Differences in Chromatin Texture and Nuclear Fractal Dimension Between Hashimoto's and Lymphocytic Thyroiditis Lymphocytes.

Previous evidence suggested that lymphocytic thyroiditis (LT) was a variant of Hashimoto's thyroiditis (HT), thus the aim of the current study is to quantify structural changes in histological specimens taken from HT and LT patients. A total of 600 images containing a single lymphocyte nucleus (300 nuclei per group) were obtained from 20 patients with HT and LT. In order to quantify changes in the nuclear architecture of investigated lymphocytes, the fractal dimension (FD) and some gray-level co-occurrence matrix texture parameters (angular second moment, inverse difference moment, contrast, entropy, and correlation) were calculated for each nucleus. A statistically significant difference in the FD of the "binary-outlined" nucleus and that of the corresponding "black-and-white" nucleus was detected between HT and LT lymphocyte nuclei. In addition, there was also a statistically significant difference in contrast and correlation between HT and LT lymphocyte nuclei. In conclusion, the results of this study suggested that there was a difference in structural complexity between investigated lymphocyte nuclei; additionally, LT lymphocytes possessed probably more complex texture and larger variations as well as more asymmetrical nuclei compared with HT lymphocytes. Accordingly, these findings indicate that LT is probably not a variant of HT; however, more complex studies are necessary to estimate differences between these types of thyroiditis.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

Exposure to ß-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Magnesium Effects on Nonsynaptic Epileptiform Activity in Leech Retzius Neurons.

The effects of Mg2+ on Ni(2+)-induced epileptiform bursting activity and input membrane resistance during this activity of leech Retzius neurons were examined using intracellular recordings. To induce epileptiform activity, 3 mmol/l NiCl2 was added into superfusing Ringer (Ri) saline. To test for dose-dependence of the effects of Mg2+ on the induced epileptiform activity, MgCl2 was added in concentrations from 1 mmol/l to 20 mmol/l Mg2+ to the Ni(2+)-containing Ri saline. Input membrane resistance (IMR) was measured in standard Ri, Ni2+ Ri and 20 mmol/l Mg2+Ni2+ Ri saline. Superfusion with Ni2+ Ri induced epileptiform bursting activity characterized by generation of paroxysmal depolarization shifts (PDSs). Parameters of epileptiform activity including PDS frequency, PDS duration, PDS amplitude and the number of spikes/PDS were measured. Magnesium suppressed Ni(2+)-induced epileptiform activity, significantly reducing values of all parameters observed in a concentration-dependent manner. The highest concentration applied of 20 mmol/l Mg2+ completely eliminated epileptiform activity. To test for the effect of Mg2+ on membrane conductance during bursting, IMR was measured. Magnesium significantly increased IMR during bursting suppression.

The Impact of Chronic Magnesium Deficiency on Excitable Tissues-Translational Aspects.

Neuromuscular excitability is a vital body function, and Mg2+ is an essential regulatory cation for the function of excitable membranes. Loss of Mg2+ homeostasis disturbs fluxes of other cations across cell membranes, leading to pathophysiological electrogenesis, which can eventually cause vital threat to the patient. Chronic subclinical Mg2+ deficiency is an increasingly prevalent condition in the general population. It is associated with an elevated risk of cardiovascular, respiratory and neurological conditions and an increased mortality. Magnesium favours bronchodilation (by antagonizing Ca2+ channels on airway smooth muscle and inhibiting the release of endogenous bronchoconstrictors). Magnesium exerts antihypertensive effects by reducing peripheral vascular resistance (increasing endothelial NO and PgI2 release and inhibiting Ca2+ influx into vascular smooth muscle). Magnesium deficiency disturbs heart impulse generation and propagation by prolonging cell depolarization (due to Na+/K+ pump and Kir channel dysfunction) and dysregulating cardiac gap junctions, causing arrhythmias, while prolonged diastolic Ca2+ release (through leaky RyRs) disturbs cardiac excitation-contraction coupling, compromising diastolic relaxation and systolic contraction. In the brain, Mg2+ regulates the function of ion channels and neurotransmitters (blocks voltage-gated Ca2+ channel-mediated transmitter release, antagonizes NMDARs, activates GABAARs, suppresses nAChR ion current and modulates gap junction channels) and blocks ACh release at neuromuscular junctions. Magnesium exerts multiple therapeutic neuroactive effects (antiepileptic, antimigraine, analgesic, neuroprotective, antidepressant, anxiolytic, etc.). This review focuses on the effects of Mg2+ on excitable tissues in health and disease. As a natural membrane stabilizer, Mg2+ opposes the development of many conditions of hyperexcitability. Its beneficial recompensation and supplementation help treat hyperexcitability and should therefore be considered wherever needed.

