Ante L. Padjen

Electrochemical investigations of the interaction of C-reactive protein (CRP) with a CRP antibody chemically immobilized on a gold surface

A possibility of using a range of dc and ac electrochemical techniques to probe associative interactions of C-reactive protein (CRP) with CRP antibody (aCRP) immobilized on a gold electrode surface was investigated. It was demonstrated that the investigated electrochemical techniques can be used efficiently to probe these interactions over a wide CRP concentration range, from 1.15 x 10(-5) to 1.15 mg L(-1). The measured sensitivity of the techniques is in the following decreasing order: differential pulse voltammetry, charge-transfer resistance obtained from electrochemical impedance spectroscopy (EIS), cyclic voltammetry, chronoamperometry, and double-layer capacitance deduced from EIS measurements which gave the poorest sensitivity. Measurements of kinetic parameters demonstrated that the associative interactions of CRP with the immobilized aCRP reached quasi-equilibrium after 20-30 min. The kinetics of these interactions was modeled successfully using a two-step kinetic model. In this model, the first step represents reversible CRP-aCRP associative-dissociative interactions, while the second step represents the irreversible transformation of the bound CRP into a thermodynamically stable configuration. It was demonstrated that the thermodynamically stable configuration of CRP starts prevailing after 7 min of interaction of CRP with the immobilized aCRP.

Effect of temperature on the rapid retrograde transport of microscopically visible intra-axonal organelles

Summary The effects of temperature on the retrograde axonal transport of microscopically visible intra-axonal organelles was studied in acutely isolated myelinated axons from bullfrog sciatic nerve. Dark-field microscopy, cinemicrophotography, and computer-based analytical methods were employed. More than 90% of the optically detectable particles move in the retrograde direction. In the range of 8–37°C, the average speed of these particles is highly temperature sensitive, with a Q10 of 2.5–3.5. Movement stops entirely at about 5°C. Our findings show that the effect of temperature must be taken into consideration when comparing rates of axonal transport reported for different experimental systems. The average organelle speeds in frog are similar to those reported by others in mature axons of other species, but are faster than the rates which have been measured in neurites in tissue culture. The mean speeds of the particles moving in the retrograde direction are similar to the rates of retrograde axonal transport estimated by other methods. The average particle speeds are less than half of the anterograde rapid transport rates reported in the literature. However, the fastest particles move (in the retrograde direction) at speeds which approach the anterograde rates. The standard deviation of the distribution of retrograde particle velocities also varies with temperature, but remains a constant fraction (about 36%) of the population mean at all temperatures. The same fractional standard deviation (i.e. S.D. of the mean= 0.36) has been reported by others in axons from other species and in neurites in tissue culture, and may reflect a fundamental property of the axonal transport process.

Actions of Acidic Amino Acids on the Excitability of Medial Hypothalamic Neurons in the Rat

Systemic administration of acidic amino acids produces selective neurotoxic effects on neurons in the arcuate nucleus. However, little is known on the comparative or topographical actions of various a

Further evidence in support of taurine as a mediator of synaptic transmission in the frog spinal cord

It has been reported that 6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-1, 1-dioxide (AMBD, TAG) is a specific blocker of taurine and beta-alanine responses in the central nervous system. We have re-examined the effect of AMBD on amino acid and synaptically evoked responses recorded from isolated hemisected frog spinal cords by means of the sucrose gap technique. When indirect responses were blocked by adding tetrodotoxin (0.2 microM) or manganese chloride (2 mM) to the normal Ringer solution, AMBD (0.01-0.5 mM) selectively antagonized taurine, beta-alanine, hypotaurine and kojic amine evoked depolarizations of primary afferents at their intramedullary part (dorsal root terminals, DRT) and on dorsal root ganglia (DRG), without significantly affecting responses to glutamate (on DRT), glycine (on DRT) or GABA (on DRT and DRG). Depolarizing responses to taurine and beta-alanine (1 mM) were depressed by up to 50% with 0.1 mM AMBD and often completely antagonized with 0.25 mM AMBD. In normal Ringer solution, AMBD selectively antagonized the dorsal root potential evoked by ventral root stimulation (VR-DRP, threshold at 0.02 mM AMBD, 90% block with 0.25 mM); other synaptic potentials increased in duration and/or amplitude, demonstrating a strong convulsant effect of AMBD. Thus, the depolarizing responses of taurine, beta-alanine and hypotaurine on primary afferents are pharmacologically indistinguishable from the VR-DRP. These results are in agreement with the proposal that taurine or a taurine-like substance (possibly beta-alanine or hypotaurine) is the mediator of VR-DRP in amphibian spinal cord.

