the increase of serum concentration of TNF-alpha, IL-6, and results in higher survival rate. Limited volume resuscitation is an ideal means for hemorrhagic shock resuscitation in pregnant rabbit.

Signal Transduction in TNF-alpha-induced c-jun Gene Expression.

Our previous studies demonstrated that p38 mitogen-activated protein (MAP) kinase regulated the c-jun protein expression through phosphorylation of transcription factors of myocyte enhancer factors 2 (MEF2) family. There was a MEF2 binding site in the promoter of c-jun gene. Members of the MEF2 family of trans-cription factors bound as homo- and heterodimers to this MEF2 binding site. Here the potential role of the p38 and BMK1 MAP kinases in the regulation of c-jun expression induced by TNF-alpha was examined. It was shown that p38 MAP kinase up-regulated the transcription activity of MEF2A, while BMK1 MAP kinase up-regulated not only the transcription activity of MEF2A, but also MEF2D. The p38 and BMK1 MAP kinases had coordinated effect on the regulation of c-jun transcription. TNF-alpha induced the formation of MEF2A/MEF2D hete-rodimer. Over-expression of homodimer of MEF2 proteins inhibited c-jun transcription induced by TNF-alpha, while over-expression of heterodimer MEF2A/MEF2D enhanced c-jun transcription induced by TNF-alpha. Phosphorylation of MEF2A and MEF2D by p38 and BMK1 respectively appeared very important in TNF-alpha induced MEF2A/MEF2D heterodimer formation to enhance c-jun gene expression.

[Expression of intercellular adhesion molecule-1 in different organs of the mice with endotoxic shock induced by lipopolysaccharide].

To investigate and compare the expression of intercellular adhesion molecule-1 (ICAM-1) in different organs of the mice with endotoxic shock induced by lipopolysaccharide (LPS), protein and mRNA of ICAM-1 were measured by Western blotting and RT-PCR respectively in different organs of BALB/c mice administered intraperitoneally with 5 mg/kg LPS. The results showed that the constitutive expression of ICAM-1 protein and mRNA was the greatest in the lungs, followed by the spleen, kidney and intestine. After LPS stimulation, the upregulation of ICAM-1 was still greatest in the lungs, followed by the liver, spleen, heart, kidney and intestine. Compared with the normal mice, the expression of ICAM-1 protein in endotoxic shocked mice increased by 4.5-fold in the lungs, 3.0-fold in the kidney, 1.5-fold in the spleen; the expression in the liver and heart was negative under normal condition and changed into positive during endotoxic shock; but ICAM-1 expression in the intestine did not change significantly. The expression of ICAM-1 mRNA also increased consistently. These data highlight that LPS can up-regulate ICAM-1 protein and mRNA expression in different tissues of the mice with endotoxic shock. The difference in ICAM-1 expression among the organs may lead to different sensitivity of organ damage in endotoxic shock. This suggests that inhibition of ICAM-1 expression may be a useful principle for prevention and treatment of endotoxic shock.

The ATP-sensitive K(+) channel and membrane potential in the pathogenesis of vascular hyporeactivity in severe hemorrhagic shock.

OBJECTIVE: To elucidate the mechanism of vascular hyporeactivity following severe hemorrhagic shock (HS) by studying the changes of ATP-sensitive potassium channels' (K(ATP)) properties and membrane potential of mesenteric arteriolar smooth muscle cells. METHODS: Single channel currents were studied on cell-attached and inside-out patches of enzymatically isolated mesenteric arteriolar smooth muscle cells (ASMCs). Membrane potentials of arteriolar strips and ASMCs were recorded by intracellular membrane potential recording method and confocal microscopy, respectively. RESULTS: K(ATP) channels in ASMCs were activated, which induced smooth muscle hyperpolarization following vascular hyporeactivity in HS. CONCLUSIONS: Hyperpolarizing effect of K(ATP) channel activation plays an important role in low vasoreactivity during severe hemorrhagic shock.

Effects of lipopolysaccharide on actin reorganization and actin pools in endothelial cells.

OBJECTIVE: To investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304. METHODS: F-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry. RESULTS: F-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool. CONCLUSIONS: LPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.

[Construction and expression of red fluorescent protein reporter gene vector containing human eNOS promoter].

