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.

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.

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.

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.

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.

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.

[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.

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.

Local thermal injury induces general endothelial cell contraction through p38 MAP kinase activation.

Endothelial cells (ECs) of thin-walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin-dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight-junction damage. Inhibition of the p38 mitogen-activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress-fiber formation, contraction, volume changes and tight-junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.

MK2 plays an important role for the increased vascular permeability that follows thermal injury.

We previously reported Rho kinase is involved in vessel hyper-permeability caused by burns. Here we further explore the Rho kinase downstream signaling, it is found that its specific inhibitor Y27632 significantly diminishes the activation of JNK and p38 MAPKs but not ERK that induced by serum from burned rats (burn-serum). JNK activation was found involved in the expression of HUVEC adhesion molecules following thermal injury, although not in the process of stress fiber formation. Inhibition of various MAPKs by specific inhibitors showed that SB203580 (inhibitor of p38), but neither SP600125 (inhibitor of JNK) nor PD98059 (inhibitor of ERK), abolish activation of the p38 downstream kinase MK2. Demonstration of stress fibers by fluorescent-labeled phalloidin showed that inhibition of MK2, either by its specific inhibitor or by dominant negative adeno-viral-carried constructs, significantly reduced burn-serum-induced HUVEC stress-fiber formation, while inhibition of another downstream p38 MAPK kinase, PRAK, had no such effects. Transfection of dominant negative adeno-viral MK2 (Ad-MK2(A)) significantly inhibited thermal injury-induced blood vessel hyper-permeability in rats and, moreover, prolonged the survival of burned rats beyond 72 h following thermal injury. One of the mechanisms behind these phenomena is that Ad-MK2(A) causes a significant depression of burn-serum-induced HSP27-phosphorylation, while the adeno-viral transported dominant negative PRAK (Ad-PRAK(A)) does not block. Although the effect of blockade of MK2 through its adeno-viral approach requires further study and investigation of alternatives to know for sure, we may have found a new pathway behind thermal-injury-induced blood vessel hyper-permeability, namely: Rho kinase>p38>MK2>HSP27.

Roles of mitogen-activated protein kinases in the modulation of endothelial cell function following thermal injury.

Several mitogen-activated protein kinases (MAPKs) are activated during thermal injury, and the p38 MAPK is specifically involved in endothelial cell (EC) actin and myosin rearrangement (stress-fiber formation) with ensuing cellular contraction and enhanced vessel permeability. Inhibition of p38 MAPK and extracellular signal-related kinase MAPK by their inhibitors SB203580 and PD98059, respectively, significantly reduces burn serum-induced EC stress-fiber formation, whereas SB203580 also inhibits burn serum-induced EC tight-junction damage and thereby general blood vessel hyperpermeability. The JNK MAPK inhibitor, SP600125, on the contrary, influences neither stress-fiber formation nor EC tight-junction damage. Extracellular signal-related kinase MAPK inhibition significantly decreases burn serum-induced Monocyte chemotactic protein-1 (MCP-1) release, whereas SB203580 and SP600125 have only limited such effects. Western blotting, real-time reverse transcriptase-polymerase chain reaction, and confocal laser scanning microscopy proved that SP600125 significantly inhibits burn serum-induced intercellular adhesion molecule 1 expression, whereas SB203580 depresses the expression of P selectin. In vivo studies, using the dominant negative adenoviral approach of MAPK kinase 3b and MAPK kinase 6b to block p38 MAPKs, and MKK4 and MKK7 to block JNK MAPKs, show that the latter MAPKs are involved in the regulation of P selectin and intercellular adhesion molecule 1 expression, respectively, following thermal injury. Taken together, the results suggest that several MAPKs play important, although different, roles in general EC alterations following burn injuries.

[Alteration in intestinal epithelial permeability and its role in the pathogenesis of burn shock].

The intestinal epithelial barrier serves a dual role: to keep harmful external agents out of the body and to allow beneficial nutrients to enter the body. Tight junction (TJ) is of crucial importance for the barrier function. Over the past 15 years, some of the molecular events underlying the epithelial barrier regulation have been described. This forum introduces briefly the molecular structure of TJ and its regulation in gut barrier. It was shown that gut barrier function was impaired as early as 5 minutes post burn and became worst by 4 hours. In this forum the mechanism of gut barrier injury in burns is described, and it includes 4 aspects: the phosphorylation of TJ protein and perijunctional actin-myosin ring, the reduction of TJ proteins expression, the endocytosis of TJ proteins, and the apoptosis and necrosis of the epithelial cells. It is well known that the increase in gut permeability promotes bacterial translocation in burns. Moreover, a new auto-digestion theory of gut in shock and MODS was recently raised. Therefore, protection against gut barrier damage has again been recognized as a therapeutic target in shock and MODS treatment.

