Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis

BACKGROUND: We report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever. METHODS: The major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1(B. recurrentis A1) gene in both this and other isolates. RESULTS: This isolate was found to carry silent and expressed copies of the vlp1(B. recurrentis A1) gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1(B. recurrentis A17) on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters. CONCLUSION: Taken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates

Nitric Oxide Signalling Augments Neuronal Voltage-Gated L-Type (CaV1) and P/Q-Type (CaV2.1) Channels in the Mouse Medial Nucleus of the Trapezoid Body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (CaV) within this nucleus. Whole-cell patch recordings were made from brain slices from P13–15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 µM) and pharmacological blockers used to isolate Ca2+ current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 µM) or Diethyl-ammonium-nonoate (DEA, 100 µM) were made to elucidate NO-dependent modulation of the expressed CaV subtypes. A differential effect of NO on the calcium channel subtypes was observed: CaV1 and CaV2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (CaV2.2) and R-type (CaV2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36±0.04 nA to 0.64±0.11 nA and P/Q+R-type from 0.55±0.09 nA to 0.94±0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways

Characterisation of silent and active genes for a variable large protein of <it>Borrelia recurrentis</it>

<p>Abstract</p> <p>Background</p> <p>We report the characterisation of the variable large protein (<it>vlp</it>) gene expressed by clinical isolate A1 of <it>Borrelia recurrentis</it>; the agent of the life-threatening disease louse-borne relapsing fever.</p> <p>Methods</p> <p>The major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1_{<it>B. recurrentis A1</it>} gene in both this and other isolates.</p> <p>Results</p> <p>This isolate was found to carry silent and expressed copies of the <it>vlp1</it>_{<it>B. recurrentis A1</it>} gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent <it>vlp1</it>_{<it>B. recurrentis A17</it>} on a 54 kbp plasmid. Silent and expressed <it>vlp1</it> have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of <it>vlp1</it> is transcribed in the A1 isolate of <it>B. recurrentis</it>, yet both 5' upstream sequences of this <it>vlp1</it> gene possess features of bacterial promoters.</p> <p>Conclusion</p> <p>Taken together these results suggest that antigenic variation in <it>B. recurrentis</it> may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other <it>B. recurrentis</it> isolates.</p