Explant cultures of adult amphibian sympathetic ganglia: electrophysiological and pharmacological investigation of neurotransmitter and nucleotide action.

Explant cultures from adult bullfrog sympathetic ganglia can be maintained in vitro for several months and are suitable for electrophysiological recording. The cultured neurons display morphological, electrophysiological and pharmacological characteristics similar, in most respects, to those reported for acutely isolated sympathetic ganglia. Individual cells were visualized by Nomarski optics and impaled with a glass micropipette, which was used for voltage recording and current injection. The average specific membrane properties, calculated from cell dimensions and responses to current injection, were Vm = -46 mV, Rin = 27 M omega, Rm = 1665 omega cm2, tau m = 5 msec, and Cm = 3.2 microF/cm2. Bath perfusion of the cholinergic agonist muscarine depolarized most neurons with an increase in input resistance, while carbachol depolarized neurons with both increases and decreases in input resistance. GABA depolarized all neurons tested with a decreased resistance. High concentrations of catecholamines (2-5 mM) generally hyperpolarized explanted neurons, usually in association with an increased resistance. Extracellularly or intracellularly applied cyclic AMP and two other analogues produced weak and inconsistent hyperpolarizations. In contrast, perfusion or iontophoresis of most non-cyclic purine and pyrimidine nucleotides markedly depolarized most neurons in association with an increased input resistance. UTP and UDP were most potent, revealing threshold concentrations of about 10(-8) to 5 x 10(-8) M. The related nucleosides were largely ineffective. The nucleotide-evoked depolarizations were similar to the muscarine responses but were not blocked by atropine. These results suggest that the purine or pyrimidine nucleotides should be considered for a possible involvement in neurotransmission in sympathetic ganglia.

Some characteristics of baclofen-evoked responses of primary afferents in frog spinal cord

Baclofen has been shown to be a selective agonist for a subclass of GABA receptors (GABAB) in many regions of the vertebrate nervous system. On the intraspinal terminals of dorsal roots (DRT), it evokes a pure hyperpolarizing response. We have previously shown that the response of DRT to GABA and some of its analogs (e.g. kojic amine) in isolated frog spinal cord is dual in nature, consisting of a bicuculline-sensitive depolarizing component and a bicuculline-resistant hyperpolarizing component. Under the working hypothesis that the hyperpolarizing component of the GABA-evoked response is mediated by the activation of GABAB receptors, we have examined, using the sucrose gap technique, some characteristics of the response of DRT to baclofen. We have found that this response is stereospecific (L-baclofen being about 100 times more potent than D-baclofen), dependent on [K]o (response amplitude inversely related to [K]o), blocked by barium (0.5 mM causing a reduction of the response amplitude to 37% of control), and is not significantly affected by 4-aminopyridine, nor by inorganic calcium channel blockers (manganese, cobalt, cadmium). Some proposed GABAB antagonists (delta-aminovaleric acid, delta-aminolaevulinic acid, phaclofen) are also rather ineffective at blocking it. These results are therefore consistent with the notion that the baclofen-evoked response of DRT is mediated by an increase in conductance to potassium ions.