Environmental Neurotoxin ß-N-Methylamino-L-alanine (BMAA) as a Widely Occurring Putative Pathogenic Factor in Neurodegenerative Diseases.

In the present review we have discussed the occurrence of ß-N-methylamino-L-alanine (BMAA) and its natural isomers, and the organisms and sample types in which the toxin(s) have been detected. Further, the review discusses general pathogenic mechanisms of neurodegenerative diseases, and how modes of action of BMAA fit in those mechanisms. The biogeography of BMAA occurrence presented here contributes to the planning of epidemiological research based on the geographical distribution of BMAA and human exposure. Analysis of BMAA mechanisms in relation to pathogenic processes of neurodegeneration is used to critically assess the potential significance of the amino acid as well as to identify gaps in our understanding. Taken together, these two approaches provide the basis for the discussion on the potential role of BMAA as a secondary factor in neurodegenerative diseases, the rationale for further research and possible directions the research can take, which are outlined in the conclusions.

Cadmium significantly changes major morphometrical points and cardiovascular functional parameters during early development of zebrafish.

Living organisms are commonly exposed to cadmium and other toxic metals. A vast body of research has shown the significant effects of these toxic metals on developmental processes. In order to study the role of toxic metals on early developmental stages of eukaryotes, we explored the effect of cadmium (Cd2+) contaminant on zebrafish. Thus, zebrafish embryos were exposed to 3 mg/L (16.7 µM) Cd2+ for 96 h and imaged every 24 h from the exposure onwards. Hatching rates of the eggs were determined at 72 h, followed by analyses at 96 h for: survival rate, morphometrical factors, and functional parameters of the cardiovascular system. Interestingly enough, significant hatching delays along with smaller cephalic region and some morphological abnormalities were observed in the treatment group. Moreover, substantial changes were noticed in the length of notochord and embryo, absorption of yolk sac with shorter extension, area of swimming bladder, as well as pericardium sac after Cd2+ treatment. Cadmium also caused significant abnormalities in heart physiology which could be the leading cause of mentioned morphological deformities. Herein, our results shine light on systematic acute embryological effects of cadmium in the early development of zebrafish for the first time.

Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons.

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons' membrane potential is dependent on sodium ions. Depolarizing effect of 5·10-3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ±â€¯0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ±â€¯1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect - activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10-3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ±â€¯0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10-3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ±â€¯2.22 mV to 16.28 ±â€¯2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Augmentation and ionic mechanism of effect of beta-N-methylamino-L-alanine in presence of bicarbonate on membrane potential of Retzius nerve cells of the leech Haemopis sanguisuga.

The role of neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (L-BMAA) as a putative causative agent of Western pacific amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC) has recently been reinvigorated. In view of this data we have investigated the strength and mechanism of effect of L-BMAA in presence of 20 mmol/L bicarbonate (a cofactor for BMAA) on membrane potential of the Leech Haemopis sanguisuga. Our results show that L-BMAA has excitatory effect in bicarbonate containing solution, which is more potent than in nominally bicarbonate free solution. This potentiation by bicarbonate is L-BMAA specific, as it was not exhibited by beta-N-oxalylamino-L-alanine. The effect of L-BMAA was partially blocked by non-NMDA receptor antagonist CNQX. Application of L-BMAA caused a decrease in input membrane resistance, an increase of intracellular sodium activity, and a decrease of intracellular potassium activity. Present findings indicate that BMAA could initiate excitotoxicity through activation of non-NMDA ionotropic glutamate receptors.

