G117C MelB, a mutant melibiose permease with a changed conformational equilibrium.

Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displacements after substrate concentration jumps are strongly reduced and the fluorescence changes disappear. Together with its maintained transport activity this indicates that substrate translocation in G117C melibiose permease is not impaired but that the initial conformation of the mutant transporter differs from that of the wild type permease. A kinetic model for the G117C melibiose permease based on a rapid dynamic equilibrium of the substrate free transporter is proposed. Implications of the kinetic model for the transport mechanism of the wild type permease are discussed.

Charge translocation during cosubstrate binding in the Na+/proline transporter of E.coli.

Charge translocation associated with the activity of the Na(+)/proline cotransporter PutP of Escherichia coli was analyzed for the first time. Using a rapid solution exchange technique combined with a solid-supported membrane (SSM), it was demonstrated that Na(+)and/or proline individually or together induce a displacement of charge. This was assigned to an electrogenic Na(+)and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k>50s(-1) which preceeds the rate-limiting step. Based on the kinetic analysis of our electrical signals, the following characteristics are proposed for substrate binding in PutP. (1) Substrate binding is electrogenic not only for Na(+), but also for the uncharged cosubstrate proline. The charge displacement associated with the binding of both substrates is of comparable size and independent of the presence of the respective cosubstrate. (2) Both substrates can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails, while at sufficiently high concentrations, each substrate can bind in the absence of the other. (3) Both substrate binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate. (4) Proline binding proceeds in a two-step process: low affinity (approximately 1mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition.

Persistent Sweet's syndrome occurring in a child with a primary immunodeficiency.

Sweet's syndrome (SS) occurs most commonly in association with inflammatory or neoplastic disorders. Only rarely has it been associated with immunodeficiency disorders. We describe a child with a T-cell immunodeficiency who had a persistent neutrophilic dermatosis that was histologically and clinically consistent with SS. SS associated with immunodeficiencies may occur as a reaction to an underlying infection or a defect in immunoregulation. Such patients, however, may not be able to produce the classic fever and neutrophilia associated with SS. They may fail to respond to standard treatment for SS and may suffer a prolonged and persistent course.

Immunologic bullous diseases: blisters without a precipitating event.

Although the overall incidence of immunologic bullous diseases is comparatively low, they are nonetheless potentially lethal dermatologic disorders that occur most commonly in individuals age 55 and older. Thus familiarity with the signs, symptoms, and treatments will be valuable to the primary care physician. For bullous diseases in general, the challenge is to differentiate between those arising from immunologic rather than exogenous causes, such as drug-induced or drug-triggered pemphigus. Treatment goals include screening for associated malignancies, managing the lesions, and minimizing the morbidity and mortality associated with the disease. Collaboration with a dermatologist can aid in achieving these objectives.

Reflex sympathetic dystrophy with mutilating ulcerations suspicious of a factitial origin.

Reflex sympathetic dystrophy (RSD) is a poorly understood posttraumatic pain syndrome associated with dysfunction of the sympathetic nervous system. Pain is often out of proportion to the extent of injury. Progression of the disease may lead to dystrophic and atrophic changes resulting in total disability of an affected limb. Skin findings are highly variable and nonspecific and may rarely include bullae and ulcerations. We describe a mutilating case of RSD with unusual and severely disfiguring ulcerations that necessitated amputation of the right arm. Shortly after the amputation, ulcerations began appearing on the left arm. We suspect a factitial component but have been unable to prove or disprove it. We propose that some ulcerations in patients with RSD may be factitial in origin.

Dexamethasone binding to chromatin, inhibition of in vitro RNA synthesis, and therapeutic effect on human lymphosarcoma.

A very high degree of specific dexamethasone binding to chromatin and a marked in vitro inhibition of RNA-synthesizing capacity of purified lymphoma cell nuclei was found to correlate closely with the very strong cytolethal effect of glucocorticoids on lymphoma cells (in this case neither B- nor T-cells) of a young patient, whose condition rapidly and unexpectedly deteriorated after a total dose fo 90 mg of Prednisone given during a two day period; and in spite of prophylactic antihyperuricaemia treatment, the patient subsequently died. In this case the amount of glucocorticoid bound specifically by lymphoma chromatin was about 400% larger than is normally found in chromatin isolated from normal human thymus cells in persons on this age. The in vitro inhibition of RNA-Synthesizing activity measured with the aggregate enzyme and with isolated nuclei from lymphoma cell-nuclei by dexamethasone correlates closely to the specific dexamethasone binding capacity of chromatin. Thus a better prediction of the therapeutic effect of such lymphosarcoma cells to glucocorticoid may be possible.

Orotic acid: its influence on nuclear RNA and microsomal protein synthesis in brain cells of undernourished rat pups.

Malnourished rat pups were supplemented with orotic acid. RNA-synthesizing activity of isolated brain cell nuclei and microsomal protein synthesis was measured in vitro. A marked increase was found in the activity of nuclear RNA synthesis but not in microsomal protein synthesis after orotic acid treatment for 7 days.

RNA synthesizing activities and glucocorticoid binding capacities in thymus cells during rat development.

This investigation was designed to study specific glucocorticoid binding to cytoplasmic fraction and nuclei of thymus cells during rat development and to find out whether these data can be correlated to changes of RNA synthesizing activity of nuclei and cytoplasm. The dexamethasone binding capacity of cytoplasm rose rapidly in rats weighing up to 125 g and decreased significantly in animals weighing more than 160 g. Hormone binding to nuclei revealed similar but less pronounced changes. RNA synthesizing activity of nuclei measured by [3H]uridine incorporation in vitro decreased from 57 +/- 4.6 dpm/microng DNA in young to 23 +/- 2.2 dpm/microng DNA in adult rats. RNA synthesizing activity of the cytoplasmic fraction fell 21.6% and that of purified polymerase IIIB fell 27.8% during development. Inhibition of RNA synthesis by dexamethasone applied in vivo and in vitro showed age-dependent differences. The RNA synthesizing capacity of nuclei was inhibited up to 39% in animals weighing 130 g and only 9% in aging rats. Similar changes were observed by incubation of intact thymocytes with and without hormone. The observed inhibitory effect of dexamethasone on RNA synthesis is well correlated to changes of cytoplasmic hormone receptor capacities during development.

[Template activity of chromatin and DNA-dependent RNA polymerase III during postnatal development (author's transl)]. / Chromatin-Matrizenaktivität und Verhalten DNA-abhängiger RNA-Polymerase III in der Peri- und Postnatalperiode

The template-activity of chromatin isolated from liver nuclei of developing rats increases sharply at birth and decreases remarkable until the 10th day. Between the 10th and the 40th day a slow increase was shown in template activity of chromatin tested with purified E.-coli RNA-polymerase (EC 2.7.7.6). We correlated these findings with changes of RNA-polymerase III-activity in rat liver during development.