Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.     

Prevalence of extracranial carotid and vertebral artery disease in Chinese patients with coronary artery disease

Electrotonic responses recorded extra- or intracellularly from peripheral nerve preparations show a "sag" to hyperpolarizing current pulses. The biophysical nature of this "inward rectification" is still under discussion since the phenomenon has not been noted at voltage-clamped single nerve fibres, and since Cs+, which reduces inward rectification, is not a specific ion channel blocker. In this study, we found that low micromolar concentrations of ZD 7288, a specific blocker of the hyperpolarization-activated cationic current (Ih) in the soma of central mammalian neurons, result in a complete block of inward rectification in the electrotonic responses of isolated rat spinal dorsal roots. In addition, ZD 7288 enhanced the activity-dependent slowing of conduction seen in compound C fibre action potentials of isolated rat vagus nerves and augmented the post-tetanic hyperpolarization following trains of action potentials in unmyelinated and myelinated axons. The data suggest that ZD 7288 is a potent blocker and a useful research tool for the study of hyperpolarization-activated inward rectification (Ih) of peripheral nerve preparations.     

FBC中含S和Cl煤燃烧下的碳钢退化研究

在实验室规模的流化床(FBC)中分别燃烧低S(097 m ass％）与低Cl(0026 mass％)，中S(168 ss％）与高Cl(042 mass％)，高S(448 mass％）与高Cl(041 mass％)三种煤各1000小时后，对放置于接近沸腾床位置的 A210-C碳钢管的退化情况进行了研究.结果表明，(1)碳钢退化明显受温度影响，随表面温度的升高显著加快；(2)碳钢退化明显受飞行粒子带来的冲蚀影响；（3)燃烧低S低Cl煤对碳钢的耗损影响最小，而燃烧高S高Cl煤对碳钢耗损影响最大.同时根据对腐蚀产物的观测分析，对不同煤种燃烧产生的环境中碳钢的退化机制进行探讨.     

Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells

A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.     

Glucosylation of glycosylphosphatidylinositol membrane anchors: identification of uridine diphosphate-glucose as the direct donor for side chain modification in Toxoplasma gondii using carbohydrate analogues

Toxoplasma gondii is an obligate intracellular parasite of the phylum apicomplexa and a common and often life-threatening opportunistic infection associated with AIDS. A family of parasite-specific glycosylphosphatidylinositols containing a novel glucosylated side chain has been shown to be highly immunogenic in humans (Striepen et al. (1997) J. Mol. Biol. 266, 797-813). In contrast to trypanosomes in T. gondii side chain modification takes place before addition to protein in the endoplasmic reticulum. The biosynthesis of these modifications was studied in an in vitro system prepared from hypotonically lysed T. gondii parasites. Radiolabeled glucose-containing glycosylphosphatidylinositol precursors were synthesized by T. gondii membrane preparations upon incubation with uridine diphosphate-[3H]glucose. Synthesis of glucosylated glycolipids took place only in the presence of exogenous uridine diphosphate-glucose and was stimulated by unlabeled uridine diphosphate-glucose in a dose-dependent manner. In contrast to glycosylphosphatidylinositol mannosylation, glucosylation was shown to be insensitive to amphomycin treatment. In addition, the glucose analogue 2-deoxy-D-glucose was used to trace the glycosylphosphatidylinositol glucosylation pathway. Detailed analysis of glycolipids synthesized in vitro in the presence of UDP and GDP derivatives of D-glucose and 2-deoxy-D-glucose ruled out an involvement of dolichol phosphate-glucose and demonstrates direct transfer of glucose from uridine diphosphate-glucose.     