Electrophysiological analysis of peptide actions in neural tissue

The functional characterisation of endogenous neural substances demands the concerted efforts of investigators in numerous disciplines, a point that is adequately illustrated in the proceedings of this symposium. Over the past 20 years, electrophysiological techniques have provided a most valuable insight into the function of diverse excitable tissues, especially the nervous system. Because of its excellent time resolution, relatively simple technology and often unequivocal interpretation, electrophysiology has continued to be a most useful method for real-time monitoring of neuronal activity especially at the cell and/or membrane level. Electrophysiological techniques have been used to elucidate basic mechanisms of synaptic transmission, and continue to be of major importance in the sometimes frustrating analysis of complex neural organisation and behaviour. A large portion of our knowledge of central nervous system (CNS) pharmacology is derived from the analysis of drug effects on the electrical activity of the nervous system.

Translation of Pre-Clinical Studies into Successful Clinical Trials for Alzheimer's Disease: What are the Roadblocks and How Can They Be Overcome?

Preclinical studies are essential for translation to disease treatments and effective use in clinical practice. An undue emphasis on single approaches to Alzheimers disease (AD) appears to have retarded the pace of translation in the field, and there is much frustration in the public about the lack of an effective treatment. We critically reviewed past literature (1990-2014), analyzed numerous data, and discussed key issues at a consensus conference on Brain Ageing and Dementia to identify and overcome roadblocks in studies intended for translation. We highlight various factors that influence the translation of preclinical research and highlight specific preclinical strategies that have failed to demonstrate efficacy in clinical trials. The field has been hindered by the domination of the amyloid hypothesis in AD pathogenesis while the causative pathways in disease pathology are widely considered to be multifactorial. Understanding the causative events and mechanisms in the pathogenesis are equally important for translation. Greater efforts are necessary to fill in the gaps and overcome a variety of confounds in the generation, study design, testing, and evaluation of animal models and the application to future novel anti-dementia drug trials. A greater variety of potential disease mechanisms must be entertained to enhance progress.

Intra-axonal recording from large sensory myelinated axons: Demonstration of impaired membrane conductances in early experimental diabetes

Aim/hypothesisDiabetic neuropathy is accompanied by a range of positive (paresthaesia, dysesthaesia, pain) and negative (hypesthaesia, anesthaesia) neurological symptoms suggesting widespread alterations in axonal excitability. The nature and the mechanisms underlying these alterations in axonal excitability are not well understood. The aim of this study was to examine the extent of changes in membrane properties of an identified neuronal structure—the large myelinated sensory axons in early experimental diabetes in rats.MethodsIntra-axonal microelectrode recordings from large sensory myelinated axons from the isolated sural nerve in short-term streptozotocin-induced diabetic rats were used to study membrane properties using standard current-clamp technique.ResultsIn addition to decreased conduction velocity we found several differences in physiological properties of sensory axons from diabetic rats: decreased resting membrane potential, decreased single action potential amplitude associated with slower rate of rise and decrease in inward rectification associated with slight alteration in outwardly rectifying conductances indicating impaired potassium conductances.Conclusion/InterpretationThese results extend previous indirect evidence that potassium and sodium ionic conductances, most notably the inward rectifier (IR, Ih), are altered in large sensory axons of diabetic rats. The depression of IR could underly clinical neurological findings in diabetic patients.

Higher sensitivity of CA1 synapses to aglycemia in streptozotocin-diabetic rats is age-dependent.

We studied conduction velocity in peripheral nerves and the block of synaptic transmission produced by lack of glucose in hippocampal slices from 4- and 12-month-old streptozotocin-induced diabetic rats and their age-matched controls. In sural nerves of young and old diabetic rats, the conduction velocity was reduced by 30-35%. In slices from young diabetics, CA1 synaptic transmission was more sensitive to aglycemia than in control slices. However, all slices from older rats showed comparable increases in CA1 synaptic sensitivity to aglycemia. We conclude that the cerebral adaptation to diabetic hyperglycemia apparent in the hippocampus of young rats is masked in older rats by an age-dependent increase in sensitivity to lack of glucose.