Synthesis and glutathione S-transferase structure-affinity relationships of nonpeptide and peptidase-stable glutathione analogues

A series of nonpeptidic glutathione analogues where the peptide bonds were replaced by simple carbon-carbon bonds or isosteric E double bonds were prepared. The optimal length for the two alkyl chains on either side of the mercaptomethyl group was evaluated using structure-affinity relationships. Affinities of the analogues 14a-f, 23, and 25 were evaluated for a recombinant GST enzyme using a new affinity chromatography method previously developed in our laboratory. Analysis of these analogues gives an additional understanding for GST affinity requirements: (a) the carbon skeleton must conserve that of glutathione since analogue 14a showed the best affinity (IC50 = 5.2 microM); (b) the GST G site is not able to accommodate a chain length elongation of one methylene group (no affinity for analogues 14c,f); (c) a one-methylene group chain length reduction is tolerated, much more for the "Glu side" (14d, IC50 = 10.1 microM) than for the "Gly side" (14b, IC50 = 1800 microM); (d) the mercaptomethyl group must remain at position 5 as shown from the null affinity of the 6-mercaptomethyl analogue 14e; (e) the additional peptide isosteric E double bond (25) or hydroxyl derivative (23) in 14e did not help to retrieve affinity. This work reveals useful information for the design of new selective nonpeptidic and peptidase-stable glutathione analogues.     

Homology and functional similarity of an hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato

The "disease-specific" (dsp) region next to the hrp gene cluster of Erwinia amylovora is required for pathogenicity but not for elicitation of the hypersensitive reaction. A 6.6-kb apparent operon, dspEF, was found responsible for this phenotype. The operon contains genes dspE and dspF and is positively regulated by hrpL. A BLAST search revealed similarity in the dspE gene to a partial sequence of the avrE locus of Pseudomonas syringae pathovar tomato. The entire avrE locus was sequenced. Homologs of dspE and dspF were found in juxtaposed operons and were designated avrE and avrF. Introduced on a plasmid, the dspEF locus rendered P. syringae pv. glycinea race 4 avirulent on soybean. An E. amylovora dspE mutant, however, elicited a hypersensitive reaction in soybean. The avrE locus in trans restored pathogenicity to dspE strains of E. amylovora, although restored strains were low in virulence. DspE and AvrE are large (198 kDa and 195 kDa) and hydrophilic. DspF and AvrF are small (16 kDa and 14 kDa) and acidic with predicted amphipathic alpha helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens.     

Oncocytic cancers of the thyroid. Hürthle cell cancers

Nonvalvular atrial fibrillation is a frequent finding in elderly patients; the risk of thromboembolic complications is comparable to that reported in patients with rheumatic atrial fibrillation. Recently, 6 multicenter clinical trials (5 primary prevention, 1 secondary prevention trail) have been published which demonstrate equivocally that oral anticoagulation therapy significantly reduces the embolic risk in patients with nonvalvular atrial fibrillation by about 67% to 87%. The target INR of anticoagulation with warfarin in 2 of these trials was 1.4 to 2.8 ("low-dose" warfarin); interestingly, the magnitude of risk reduction was similar to these 2 studies with "low-dose" warfarin as it has been reported by the others using full-dose warfarin with an INR target between 2.0 and 4.5. Side effects of oral anticoagulation (severe bleeding complications) were low in these trials. Thus, the benefit-risk ratio in these 6 clinical trials encourage the use of oral anticoagulation in patients with nonvalvular atrial fibrillation. It will be a challenge to transfer these results to clinical practice, and to define in more detail the risk-benefit ratios for subgroups of patients with atrial fibrillation, e.g. patients > 75 years of age, or patients with "lone" or paroxysmal atrial fibrillation. It is well established that patients with chronic atrial fibrillation undergoing medical or DC-cardioversion are at risk for thromboembolic complications. In previous studies, this risk appears to be in the range of 2% without concomitant anticoagulation, but only 0.33% in those patients with concomitant anticoagulation. Thus, it is widely accepted that patients should be anticoagulated for at least 2 weeks prior and after planned cardioversion. Recently, an alternative concept has been proposed omitting anticoagulation before cardioversion; instead, transesophageal echocardiography is used to exclude intracardiac thrombi. Because it is known that mechanical function of the left atrium and appendage is still impaired after cardioversion, this concept of echocardiographic-guided cardioversion does not assign the necessity of anticoagulation at the day of cardioversion, and 2 weeks afterwards. The safety aspects of this concept of echocardiographic-guided cardioversion is under current investigation.     

Rerouting of the intratemporal facial nerve: an analysis of the literature

Anterior rerouting of the intratemporal facial nerve in the infratemporal fossa approach is employed to access to the jugular bulb, hypotympanum, and lateral skull base, whereas posterior rerouting of the facial nerve, as employed in the transcochlear craniotomy, is most frequently used for surgery of the posterior fossa, cerebellopontine angle, prepontine region, and petrous apex. Facial nerve rerouting may lead to facial paresis or paralysis. This review of the literature is intended to define the physiologic "cost" of these procedures, so that the neurotologic surgeon can determine if the morbidity incurred in these techniques is worth the resultant exposure. Inconsistencies in reporting facial function places into question the validity of some of the cumulative data reported. Postoperatively, grades I-II facial nerve function was seen in 91% of patients undergoing short anterior rerouting, 74% of patients undergoing long anterior rerouting, and 26% of patients undergoing posterior complete rerouting. Although facial nerve rerouting allows unhindered exposure to previously inaccessible regions, it is achieved at the cost of facial nerve function. Facial nerve dysfunction increases with the length of facial nerve rerouted.     

