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- Allander, T, et al.
(författare)
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Recombinant human monoclonal antibodies against different conformational epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibit its interaction with CD81
- 2000
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Ingår i: Journal of General Virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 81:10, s. 2451-2459
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Tidskriftsartikel (refereegranskat)abstract
- The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81-E2 interaction in vitro; all four were positive at 0.3-0.5 microg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2-CD81 interaction, of which one appeared immunodominant in this donor.
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- Cangiano, L, et al.
(författare)
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Fast and slow locomotor burst generation in the hemispinal cord of the lamprey
- 2003
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Ingår i: Journal of neurophysiology. - : American Physiological Society. - 0022-3077 .- 1522-1598. ; 89:6, s. 2931-2942
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Tidskriftsartikel (refereegranskat)abstract
- A fundamental question in vertebrate locomotion is whether distinct spinal networks exist that are capable of generating rhythmic output for each group of muscle synergists. In many vertebrates including the lamprey, it has been claimed that burst activity depends on reciprocal inhibition between antagonists. This question was addressed in the isolated lamprey spinal cord in which the left and right sides of each myotome display rhythmic alternating activity. We sectioned the spinal cord along the midline and tested whether rhythmic motor activity could be induced in the hemicord with bath-applied d-glutamate or N-methyl-d-aspartate (NMDA) as in the intact spinal cord or by brief trains of electrical stimuli. Fast rhythmic bursting (2–12 Hz), coordinated across ventral roots, was observed with all three methods. Furthermore, to diminish gradually the crossed glycinergic inhibition, a progressive surgical lesioning of axons crossing the midline was implemented. This resulted in a gradual increase in burst frequency, linking firmly the fast hemicord rhythm [6.6 ± 1.7 (SD) Hz] to fictive swimming in the intact cord (2.4 ± 0.7 Hz). Ipsilateral glycinergic inhibition was not required for the hemicord burst pattern generation, suggesting that an interaction between excitatory glutamatergic neurons suffices to produce the unilateral burst pattern. In NMDA, burst activity at a much lower rate (0.1–0.4 Hz) was also encountered, which required the voltage-dependent properties of NMDA receptors in contrast to the fast rhythm. Swimming is thus produced by pairs of unilateral burst generating networks with reciprocal inhibitory connections that not only ensure left/right alternation but also downregulate frequency.
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- Cangiano, L, et al.
(författare)
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Role of apamin-sensitive k(ca) channels for reticulospinal synaptic transmission to motoneuron and for the afterhyperpolarization
- 2002
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Ingår i: Journal of neurophysiology. - : American Physiological Society. - 0022-3077 .- 1522-1598. ; 88:1, s. 289-299
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Tidskriftsartikel (refereegranskat)abstract
- Single motoneurons and pairs of a presynaptic reticulospinal axon and a postsynaptic motoneuron were recorded in the isolated lamprey spinal cord, to investigate the role of calcium-dependent K+ channels (KCa) during the afterhyperpolarization following the action potential (AHP), and glutamatergic synaptic transmission on the dendritic level. The AHP consists of a fast phase due to transient K+ channels (fAHP) and a slower phase lasting 100–200 ms (sAHP), being the main determinant of spike frequency regulation. We now present evidence that the sAHP has two components. The larger part, around 80%, is abolished by superfusion of Cd2+ (blocker of voltage-dependent Ca2+ channels), by intracellular injection of 1,2-bis-( 2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid (BAPTA; fast Ca2+ chelator), and by apamin (selective toxin for KCa channels of the SK subtype). While 80% of the sAHP is thus due to KCa channels, the remaining 20% is not mediated by Ca2+, either entering through voltage-dependent Ca2+ channels or released from intracellular Ca2+ stores. This Ca2+-independent sAHP component has a similar time course as the KCa portion and is not due to a Cl− conductance. It may be caused by Na+-activated K+ channels. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by single reticulospinal axons give rise to a local Ca2+ increase in the postsynaptic dendrite, mediated in part by N-methyl-d-aspartate (NMDA) receptors. The Ca2+ levels remain elevated for several hundred milliseconds and could be expected to activate KCa channels. If so, this activation should cause a local conductance increase in the dendrite that would shunt EPSPs following the first EPSP in a spike train. We have tested this in reticulospinal/motoneuronal pairs, by stimulating the presynaptic axon with spike trains at different frequencies. We compared the first EPSP and the following EPSPs in the control and after blockade with apamin. No difference was observed in EPSP amplitude or shape before and after apamin, either in normal Ringer or in Mg2+-free Ringer removing the voltage-dependent block of NMDA receptors. In conclusion, the local Ca2+ entry during reticulospinal EPSPs does not cause an activation of KCa channels sufficient to affect the efficacy of synaptic transmission. Thus the integration of synaptic signals at the dendritic level in motoneurons appears simpler than would otherwise have been the case.
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