Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides

The specific recognition by Escherichia coli RNA polymerase of single-stranded oligodeoxyribonucleotides (oligos) with the sequence of the -10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopurine residues incorporated in the oligos. The effects of salt on the rates of formation and dissociation of RNA polymerase.oligo complexes are relatively small, from which we conclude that electrostatic interactions contribute minimally to the favorable binding free energy. From the convex temperature dependence of ln Ka (Ka is the equilibrium association constant), we infer that a large apparent negative heat capacity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is interpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is more than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change in the RNA polymerase. Oligo binding was shown to compete with the interaction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase     

Carbocyclic analogues of the potent cytidine deaminase inhibitor 1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine)

Three carbocylic analogues of the potent cytidine deaminase inhibitor (CDA) zebularine [1-(beta-D-ribofuranosyl)-1, 2-dihydropyrimidin-2-one, 1a] were synthesized. The selected pseudosugar templates correspond, respectively, to the cyclopentenyl moiety of neplanocin A (compound 4), the cyclopentyl moiety of aristeromycin (compound 5), and a newly designed, rigid bicyclo[3.1. 0]hexane moiety (compound 6). These three carba-nucleoside versions of zebularine were fashioned to overcome the inherent instability of the parent drug. Each target compound was approached differently using either convergent or linear approaches. The immediate precursor to the cyclopentenyl analogue 4 was obtained by a Mitsunobu coupling of pseudosugar 7 with 2-hydroxypyrimidine. The cyclopentyl analogue 5 was linearly constructed from carbocyclic amine 17, and the final target 6 was similarly constructed from the carbobicyclic amine 27. Of the three target compounds, only 5 showed a significant level of inhibition against human CDA, but it was 16 times less potent than zebularine (Ki = 38 microM vs Ki(apparent) = 2.3 microM). Although these carbocyclic analogues appeared to be more stable than zebularine, replacement of the electronegative CO4' oxygen for the less electronegative carbon in 4-6 presumably reduces the capacity of the pyrimidin-2(1H)-one ring to form a covalent hydrate, a step considered crucial for the compound to function as a transition-state inhibitor of the enzyme.     

The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcgamma binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.     

Cerebral microembolism in patients with retinal ischemia

BACKGROUND AND PURPOSE: We investigated the frequency of cerebral microembolism detected by transcranial Doppler ultrasonography in patients with clinical evidence of retinal ischemia, including transient monocular blindness, central and branch retinal artery infarction, and ischemic oculopathy, and assessed its correlation with carotid artery stenosis. METHODS: Records of 331 consecutive patients examined during a 47-month period at the Neurovascular Laboratory were reviewed. Of the original 453 intracranial arteries, 186 middle cerebral arteries (MCAs) satisfied qualifying criteria that excluded patients with cardiac embolic sources. Forty-five MCAs ipsilateral to the symptomatic eye constituted the study group. The control group consisted of 141 asymptomatic MCAs. Microembolus detection studies were performed on transcranial Doppler instruments equipped with special software, and the degree of carotid artery stenosis was measured by cerebral or MR angiography or by color duplex studies. RESULTS: Microembolism was detected in 40.0% of study MCAs and 9.2% of controls (P < 0.001). In the study group, microembolic signals were detected in 61.9% of MCAs tested within a week of symptom onset and 20.8% of those tested afterward (P < 0.001). Severe (> or = 70%) carotid stenosis or occlusion was more frequent in the study group (P < 0.001). Microembolic signals were detected in 25.3% and 11.2%, respectively, of MCAs distal to carotid arteries with 70% to 100% and 0% to 69% stenosis (P = 0.013). CONCLUSIONS: In patients without cardiac embolic sources, cerebral microembolism is frequently present on the side of retinal ischemia, particularly during the week after onset of symptoms. It is often associated with severe stenosis or occlusion of the ipsilateral carotid artery.     

Relationship of somatic cell count and cell volume analysis of goat's milk to intramammary infection with coagulase-negative staphylococci

The prevalence of intramammary infection in 4 commercial goat herds was studied in conjunction with electronic somatic cell count and volume analysis determined using a Coutler Counter and volume analyser. Neither streptococci nor mycoplasma were isolated from any half and the prevalence of intramammary infection with Staphylococcus aureus ranged from 0 to 3% between herds. For coagulase-negative staphylococci the range from infected halves was 36-71%. There was no significant difference between the mean total microscopic somatic cell count for halves infected with coagulase-negative staphylococci and those free from infection. A similar trend was observed for electronic somatic cell counts although the mean electronic cell count was greater than the mean total microscopic count on the 2 occasions that they were compared. The correlation coefficients between the 2 cell counting methods were 0.86 and 0.94. Between herds there were significant differences in mean electronic somatic cell count, with herd means ranging from 438 x 103 to 1684 x 103 cells/ml. In 2 of the 4 herds studied, milk samples from halves infected with coagulase-negative staphylococci had a significantly higher prevalence of cell volume distributions with a modal cell volume between 65 mu 3 and 100 mu 3. This was attributed to a higher proportion of polymorphonuclear neutrophils. Use of electronic somatic cell count and cell volume analysis were considered of little value in predicting infection caused by coagulase-negative staphylococci as there was a high proportion of false negative and false positive predictions.     

Die Zukunft von Information und Kommunikation

Ohne Zusammenfassung Kurzfassung eines Vortrags von Vors.-Dir. Dipl.-Ing. Eduard Uhl anl?sslich einer Siemens-Kundentagung in Alphach.     

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Ohne Zusammenfassung