Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.     

Word completion in chronic pain: evidence for schematic representation of pain?

Schematic representation of pain information was investigated in chronic pain patients, health professionals, and nonpatient controls. Under the guise of an English-language experiment, Ss were presented with 12 word stems to be completed with the first 2 English words that came to mind. Four of the stems could be completed with sensory pain words, 4 with effective, and 4 with words associated with pain or illness. All could be completed with at least 3 other nonpain words of equal or greater frequency. Results indicate that chronic pain Ss produced significantly more pain-related completions than control Ss and that in all 3 groups the types of pain words produced were related to the extent of personal experience of pain. The theoretical implications of these findings are discussed in relation to the organization of schema, implicit memory, and the activation of mental representations of pain (schema).     

Quantification of device adherent, circulating, and organ pool of thrombin and fibrinogen after cardiopulmonary bypass in a pig model

The pool of thrombin and fibrinogen in circulation, in organs, and on cardiopulmonary bypass devices was quantified during and after cardiopulmonary bypass in four groups of 24 Yorkshire pigs (weight, 30-35 kg); two groups of 10 unoperated pigs were used as controls. Thrombin-alpha and fibrinogen were iodinated with 125iodide using an iodogen transfer technique; 250-300 microCi of these tracers were injected intravenously 1 hr before cardiopulmonary bypass. All pigs were systematically heparinized (activated clotting time > 400 sec); cardiopulmonary bypass was performed at 2.5-3.5 L/min at 28 degrees C using a centrifugal pump, oxygenator (Bentley Univox 1.8 m2; Bentley Inc., Irvine, CA), arterial filter (0.25 m2), and cardiotomy reservoir (BMR 3500) for 90 min, followed by a 90 min reperfusion and 180 min of cardiopulmonary bypass. Iodinated thrombin-alpha and fibrinogen in intact organs and samples of blood, organs, tissues, and oxygenator-arterial filter-cardiotomy reservoir were quantified with an ion chamber and a gamma counter, respectively. The percent of injected iodinated thrombin-alpha and fibrinogen dose (mean +/- SD) in organs and cardiopulmonary bypass devices of all groups of cardiopulmonary bypass pigs was calculated. Thrombin generated at the small area of surgical wounds (0.016-0.038 m2), and fibrin deposited on surfaces of cardiopulmonary bypass devices (2.59 m2), initiate and propagate thrombus formation and embolization. The protein level reached saturation values on all cardiopulmonary bypass devices at 180 min. High levels of thrombin and fibrinogen-fibrin circulate in blood and organs, and are adsorbed on cardiopulmonary bypass devices; this large blood pool of pro-coagulants in the cardiac cradle, tissues, and perfused organs may account for thrombi and emboli during and after cardiopulmonary bypass.     

Portal hypotensive effects of DL-028 and prazosin on portal hypertensive rats

The portal hypotensive effects of prazosin and DL-028 (chem- ical name: 3-[[4-(2-methoxyphenyl)piperazin-1-yl]methyl]- 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-one(27b)), a synthetic alpha1-adrenoceptor antagonist, were assessed in portal hypertensive rats. Portal hypertension was induced by partial portal vein ligation in Sprague-Dawley rats. Two weeks after ligation, when the hyperdynamic state was stabilized the rats were anesthetized after an overnight fast and cannulated for measuring mean arterial pressure (MAP), portal venous pressure (PVP), cardiac index (CI) and heart rate (HR). Both DL-028 and prazosin (1, 3.3 and 10 microgram/kg) induced dose-dependent decreases of PVP and MAP after intravenous infusion, with effects lasting for longer than 30 min. The maximum percentage reduction of PVP after DL-028 was 10, 10 and 15%, respectively, for the dosages given (1, 3.3 and 10 microgram/kg), and 5, 12 and 25%, respectively, after prazosin. CI was not changed by either drug. HR was not changed by either drug except DL-028 at 10.0 microgram/kg with a bradycardiac effect. Our results showed that both DL-028 and prazosin reduced PVP in portal hypertensive rats.     

Antibody catalysis of peptidyl-prolyl cis-trans isomerization in the folding of RNase T1

An antibody generated to an alpha-keto amide containing hapten 1 catalyzes the cis-trans isomerization of peptidyl-prolyl amide bonds in peptides and in the protein RNase T1. The antibody-catalyzed peptide isomerization reaction showed saturation kinetics for the cis-substrate, Suc-Ala-Ala-Pro-Phe-pNA, with a kcat/Km value of 883 s-1.M-1; the reaction was inhibited by the hapten analog 13 (Ki = 3. 0 +/- 0.4 microM). Refolding of denatured RNase T1 to its native conformation also was catalyzed by the antibody, with the antibody-catalyzed folding reaction inhibitable both by the hapten 1 and hapten analog 13. These results demonstrate that antibodies can catalyze conformational changes in protein structure, a transformation involved in many cellular processes.     

Chromosome-specific aneusomy in carcinoma of the breast

Fluorescence in situ hybridization was performed on touch preparations from 55 primary infiltrating ductal carcinomas of the breast to determine numeric chromosome abnormalities. The frequency of aneusomy, measured by both nondisomy and chromosomal gain, was determined for chromosomes X, 4, 6-12, 17, and 18 with the use of chromosome-specific, alpha-satellite DNA probes. The presence of chromosome-specific numeric abnormalities was correlated with established clinicopathological parameters, including tumor size, lymph node involvement, tumor grade, estrogen receptor level, and menopause status. In addition, a case-control study was performed to explore a possible association between chromosome-specific aneusomy and recurrence in lymph-node-negative patients. Although chromosomes 8 and 6 were most frequently aneusomic, numeric abnormalities of chromosomes 4 and 11 were most strongly associated with established prognostic factors. For chromosomes 4 and 11, strong associations were found with tumor involvement of lymph nodes and increased tumor size, along with a weaker association with tumor grade. In addition, numeric abnormalities of the following chromosomes were associated with the corresponding prognostic factors: chromosomes X, 7, and 12 with lymph node status; chromosomes 10, 17, and 6 with tumor size; and chromosomes 7, 12, 17, and X with tumor grade. No correlations were observed with estrogen receptor level or menopause status. In the case-control study performed on isolated nuclei of paraffin-embedded tissue from lymph node-negative breast cancer patients (19 cases and 19 controls), the gain of chromosome 4 was correlated with disease progression. These findings suggest that chromosome-specific aneusomy is associated with certain established prognostic factors and may be associated with disease progression.     

Anatomic correction of transposition of the great vessels

We present a new approach for anatomic correction of transposition of the great arteries. The two coronary arteries, with a piece of the aortic wall attached, are transposed to the posterior artery. The two aortic openings are closed with a patch. The aorta and pulmonary artery are transected, contraposed, ant then anastomosed. The interventricular septal defect is closed with a patch, through a right ventriculotomy approach, because the right ventricle is no longer part of the systemic circulation. Two patients, aged 3 months and 40 days weighing 4,200 and 3,700 grams, respectively, were operated upon with deep hypothermia and total circulatory arrest. There was good recovery from the operation, with normal cardiocirculatory conditions. Renal failure developed in the first patient, and she died on the third postoperative day. During this time the cardiocirculatory conditions were good. The second patient made an uneventful recovery. Hemodynamic studies 20 days after the operation showed complete correction of the malformation. Five and one-half months after the operation, he weighs 7,500 grams, and his development is very good. We believe that this operation will be reproducible by most cardiovascular septal defect and pulmonary hypertension.     

A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces

The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.