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Characterization of Moraxella bovis Aspartate Transcarbamoylase

Description: Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.
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Date: December 2001
Creator: Hooshdaran, Sahar

Characterization of the Aspartate Transcarbamoylase that is Found in the pyrBC’ Complex of Bordetella Pertussis

Description: An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.
Date: December 2001
Creator: Dill, Michael T

Investigations of neuronal network responses to electrical stimulation in murine spinal cultures.

Description: Spontaneous activity in neuronal networks in vitro is common and has been well documented. However, alteration of spontaneous activity in such networks via conditioning electrical stimulation has received much less experimental attention. Two different patterns of electrical stimulation were used to enhance or depress the level of spontaneous activity in spinal cord cultures. High-frequency stimulation (HFS), a method routinely shown to increase the efficacy of synaptic transmission, was employed to augment spontaneous activity. Low-frequency stimulation (LFS), the technique often applied to depress synaptic efficacy, was employed to decrease spontaneous activity. In addition, LFS was used to reverse the effect of HFS on spontaneous activity. Likewise, HFS was applied to counter the effect of LFS. Because these networks were grown on multi-microelectrode plates (MMEPs), this allowed the simultaneous stimulation of any combination of the 64 electrodes in the array. Thus, the possible differences in response to single versus multi-electrode stimulation were also addressed. Finally, test-pulses were delivered before and after the conditioning stimulation on the same stimulation electrode(s) in order to assess the change in mean evoked action potentials (MEAPs). Dissociated spinal tissue from embryonic mice was allowed to mature into self-organized networks that exhibited spontaneous bursting activity after two weeks of incubation. Spontaneous activity was monitored from up to 14 recording channels simultaneously. Although uniform responses to stimulation across all recording electrodes were rarely observed, a large majority of the recording channels had similar responses. Spontaneous activity was increased in 52% of 89 HFS trials, whereas activity was decreased in 35% of 75 LFS trials. The duration of most of these increases was less than 5 minutes. When there were substantial and long-term (> 15 min) changes in spontaneous activity, the opposing stimulation pattern successfully reversed the effect of the previous stimulation. The percent change in MEAPs following conditioning stimulation ...
Date: December 2001
Creator: Sparks, Christopher A.