Steroid-responsive encephalomyelitis in childhood

The syndrome of parainfectious encephalomyelitis evolves from an antecedent infection. Several etiologic agents have been associated with this complication, although the pathogenesis in each instance may prove to be more uniform. Considerable evidence suggests that the syndrome is mediated immunologically. The seven cases reported here were clinically similar, although the infectious etiologies were diverse. Leptospirosis antedated the neurologic syndrome in two cases, and a "viral" illness preceded the other five cases. The evolution of the syndrome was slowly progressive in each case, and six patients had prominent involvement of rhombencephalic structures. The progressive course was reversed rapidly with eventual full recovery in each instance after initiation of corticosteroid therapy. Our experience with these cases coupled with a review of the literature suggests that corticosteroid therapy should be considered in the subacute or chronic cases of parainfectious encephalomyelitis.     

Measurement of thrombus formation on intravascular catheters

War is a complex social situation due to the interplay of multiple factors. Economical and political ones are of utmost importance, but human attitudes and motivations must be also taken into account. Being desirable to modify human transactions in such a way that they do not interfere with the basic right of everyone to a condition of physical and mental well-being, war must be abolished. The author contends that an understanding of behaviour through Psychology can be helpful in that aim. Aggression is considered the principal psychological cause of war. It is worth while to differentiate between aggression as an instrument for attaining a special end, and as pure hostility. Only in the first form, it is held here, does it play an important role in war. Psychogists must deal also with a wide range of states of mind that can be "served" by aggression: feelings of inferiority or insecurity, fear, greed, projections, compensations, rationalizations, etc. Scientific approach is not the only one Physiology brings to war prevention. It is equally important the wide dissemination of its conclusions among the general public. Information on the dynamics that prompt people to decide war would make it easier to control. This applies not only to political or military leaders, but also to civil populations. Concerning those two possible contributions of psychologists, scientific and educative, it is suggested the extensive use of psychodramatic techniques. Their richness lie in the lifelike experiences they convey to the participants, and particular aptitude to promote changes of attitudes. Investigation and information on the psychological processes related to war should be undertaken by international organizations of social scientists, acting simultaneously in several countries. Some of the initial steps they could further: 1) that prevention of war be a current subject matter in psychological courses; 2) that the World Health Organization take interest in this subject; 3) that psychologists participate in international political and economical bodies in order to clarify the psychological factors leading to war.     

The effects of VIP on intestinal motility: study on ex vivo perfused isolated canine jejunal loops

The effects of VIP on intestinal motility were studied on isolated canine jejunal loops ex vivo perfused at normothermia, under pulsatile flow with heparinized, oxygenated and nonrecirculated canine whole blood, by means of an intraluminal balloon. VIP was administered intraarterially either by 1 min injections or by long-time infusions. The results showed that for arterial concentrations of the polypeptide ranging between 25 pg/ml and 300-500 pg/ml a fast but short-lasting relaxant effect was observed. For higher concentrations VIP usually produced a biphasic response: The relaxant effect is followed by an increase of the basal muscular tone often accompanied, for concentrations higher than about 25 ng/ml, by a marked and transient increase in amplitude of the intestinal rhythmic contractions. During long-time infusions a biphasic response was also observed but both effects were of short duration. A cholingeric origin of the secondary contracting phase was expected but could not be demonstrated because, at blood concentrations at which atropine affected the biphasic response, not only was the contractile effect abolished but also the initial relaxing phase. It is suggested that the secondary contraction may be a "rebound excitation" of myogenic nature or a result of noncholingeric excitatory fiber stimulations. The short-lasting relaxant effect observed under the present experimental conditions, even during long-time infusion of the polypeptide, fails to argue for an important physiological role of VIP as an hormonal inhibitor of intestinal motility. The biphasic response, however, might have a physiological significance in so far as the aboral propulsion of the intestinal content requires a muscular inhibition which rapidly changes to contraction.     

A randomized controlled trial of home care: clinical outcome for five surgical procedures

The clinical function of patients receiving home care after five surgical procedures was assessed. Hospital patients who normally would have received minimal nursing care at the end of their hospital stay were randomly assigned to an experimental home-care group or a control group who were discharged from hospital after the normal length of stay. Comprisons of "untoward events" (discomfort, infection, delayed healing, or complications) are reported for the two groups in five surgical categories (varicose vein stripping, herniorrhaphy, cholecystectomy, anal and rectal operations and abdominal hysterectomy) where the home-care program operated efficientyl. No apparent differences in the rates of untoward events were noted between hospital and home-care groups. It is concluded that home care should be considered for reasons other than clinical function, such as socio-economic functioning, the wishes of the patient, or more efficient use of hospital space.     

Hierarchical bayesian models for regularisation in sequential learning

We show that a hierarchical Bayesian modeling approach allows us to perform regularization in sequential learning. We identify three inference levels within this hierarchy: model selection, parameter estimation, and noise estimation. In environments where data arrive sequentially, techniques such as cross validation to achieve regularization or model selection are not possible. The Bayesian approach, with extended Kalman filtering at the parameter estimation level, allows for regularization within a minimum variance framework. A multilayer perceptron is used to generate the extended Kalman filter nonlinear measurements mapping. We describe several algorithms at the noise estimation level that allow us to implement on-line regularization. We also show the theoretical links between adaptive noise estimation in extended Kalman filtering, multiple adaptive learning rates, and multiple smoothing regularization coefficients.     

In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.