OBJECTIVE: To construct the red fluorescent protein reporter gene vector containing human eNOS promoter sequence to study the mechanism of regulating the expression of eNOS gene by vessel wall shear stress. METHODS: The genomic DNA of endothelial cells from fetal umbilical vein was drawn. The gene sequence of eNOS promoter gene therein was cloned by PCR technique and constructed into the red fluorescent protein vector, pDsRed-1. The recombinant vector, pDseNOSRed was then transfected into 293 cells, human fetal renal epithelial cells. Blank vector, pDsRed-1, was transfected into 293 cells as controls. The expression and distribution of the reporter gene were observed by fluorescent microscopy. RESULTS: PCR and double restriction enzyme digestion showed that the recombinant vector, pDseNOSRed, was constructed correctly. This vector was highly expressed in the 293 cells. Expression of red fluorescence, evenly distributed in whole cells, occurred since 12 hours after transfection, reached the peak concentration 3648 h after transfection, and dissappeared almost completely 120 h after. No red fluorescence was observed in the control cells. CONCLUSION: A red fluorescent protein reporter gene vector containing human eNOS promoter sequence and expressed highly in mammalian cells has been constructed successfully, thus providing an important and convenient tool to study the mechanism mechanism of regulating the expression of eNOS gene by vessel wall shear stress.

Effect of SOD and NaHCO3 on the vascular hyporeactivity of rats after severe hemorrhagic shock.

OBJECTIVE: To investigate the effect of SOD and NaHCO3 on the vascular hyporeactivity after severe hemorrhagic shock in rats. METHODS: Twenty eight Sprague-Dawley rats were divided randomly into 4 groups of 7 each: SOD-treated group, NaHCO3-treated group, SOD+NaHCO3-treated group and normal saline-treated control group. The responses of arterioles to norepinephrine (NE), the effects of dopamine on blood pressure, blood flow velocity in arterioles, the mean arterial pressure (MAP), and the survival time within 24 h were measured. RESULTS: Two hours after shock, the arteriolar vasoreactivity was significantly reduced and threshold concentration of NE was increased by 24 to 27 folds. After beting treated separately with SOD, NaHCO3 and SOD+NaHCO3, arteriolar vasoreactivity was restored to some extent, among which the SOD+NaHCO3 group obtained a most significant effect with decreased threshold concentration of NE to 21% of its normal level. In SOD+NaHCO3-treated group, injection of dopamine indicated a most significant effect on MAP with 1.9 times more than that in control group, and the enhanced MAP was maintained at a level of more than 13.33 kPa after reinfusion of shed blood. The blood flow in arteriole was 2.54-fold higher than that in control group after 2 h resuscitation. The average survival time in SOD+NaHCO3- treated group was 2.9 times longer than that in control group. CONCLUSIONS: SOD and NaHCO3 could both recover the lower vasoreactivity and increase the enhancing effect of dopamine on blood pressure of rats in severe hemorrhagic shock, suggesting that coadministration of SOD and NaHCO3 could be a new approach to the treatment of severe hemorrhagic shock.

[Properties of large-conductance calcium-activated potassium channel in rat mesenteric arteriole smooth muscle cells].

OBJECTIVE: To investigate the electrophysiological properties of large-conductance calcium-activated potassium channel (BKCa) in mesenteric arteriole smooth muscle cells of rat. METHOD: Mesenteric arteriolar smooth muscle cells from rats were isolated and inside-out patch clamp technique was used to study BKCa. RESULTS: The single channel conductance of BKCa recorded was 221+/-6 pS and the reversal potential was -0.12 mV in the presence of high potassium in the bathing and pipette solutions. The open probability (NPo) of the channel was both voltage- and intracellular calcium dependent, whereas the amplitude of the channel was not calcium-dependent. Prolonged closed state could be observed at intracellular calcium concentrations of 1 micromol/L or above, known as calcium-dependent inactivation. CONCLUSION: BKCa in rat mesenteric arterial smooth muscle cells has the properties of large conductance, high potassium selectivity, steep voltage dependence and high calcium sensitivity. The high sensitivity to calcium of BKCa may be related to the compensatory regulation of the smooth muscles in pathophysiological conditions.