Improved CD4 lymphocyte outgrowth in response to effective antiretroviral therapy

PURPOSE: Embolism is believed to be the major cause of end-organ damage after angioplasty and endoluminal procedures. Recently, Doppler ultrasound scanning has been used to detect asymptomatic cerebral emboli. We determined whether asymptomatic embolic signals (ES) could be detected distal to a significant iliac artery stenosis of >60% both before and soon after iliac percutaneous transluminal angioplasty (PTA). METHODS: A 2-MHz Doppler scan probe was used to monitor for ES in the common femoral artery before and after 10 successful iliac artery PTAs and at various standardized times in the following 24 hours. The same protocol was used to study 10 patients in the control group after renal PTA. In addition, a single recording was performed in a second nonoperative control group of 10 patients who had no evidence of peripheral vascular disease. The Doppler scan signals were recorded on tape for a later blinded analysis. RESULTS: In the 24 hours before iliac PTA, asymptomatic ES were detected in four of 10 patients during a 1-hour recording but in no controls (P =.025). After iliac PTA, ES were detected at 30 minutes in nine of 10 iliac subjects but in only one of 10 renal subjects (P =.0003) and at 2 hours in eight of 10 iliac subjects but in only one of 10 renal subjects (P =.001). The occurrence of ES became less frequent, and ES were present at a lower frequency in eight of 10 iliac PTA subjects at 4 hours and in five of 10 at 24 hours but in no renal PTA subjects at these time points. CONCLUSIONS: ES can be detected in the common femoral artery with Doppler ultrasound scanning in patients with iliac artery stenosis both before and soon after iliac PTA despite preangioplasty aspirin and intra-angioplasty heparin therapies. The occurrences of ES were particularly frequent in the 2 hours after PTA. This technique can be used further to study factors that control plaque stability and to evaluate the effect of therapeutic interventions.     

The use of the flow-volume loop in assessing the results of laryngotracheal reconstruction

Wild fungal strains isolated from litter and soil were inoculated in test tubes containing a synthetic medium with 0.5% sodium carboxymethylcellulose (CMC) as sole carbon source. After the incubation time, cylindrical probes were taken from each tube using a borer and stained with Congo red 0.1% to reveal the remaining CMC. The relative cellulase activity was estimated by measuring the deepness of the clearing zone, and the growth by the limit of mycelial penetration in the medium. The ratio: hydrolysis zone/depth of growth, provides additional information to compare strains. A test using culture supernatants of liquid media growing strains was developed for enzyme assay using a modification of the cylinder-plate method. Petri dishes were filled with agar-CMC medium and cups were cut off with a steel punch. Such cups were filled with culture supernatants. After incubation, the plated were stained with Congo red, and the diameter of each cleared zone was recorded. Comparative studies of strains could be performed as well as quantitation of enzyme activity using a standard cellulase solution and computing the area of the cleared zones. This method may be useful when a large number of strains must be tested for cellulolytic activity or when conventional tests fail for assaying enzymatic induction in vitro.     

Histone composition of nucleosomes isolated from cultured Chinese hamster cells

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.     

Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.     

A prospective randomized trial of the surgical blood order equation for ordering red cells for total hip arthroplasty patients

BACKGROUND: The majority of crossmatched blood is for surgical patients, and most of it is never transfused. An alternative system for ordering red cell (RBC) units, called the surgical blood order equation (SBOE), which incorporates specific patient variables for surgical patients, has been developed. STUDY DESIGN AND METHODS: A prospective double-blind randomized trial compared the SBOE with the maximal surgical blood order schedule (MSBOS) system for ordering allogeneic RBC units in 60 patients undergoing total hip arthroplasty. Autologous RBCs were available for none of the patients. RESULTS: There were no differences in patient demographic, surgical, or laboratory variables at any time. The median number (range) of allogeneic RBC units ordered was 2 (2-3) for the MSBOS and 0 (0-3) for the SBOE (p<0.0001). The SBOE ordered the correct number of RBC units for 58 percent of patients, while the MSBOS did so for 7 percent (p<0.0001). The SBOE had a lower crossmatch-to-transfusion ratio than the MSBOS (0.83 vs. 4.12). Costs were also lower with the SBOE. CONCLUSION: Incorporation of patient factors in the use of the SBOE system resulted in increased efficiency of blood-ordering practices for total hip arthroplasty.