Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Concerted loss of cyclooxygenase and peroxidase activities from prostaglandin H synthase upon proteolytic attack

Prostaglandin H synthase has two distinct enzymatic activities: a cyclooxygenase that forms PGG2 from arachidonate and a peroxidase that can reduce hydroperoxides, such as PGG2, to the corresponding alcohols. The relative sensitivities of the two synthase activities to proteolytic attack have been examined, using trypsin, chymotrypsin, and proteinase K, all known to attack the native apoprotein in the arg 253 region. The relation between the specific activity of the synthase and the loss of the two activities and the cleavage of the synthase subunit during trypsin digestion was also examined. The cyclooxygenase and peroxidase activities declined in concert throughout room temperature digestions with each of the three proteases. There was no indication of a selective loss of either activity in any of the digestions. In separate digestions with the same preparation of synthase, 3.3% (w/w) proteinase K resulted in more extensive loss of activity (90% decrease after 90 min) than did 3% (w/w) trypsin (70% decrease after 120 min) or 5% (w/w) chymotrypsin (60% decrease after 135 min). In tryptic digestions of synthase preparations with cyclooxygenase specific activity between 16 and 125 k units/mg protein, the fractional loss of cyclooxygenase activity was, within experimental error, the same as that of peroxidase activity. The extent of cleavage of the 70 kDa synthase subunit was greater than the loss of enzymatic activity, with the discrepancy being larger for synthase preparations with lower specific activity. The presence of a variable amount of catalytically-inactive, protease-sensitive, synthase protein could account for the difference between surviving activity and intact subunit in six out of the seven synthase preparations examined. Thus, it is likely that the cyclooxygenase and peroxidase activities are destroyed together during proteolytic attack on the arg 253 region of the native synthase apoprotein.     

Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid

Prostaglandin H synthase catalyzes the formation of prostaglandin (PG) G2 from arachidonic acid (cyclooxygenase activity), and also the reduction of PGG2 to PGH2 (peroxidase activity). The ability of the pure synthase to accumulate the hydroperoxide, PGG2, under conditions allowing the concurrent function of both catalytic activities was investigated. The peroxidase velocity was continuously determined from the absorbance increases at 611 nm that accompanied oxidation of a peroxidase cosubstrate, N,N,N',N'-tetramethylphenylenediamine, and PGG2 concentrations were calculated from the peroxidase velocities and the peroxidase Vmax and Km values. Cyclooxygenase velocities were then calculated from the changes in PGG2. Parallel reactions monitored by the use of radiolabelled arachidonate or with a polarographic oxygen electrode were used to confirm the calculated PGG2 levels and the cyclooxygenase velocities. The concentration of PGG2 was found to follow a transient course as the reaction of the synthase progressed, rapidly rising to a maximum of 0.7 microM in the first 10 s, and then declining slowly, reaching 0.1 microM after 60 s. The maximal level of PGG2 achieved during the reaction was constant at about 0.7 microM with higher amounts of added cyclooxygenase capacity (0.3-0.6 microM PGG2/s) but was only about 0.4 microM when the added cyclooxygenase capacity was 0.1 microM PGG2/s. The peroxidase was found to lose only 30% of its activity after 90 s, a point where the cyclooxygenase was almost completely inactive. These results support the concept of a burst of catalytic action from the cyclooxygenase and a reactive, more sustained, catalytic action from the peroxidase during the reaction of the synthase with arachidonic acid.     

Echocardiographic assessment and percutaneous closure of multiple atrial septal defects

Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction. We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization. The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection.     

Attachment of substrate metabolite to prostaglandin H synthase upon reaction with arachidonic acid

Prostaglandin H synthase was incubated with [14C]arachidonate and then analyzed by polyacrylamide gel electrophoresis under denaturing conditions and by high pressure liquid chromatography. A maximum of 1 mol of arachidonate metabolite was found to become attached per mol of synthase subunit in a time-dependent process that was much slower than the rate of self-catalyzed inactivation of the cyclooxygenase activity. Incubation of a mixture of the synthase and ovalbumin with [14C]arachidonate resulted in a selective attachment of radiolabel to the synthase. These results suggest the presence of a single site on the synthase that is susceptible to reaction with an arachidonate metabolite.     

Patterns of inflammation and the use of reversibility testing in smokers with airway complaints

Background

Although both smoking and respiratory complaints are very common, tools to improve diagnostic accuracy are scarce in primary care. This study aimed to reveal what inflammatory patterns prevail in clinically established diagnosis groups, and what factors are associated with eosinophilia.

Method

Induced sputum and blood plasma of 59 primary care patients with COPD (n = 17), asthma (n = 11), chronic bronchitis (CB, n = 14) and smokers with no respiratory complaints ('healthy smokers', n = 17) were collected, as well as lung function, smoking history and clinical work-up. Patterns of inflammatory markers per clinical diagnosis and factors associated with eosinophilia were analyzed by multiple regression analyses, the differences expressed in odds ratios (OR) with 95% confidence intervals.

Results

Multivariately, COPD was significantly associated with raised plasma-LBP (OR 1.2 [1.04–1.37]) and sTNF-R55 in sputum (OR 1.01 [1.001–1.01]), while HS expressed significantly lowered plasma-LBP (OR 0.8 [0.72–0.95]). Asthma was characterized by higher sputum eosinophilic counts (OR 1.3 [1.05–1.54]), while CB showed a significantly higher proportion of sputum lymphocytic counts (OR 1.5 [1.12–1.9]). Sputum eosinophilia was significantly associated with reversibility after adjusting for smoking, lung function, age, gender and allergy.

Conclusion

Patterns of inflammatory markers in a panel of blood plasma and sputum cells and mediators were discernable in clinical diagnosis groups of respiratory disease. COPD and so-called healthy smokers showed consistent opposite associations with plasma LBP, while chronic bronchitics showed relatively predominant lymphocytic inflammation compared to other diagnosis groups. Only sputum eosinophilia remained significantly associated with reversibility across the spectrum of respiratory disease in smokers with airway complaints.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.