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.

[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.

Downregulation of human endothelial nitric oxide synthase promoter activity by p38 mitogen-activated protein kinase activation.

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. eNOS gene expression may be upregulated by a signaling pathway, including PI-3Kgamma--> Jak2--> MEK1 --> ERK1/2--> PP2A. It remains unclear whether other mitogen-activated protein kinase (MAPK) family members, such as JNK, p38 kinase, and ERK5/BMK1, also modulate eNOS gene expression. Our purpose, therefore, is to shed light on the effect of the p38 MAPK signaling pathway on the regulation of eNOS promoter activity. The results showed that a red fluorescent protein reporter gene vector containing the full length of the human eNOS promoter was first successfully constructed, expressing efficiently in ECV304 cells with the characteristics of real time observation. The wild-types of p38alpha, p38beta, p38gamma, and p38delta signal molecules all markedly downregulated promoter activity, which could be reversed by their negative mutants, including p38alpha (AF), p38beta (AF), p38gamma (AF), and p38delta (AF). Promoter activity was also significantly downregulated by MKK6b (E), an active mutant of an upstream kinase of p38 MAPK. The reduction in promoter activity by p38 MAPK could be blocked by treatment with a p38 MAPK specific inhibitor, SB203580. Moreover, the activation of endogenous p38 MAPK induced by lipopolysaccharide resulted in a prominent reduction in promoter activity. These findings strongly suggest that the activation of the p38 MAPK signaling pathway may be implicated in the downregulation of human eNOS promoter activity.

[Effect of Bay K 8644 on arteriole smooth muscle cell membrane potential in rats with severe hemorrhagic shock].

OBJECTIVE: To investigate the effect of Ca(2+) influx through L-type Ca(2+) channels on normal and hyperpolarized membrane potential of arteriole smooth muscle cells (ASMCs) in rats. METHODS: The ASMCs isolated from normal rats and those with severe hemorrhagic shock were labeled with DiBAC4 (3) for membrane potential detection. RESULTS: Ca(2+) influx caused hyperpolarization of the membrane potential in the normal ASMCs but depolarization in the cells from rats with hemorrhagic shock, and this effect could be inhibited by TEA. CONCLUSION: Ca(2+)-activated potassium channels activated by Ca(2+) influx through L-type Ca(2+) channels in normal ASMCs to cause hyperpolarization but leads directly to membrane potential depolarization in ASMCs from rats with severe hemorrhagic shock. This finding can be meaningful for treatment of vascular hyporeactivity in advanced stage of severe shock.

Role of AP1 element in the activation of human eNOS promoter by lysophosphatidylcholine.

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. It, therefore, is very essential to elucidate the regulation of it. In the current study, a red fluorescent protein (RFP) reporter system containing human eNOS promoter was first constructed, being characteristics of real time morphologic and quantitative analysis for the same sample. It was observed by DNA sequence deletion that 68% of the basal activity of the promoter was controlled by the region from -1 to -166 bp, and 32% of it was dependent on the region from -1,033 to -1,600 bp. The mutation of SSRE element (-999 to -994 bp) and wild-type SSRE decoy oligodeoxynucleotides (ODN) did not alter the basal activity and the stimulating activity by lysophosphatidylcholine (LPC). The mutation of upstream AP1 element (-1,530 to -1,524 bp) did not affect the basal activity, but resulted in near 30% reduction in the stimulating activity by LPC. Moreover, wild-type AP1 decoy ODN also remarkably attenuated it. It was proved by EMSA analysis that LPC indeed enhanced the activity of AP1 transcriptional factor binding to AP1 element. However, the role of AP1 was dependent on the presence of SP1, which was proved by the combining mutation of AP1 with SP1. The mutation of downstream AP1 element (-662 to -656 bp) had no influence on the basal and stimulating activities by LPC. These results strongly suggest that the main functional region of the promoter is from -1 bp to -166 bp, that the upstream AP1 participates in the activation of the promoter by LPC on the premise of the presence of SP1, and that the downstream AP1 and SSRE do not involve the basal and stimulating activity by LPC.