Ethanol and magnesium suppress nickel-induced bursting activity in leech Retzius nerve cells.

In the present study we have examined effects of ethanol and magnesium on Ni(2+)-induced bursting of leech Retzius cells. Saline with 3 mmol/l NiCl2 induced spontaneous bursting activity, characterized by rapid depolarizations to a plateau level during which bursts of action potentials occurred. To test for the mechanism of bursting initiation external Na+ was completely removed. Removal of external Na(+) in presence of 3 mmol/l NiCl2 terminated the bursting activity. Application of 2% ethanol solution significantly decreased the bursting frequency, duration and amplitude of depolarization plateaus, and the number of spikes per plateau. Solution containing 10 mmol/l Mg2+ almost completely abolished the oscillatory activity of the neurons and completely suppressed action potential generation. We conclude that ethanol and magnesium suppress Ni(2+)-induced epileptic activity.

Comparison of effects of endogenous and exogenous excitatory amino acids on Retzius nerve cells of the leech.

In this paper we examine the effects of L-aspartate, L-glutamate, and beta-N-oxalylamino-L-alanine (Lathyrus toxin) on Retzius nerve cells of the leech Haemopis sanguisuga. The goal was to compare the electrophysiological effects of endogenous vs. exogenous amino acids, known as potent neurotoxins, through the mechanism ofexcitotoxicity. We used classical intracellular recordings on Retzius nerve cells in isolated ganglia of the leech, and plotted dose-response curves to compare potencies. Our results show that Lathyrus toxin is more than 200 times more potent in depolarizing the membrane potential on our model than L-aspartate and L-glutamate, which are approximately equipotent.

Modulation of luminal L-alanine transport in proximal tubular cells of frog kidney induced by low micromolar Cd2+ concentration.

The kidneys are recognized as a major target of cadmium-induced toxicity. However, all mechanisms that are involved in the early stages of cadmium nephrotoxicity, particularly considering low micromolar concentrations of cadmium ions (Cd2+) are still unknown. Therefore, the aim of this study was to investigate the effects of peritubular acute exposure to micromolar Cd2+ concentration (2.3 µmol/L) on the rapid depolarization and the rate of slow repolarization of peritubular membrane potential difference (PD), induced by luminal application of L-alanine in proximal tubular cells of frog kidney. The results showed that the luminal application of L-alanine rapidly depolarized the peritubular membrane PD of -42.00 ±â€¯11.68 mV by 23.89 ±â€¯4.15 mV with an average rate of slow repolarization of 5.64 ±â€¯0.81 mV/min. Additionally, peritubular acute exposure to Cd2+ induced change in rapid depolarization of peritubular membrane PD of -53.33 ±â€¯13.01 mV by 18.78 ±â€¯3.31 mV (P < 0.01) after luminal application of L-alanine. Also, peritubular acute exposure to Cd2+ led to statistically significant decrease in the rate of slow repolarization of peritubular membrane PD (3.53 ±â€¯0.35 mV/min; P < 0.05). In conclusion, these results suggest that peritubular acute exposure to low micromolar Cd2+ concentration decreased the rapid depolarization and the rate of slow repolarization of peritubular membrane PD induced by luminal application of L-alanine. This is followed by reduced luminal sodium-coupled transport of L-alanine and this change may be one of the possible mechanisms involved in the early stages of Cd2+-induced nephrotoxicity.

Electrophysiological effect of beta-N-methylamino-L-alanine on retzius nerve cells of the leech Haemopis sanguisuga.

We investigated the effect of beta-N-methylamino-L-alanine (L-BMAA) on input membrane resistance of leech Retzius nerve cells and the effect of the same substance on the membrane potential in the presence of 20 mM bicarbonate. Results of our experiments show that L-BMAA significantly reduces input membrane resistance on our model. This leads to the conclusion that L-BMAA depolarizes the cell by increasing membrane permeability and conductance. The effect of L-BMAA in 20 mM bicarbonate is significantly higher than in standard Ringer solution. These results indicate that bicarbonate increases the excitatory effect of L-BMAA in our model.