No abstract

The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence ofMycobacterium bovis by PCR. Detection by a C18-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS6110-based PCR was 94.1 and 58.8%, respectively (P < 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis.

Depolarization promotes the survival of cerebellar granule neurons via activation of the transcription factor myocyte enhancer factor 2D (MEF2D). Removal of depolarization induces hyperphosphorylation of MEF2D on serine/threonine residues, resulting in its decreased DNA binding and susceptibility to caspases. The subsequent loss of MEF2-dependent gene transcription contributes to the apoptosis of granule neurons. The kinase(s) that phosphorylates MEF2D during apoptosis is currently unknown. The serine/threonine kinase, glycogen synthase kinase-3b (GSK-3b), plays a pro-apoptotic role in granule neurons. To investigate a potential role for GSK-3b in MEF2D phosphorylation, we examined the effects of lithium, a non-competitive inhibitor of GSK-3b, on MEF2D activity in cultured cerebellar granule neurons. Lithium inhibited caspase-3 activation and chromatin condensation in granule neurons induced to undergo apoptosis by removal of depolarizing potassium and serum. Concurrently, lithium suppressed the hyperphosphorylation and caspase-mediated degradation of MEF2D. Moreover, lithium sustained MEF2 DNA binding and transcriptional activity in the absence of depolarization. Lithium also attenuated MEF2D hyperphosphorylation and apoptosis induced by calcineurin inhibition under depolarizing conditions, a GSK-3b-independent model of neuronal death. In contrast to lithium, MEF2D hyperphosphorylation was not inhibited by forskolin, insulinlike growth factor-I, or valproate, three mechanistically distinct inhibitors of GSK-3b. These results demonstrate that the kinase that phosphorylates and inhibits the pro-survival function of MEF2D in cerebellar granule neurons is a novel lithium target distinct from GSK-3b.

The publishers wish to apologize for a printing error that appeared in the above article published in J. Neurochem. 85, pp. 1488-1499. Fig. 2(b) was incorrectly printed without an immunoblot of active caspase-3. The figure is printed in full here. Approximately 500 cells from at least two fields of a culture dish were scored for apoptosis from each condition. CGNs were considered apoptotic if their nuclei were condensed and/or fragmented. The data shown represent the means ± SEM for three independent experiments, each performed in triplicate. *Significantly different from the (5K ) serum) condition in the absence of lithium (p < 0.05). (b) CGNs were incubated as described in (a) for 6 h and detergent-soluble cell lysates were electrophoresed on 15% polyacrylamide gels. Proteins were transferred to PVDF membranes and immunoblotted (IB) with a polyclonal antibody that specifically recognizes the active (cleaved) form of caspase-3, as described in Experimental procedures. The blot shown is representative of three separate experiments.