Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution. Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.     

构成信号发生器的DDS和变换器

许多设备需要使用能产生高性能、高分辩率信号的低频信号发生器.本实例提供一种能产生0～1MHz频率的电路.你利用这种电路就可产生正弦波、三角波和方波,并能达到优于0.1Hz的频率分辨率和优于0.1°的相位分辨率,从而就可以给精确的相干频率编程.这一特点对于数字调制设备和频率调谐设备来说都是很有用的.该电路使用AD μ C381和AD9834来产生所需的频率(图1).利用PC或基于Unix的工作站来给微控制器AD μ C381编程,然后再通过微控制器,利用一个三线串行接口来对AD9834编程.接口的字长为16位.     

The neurotrophic analogue of ACTH(4-9) reduces the perineuronal microglial reaction after rat facial nerve crush

Rats bled to a severe condition of volume-controlled hemorrhagic shock were randomly assigned to one of the following treatments: (1) saline, 1 ml/kg i.v.; (2) saline, 0.2 ml/kg per min i.v. for 10 min; (3) ACTH-(1-24), 160 micrograms/kg i.v.; 4) methylprednisolone, 40 mg/kg i.v.; (5) methylprednisolone, 80 mg/kg i.v.; (6) aprotinin, 10,000 KIU/kg i.v.; (7) norepinephrine, 5 micrograms/kg per min i.v. for 10 min; (8) norepinephrine, 10 micrograms/kg per min i.v. for 10 min. All rats treated with saline or with either of the two doses of methylprednisolone, and half of the rats treated with aprotinin, died within the subsequent 2 h. On the other hand, rats treated with norepinephrine, at either dose, or with ACTH-(1-24) were all still alive 2 h later, a similar improvement in cardiovascular and respiratory parameters being obtained with the two treatments. The effect of ACTH on mean arterial pressure was however more sustained throughout the observation period. These results further support the potential usefulness of ACTH-(1-24) as first-aid treatment in cases of severe blood losses.     

Principal axes and surface fitting methods for three-dimensional image registration

We evaluated the effect of the image acquisition parameters on the accuracy of the principal axes and surface-fitting techniques for three-dimensional image registration. Using two types of phantom objects, MR brain image and a mathematically defined ellipsoid, we simulated pairs of scans with known acquisition parameters, including longitudinal coverage, magnitude of mis-registration, number of sections and section thickness. Both methods are sensitive to the systematic deformation of contours. The principal axes method is also sensitive to incomplete scan coverage and to the x-axis and y-axis misangulation. Both methods are insensitive to the number of sections, section thickness and the number of points per section. Surface fitting performed well without user supervision. There is no need for routine inclusion of the scaling factors as search parameters. The results confirm the feasibility of three-dimensional multimodality registration of brain scans with accuracy 1-2 mm, with surface fitting being the method of choice.     

The affinity of an oligodeoxynucleotide-peptide conjugate for an RNA hairpin loop depends on stereochemistry at the DNA-peptide junction

Molecular models of an oligodeoxynucleotide-peptide conjugate complexed to an RNA hairpin loop were constructed to assess the effect of stereoisomerism at the point of attachment of the peptide to the oligodeoxynucleotide on the affinity of the conjugate for an RNA target. The peptide portion of the oligodeoxynucleotide-peptide conjugate, (L-lysine)8, was covalently attached to the N-allyl group of (D)- or (L)-aspartic alcohol that was incorporated into the interior of an antisense oligodeoxynucleotide. The stereocenter in the oligodeoxynucleotide interior originates from either (D)- or (L)-aspartic alcohol. The oligodeoxynucleotide portion of the oligodeoxynucleotide-peptide conjugate forms Watson-Crick base pairs with the single-stranded RNA that flanks the RNA hairpin loop. The positively charged peptide makes specific electrostatic contacts with the negatively charged phosphate backbone of the RNA hairpin loop when attached to the N-allyl of (D)-aspartic alcohol but does not have the proper orientation to make these electrostatic contacts when attached to the N-allyl of (L)-aspartic alcohol. This modelling study emphasizes the importance of stereocontrol at the point of branching in synthesizing oligodeoxynucleotide-peptide conjugates for binding of RNA hairpin loops.     

tRNA nucleotide 47: an evolutionary enigma

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.     

Calcium-sensitive calcium influx in photoreceptor inner segments

The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.     

An antinociceptive effect of the intraperitoneal injection of nifedipine in rats, measured by tail-flick test

The tail-flick (TF) technique was used to assess the antinociceptive properties of nifedipine (NIF) given intraperitoneally (i.p.). First, the most suitable intensity of the noxious stimulus (temperature of the bulb) has been ascertained and used in the main study. Male Sprague-Dawley rats received NIF, dissolved in dimethyl sulfoxide (DMSO) at the doses of 0.0, 0.5, 2, 5, 10 and 15 mg/kg, or control with no injection. For the main study, the noxious stimulus was limited to 15 sec (cut-off time) and TF latencies were recorded up to 120 min. The antinociceptive response was expressed as the area under the curve for each rat and analyzed by one-way ANOVA. The antinociceptive response to the lower doses of NIF (0.5 and 2 mg/kg) did not differ from control (no injection) and DMSO alone. Significance was found at 5, 10 and 15 mg NIF with no difference among the doses. However, there was an increasing tendency of the mean values from 0.5 to 15 mg NIF resulting in a positive correlation. The correlation coefficient was 0.32483 (p = 0.015) and regression equation Y = (19.37) x dose + 1320. Our data suggest that spinal mechanisms are involved in NIF-induced antinociception.