Dual-function reporter protein for analysis of gene expression in living cells

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.     

A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q

OBJECTIVE: To determine the effect of hemorrhagic shock on spontaneous and endotoxin-induced cytokine release from intestinal mononuclear cells compared with whole portal venous blood and splenic macrophages. DESIGN: Random assignment to either unmanipulated control group, sham operation group, or hemorrhagic shock group. SETTING: University animal laboratory. SUBJECTS: Male Wistar rats, weighing between 300 and 350 g. INTERVENTIONS: Rats were bled to 30 mm Hg for 30 mins by withdrawal/reinfusion of shed blood and Ringer's lactate equivalent to the shed blood volume. MEASUREMENTS: Rats were killed immediately, 4 hrs, or 24 hrs after reperfusion. Portal venous blood, splenic macrophages, and small bowel mononuclear cells were obtained and spontaneous (unstimulated) and endotoxin-induced supernatant tumor necrosis factor (TNF)-alpha (WEHI 164 subclone 13) and interleukin (IL)-6 (B 13-29 clone 9) release was measured by bioassay. MAIN RESULTS: Increased endotoxin-induced TNF release from gut mononuclear cells and portal venous blood was suppressed 4 and 24 hrs after reperfusion compared with sham operated animals (p < .05). TNF release from splenic macrophages could be significantly increased (p < .05) by addition of endotoxin in all groups with no difference between control, sham, and shock animals. In shock animals, endotoxin-stimulated IL-6 release was significantly greater (p < .05) than control and sham operated rats 4 hrs after reperfusion in portal blood and from splenic macrophages. In contrast to splenic macrophages, gut mononuclear cells and portal venous blood demonstrated high spontaneous IL-6 concentrations without further stimulation by endotoxin. CONCLUSIONS: Hemorrhagic shock induced a hyporesponsiveness from gut mononuclear cells and whole portal venous blood 4 and 24 hrs after reperfusion. Spontaneous and endotoxin-induced stimulation of IL-6 indicates a different modulation of cytokines in gut, portal vein and spleen. The differences of gut mononuclear cells and portal blood compared with the splenic macrophages indicate a compartmentalized cytokine response.     

数字灰尘撞击传统网络--集成于一立方毫米组件上可随意使用的传感器和通信系统

由于小型化、集成度和电源处理等方面技术的提高,完全不使用金属导线的传感器/通信系统可以集成于体积只有一个沙粒大小的组件或网络中。它的应用远到军用传感器网络,近到工业质量控制。 被称之为“聪明灰尘计划”的研究目标是探索独立传感器节点的小型化和能耗的最低界限。该设备可被用来监控气象图谱或在运货前设置一个谷物食品盒,使得仓库管理员可以测定该产品在运输途中是否暴露于风雨之中而有潮气。这些细小聪明的节点或尘埃存在的可能,是因为近期集成了的微电机系统、无线电通信和数字电路。由(美)国防高级研究计划局(阿灵顿、弗吉尼亚)资助的该研究项目正由加州伯克利大学的研究员们进行着。     

A fluid analysis of a utility-based wireless scheduling policy

In this paper, we consider packet scheduling for the downlink in a wireless network, where each packet's service preferences are captured by a utility function that depends on the total delay incurred. The goal is to schedule packet transmissions to maximize the total utility. In this setting, we examine a simple gradient-based scheduling algorithm called the U/spl dot/R-rule, which is a type of generalized c/spl mu/-rule (Gc/spl mu/) that takes into account both a user's channel condition and derived utility when making scheduling decisions. We study the performance of this scheduling rule for a draining problem, where there is a given set of initial packets and no further arrivals. We formulate a "large system" fluid model for this draining problem where the number of packets becomes large while the packet-size decreases to zero, and give a complete characterization of the behavior of the U/spl dot/R scheduling rule in this limiting regime. Comparison with simulation results show that the fluid limit accurately predicts the corresponding behavior of finite systems of interest. We then give an optimal control formulation for finding the optimal scheduling policy for the fluid draining model. Using Pontryagin's minimum principle, we show that, when the user rates are chosen from a TDM-type of capacity region, the U/spl dot/R rule is in fact optimal in many cases. Sufficient conditions for optimality are also given. Finally, we consider a general capacity region and show that the U/spl dot/R rule is optimal only in special cases.     

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.     

Hygrothermal aging studies of short carbon fiber reinforced nylon 6.6

A study of hygrothermal aging, in terms of the kinetics of moisture absorption by nylon 6.6 and its carbon fiber-reinforced composites, has been undertaken. The diffusion of water into the various materials was investigated at 100% relative humidity, by immersion of specimens in water at temperatures of between 25 and 100°C. A mathematical treatment used in analyzing the data was that of a single free phase model of diffusion, which assumed Fickian diffusion and utilized Fick's second law of diffusion. Good agreement was observed between the experimental and theoretical values. The equilibrium moisture content and the apparent diffusion coefficient of the various composites were evaluated. Hygrothermal aging has reduced the tensile properties of both unreinforced and reinforced nylon 6.6, albeit a better recoverability of the properties was achieved by the former after drying. This behavior is explained in terms of the combined action of moisture-induced plasticization and interfacial degradation. © 1994 John Wiley & Sons, Inc.     

Outcome of subarachnoid haemorrhage. An analysis of surgical variables, cognitive and emotional sequelae related to SPECT scanning

A group of 48 patients, consecutive apart from exclusions, from a 1-year series of 60 cases of aneurysmal subarachnoid haemorrhage (SAH), was reviewed, in respect of clinical and radiological features, surgical management, clinical outcome, psychological distress and psychometric status, the neuropsychological assessment being compared with a closely-matched group of controls, the postoperative assessment being accompanied by a single positron emission computed tomogram (SPECT) scan. A review of those features which might have been expected to have a bearing on cognitive outcome (CT abnormalities at the outset, angiographic vasospasm, operative aneurysmal leakage, temporary vessel occlusion) failed to show a significant difference on cognitive tests. There was, however, a cognitive deficit shown by the patient group as a whole, when compared with the controls. Thus, SAH itself, the initial insult, would appear to be the essential factor in the production of persistent cognitive deficits.     

RES, an expert system for the set-up and interpretation of a ruggedness test in HPLC method validation: Part 1: The ruggedness test in HPLC method validation

In method validation, an intralaboratory repeatability study and an interlaboratory reproducibility study can be performed as part of a precision test. In HPLC, an intralaboratory ruggedness test can be performed to detect problems that would otherwise be encountered in a reproducibility study. In a ruggedness test, variations in ambient factors that are expected to occur in practice, are simulated. Several steps determine the success of a ruggedness test. The complexity and lack of standard procedures for some of these steps is the main reason why ruggedness testing is still not widely accepted.