Mechanism of site-selective DNA nicking by the hydrodioxyl (perhydroxyl) radical.

In previous studies, the ability of the hydrodioxyl (perhydroxyl) radical [HOO., the conjugate acid of superoxide (O2.-] to "nick" DNA under biomimetic conditions was demonstrated, and a sequence selectivity was observed. A background level of nonspecific nicking also was noted. This paper provides support for 5'-hydrogen atom abstraction from the deoxyribose ring as the initial event in the sequence-selective nicking by 02.-/HOO.. Two experiments support the proposed mechanism. First, using a defined sequence 5'-32P-labeled restriction fragment as the DNA substrate, only free (unalkylated) 3'-phosphate is produced at the site of nicking. Second, using poly (dA).poly (T) as the substrate, furfural is formed in the reaction from deoxyribose ring breakdown. Both results are consistent with 5'-hydrogen atom abstraction for initiation of the site-selective nicking. Hydrogen atom abstraction at other sites of the deoxyribose ring and/or base oxidation and loss followed by strand scission likely are responsible for the nonspecific nicking. The 5'-abstraction mechanism contrasts to those elicited by other 02-derived and metal-associated oxidants, which may provide a biomarker for the reactivity of HOO. in vivo.

Evaluation of N-hydroxy-2-thiopyridone as a nonmetal dependent source of the hydroxyl radical (HO.) in aqueous systems.

N-hydroxy-2-thiopyridone (1), an established source of the hydroxyl radical (HO., Boivin, J., Crepon, E., and Zard, S. Z. (1990) Tetrahedron Lett. 31, 6869-6872), produced HO. under conditions directly applicable to biological studies. Generation of HO. by subjecting 1 to irradiation with visible light was monitored in the following "HO." assays: deoxyribose degradation, addition to dimethyl sulfoxide, and hydroxylation of salicylate and phenol. All four assays demonstrated the production of HO. from 1 (added as a sodium salt) under mild conditions in aqueous buffer systems. An improved analysis method was developed for the phenol assay. A time course analysis demonstrated that a flux of HO. is generated from 1 throughout the irradiation period, in contrast to the classical Fenton reaction of H2O2 with a transition metal in which a burst of HO. is generated in a short time period. While a thiyl radical is generated from 1 concurrent with HO. generation, this species does not contribute to, or interfere with, any of the HO. assays, suggesting that it is weakly reactive in aqueous buffers. Thus, irradiation of 1 can be used as an alternative, complementary, approach for the unequivocal generation of the biologically significant and reactive HO..

Mechanism of activation of Agrobacterium virulence genes: identification of phenol-binding proteins.

Agrobacterium tumefaciens initiates the expression of pathogenic genes (vir genes) in response to host-derived phenolic signals through a two-component regulatory system consisting of VirA and VirG. alpha-Bromoacetosyringone (ASBr) was developed as an inhibitor of this induction process and found to be a specific and irreversible inhibitor of vir gene induction in this pathogen. Formal replacement of one of the methoxy groups of ASBr with iodine gave an equally effective inhibitor that could carry an 125I label. We report here that the resulting radiolabeled inhibitor does not react with the sensory component of this system, VirA, either in vivo or in vitro. Rather, two small proteins, p10 and p21, bind labeled inhibitor in vivo in a time period that is consistent with the exposure time required for the inhibition of vir gene expression. Labeling to these proteins was protected by preexposure to ASBr but not by alpha-bromo-3,5-dimethoxyacetophenone, a compound of comparable chemical reactivity but previously shown not to inhibit vir gene expression. Our findings suggest that proteins that are not tumor-inducing plasmid-encoded mediate vir gene activation in a step prior to the VirA/VirG two-component regulatory system.

Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems.

The aglycone of the dihydrodiconiferyl alcohol glycosides, a series of phenolic growth factors able to substitute for some of the hormone requirements of tobacco cell division, are also potent inducers of virulence gene expression in Agrobacterium tumefaciens. However, these factors do not conform to the previously established structural requirements necessary for vir expression. Systematic evaluation of the structural requirements of these inducers has led to a model detailing the role of the phenolics in induction. With this model, a specific inhibitor of vir induction has been developed. This inhibitor does not affect the induction of other genes on the Ti plasmid but irreversibly blocks vir expression. The inhibitor has been used to show that the inducing phenolics must be constantly present to maintain expression of the vir regulon.

[Cell biology basis of IgE antibody regulation. Immunotherapeutic approaches]. / Zellbiologische Grundlagen der IgE-Antikörperregulation. Immuntherapeutische Ansatzpunkte.

Recent developments as to the IgE-antibody synthesis in rodents and in the human model are summarized and discussed in reference to our own data. It is by now established that the IgE-antibody response is regulated by interdependent interactions of cellular, humoral and genetic factors. The IgE-bearing B-lymphocyte develops from a surface IgM carrying B-lymphocyte. The transformation into an IgE secreting plasma cell requires T-cell help or soluble T-cell factors. Recent advances in the characterization of human lymphocytes as well as more sophisticated cell biological approaches facilitated the analysis of IgE-antibody synthesis in the human in vitro model. In most of the studies at present available the effect of the polyclonal B-cell mitogen (PWM) was analyzed. PWM either enhanced, suppressed or unlike to other Ig-classes did not affect the IgE-antibody synthesis at all. Immunotherapeutic approaches have to consider to establish the allergen induced in vitro model of IgE-antibody synthesis in humans, to modulate the interactions of T-helper and/or suppressor cells or to generate soluble suppressor factors. In any case the analysis of the IgE-antibody synthesis in humans could be a valuable tool to assess IgE-B-cell memory towards various allergens and to determine the IgE-mediated in patients.

Model of Wernicke's encephalopathy.

After a week on a thiamine-free diet and daily injections of pyrithiamine hydrobromide, a group of rats began to lose weight; soon thereafter hypothermia, piloerection, and ataxia developed, followed by convulsions and death. Neuropathologic examination disclosed hemorrhagic necrotic lesions in the thalamus, hypothalamus, collicular plate, vestibular nuclei, and inferior olives. The control groups did not show neurologic signs or neuropathologic abnormalities. The lesions in thiamine-deficient rats were similar in character and distribution to those of human Wernicke's disease. Because this experimental regimen produces neuropathologic changes rapidly and consistently, this animal model should be useful in studies designed to examine the pathophysiologic aspects of experimental Wernicke's disease in particular and CNS thiamine deficiency in general.

The expanding classroom trends and issues in postgraduate medicine. Why self-assessment?

Self-assessment can provide more than a direction for independent study and credit toward recertification. It can also provide an effective learning experience if the instruments are designed to serve an instructional purpose as well as the more traditional measurement function. Based on existing research, the characteristics of self-assessment instruments--questioning, controlling attention, elucidating objectives, evoking performance, and assessing outcomes--can enhance the learning and retention of new material.