Threshold behaviour of human axons explored using subthreshold perturbations to membrane potential

The present study explores the threshold behaviour of human axons and the mechanisms contributing to this behaviour. The changes in excitability of cutaneous afferents in the median nerve at the wrist were recorded to a long-lasting subthreshold conditioning stimulus, with a waveform designed to maximize the contribution of currents active in the just-subthreshold region. The conditioning stimulus produced a decrease in threshold that developed over 3–5 ms following the end of the depolarization and then decayed slowly, in a pattern similar to the recovery of axonal excitability following a discharge. To ensure that the conditioning stimulus did not activate low-threshold axons, similar recordings were then made from single motor axons in the ulnar nerve at the elbow. The findings were comparable, and behaviour with the same pattern and time course could be reproduced by subthreshold stimuli in a model of the human axon. In motor axons, subthreshold depolarizing stimuli, 1 ms long, produced a similar increase in excitability, but the late hyperpolarizing deflection was less prominent. This behaviour was again reproduced by the model axon and could be explained by the passive properties of the nodal membrane and conventional Na⁺ and K⁺ currents. The modelling studies emphasized the importance of leak current through the Barrett–Barrett resistance, even in the subthreshold region, and suggested a significant contribution of K⁺ currents to the threshold behaviour of axons. While the gating of slow K⁺ channels is slow, the resultant current may not be slow if there are substantial changes in membrane potential. By extrapolation, we suggest that, when human axons discharge, nodal slow K⁺ currents will be activated sufficiently early to contribute to the early changes in excitability following the action potential.     

Early transvaginal ultrasound following an accurately dated pregnancy: the importance of finding a yolk sac or fetal heart motion

Our goals were to determine the prognostic value of a yolk sac or fetal heart motion seen during an early accurately dated transvaginal ultrasound (TVU). We reviewed 225 consecutive pregnancies for fetal heart motion data. Furthermore, 63 pregnancies following in-vitro fertilization were reviewed for yolk sac information. The TVU was performed between 5 and 6 weeks following presumed conception (heart motion data) and between 22 and 32 days following in-vitro fertilization (yolk sac data). Pregnancies were followed until an ongoing pregnancy or spontaneous abortion was documented. The presence of a yolk sac between 22 and 32 days from fertilization was associated with the development of fetal heart motion in 94% of cases. The absence of the yolk sac by 32 days after fertilization was always associated with a poor outcome. In women < 36 years of age, the presence of fetal heart motion was associated with a spontaneous abortion in only 4.5% of the cases. However, the incidence of spontaneous abortion following fetal heart motion increased to 10% in women 36-39 years and 29% in women > or = 40 years of age. The presence of heart motion should not be considered a reassuring sign in the older woman. These data have implications regarding early embryology and the counselling of infertility patients.      

CD40 expression by gingival fibroblasts: correlation of phenotype with function

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non- hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine- secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up- regulates secretion of these cytokines in vitro.      

Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.      

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.