In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg^{? 1} and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg^{? 1} and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. K_i values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

Association of nicotinamide-N-methyltransferase (NNMT) gene rs694539 variant with bipolar disorder

Here we report the association of the rs694539 variant of nicotinamide-N-methyltransferase gene with bipolar disorder in a case–control study of 95 bipolar disorder patients and 201 healthy controls (χ² = 13.382, P = 0.001). With the polymerase chain reaction restriction fragment length polymorphism method we developed we were able to show the association for the first time. This new finding may provide evidence to understand the mechanism of the disease.     

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used ¹⁵N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before ¹H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.     

CYP2E1 and Parkinson’s disease in a MPTP-induced C57BL/6 mouse model

Background

A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca²⁺ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results

To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion

Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.     

Perceptual Learning of Interrupted Speech

The intelligibility of periodically interrupted speech improves once the silent gaps are filled with noise bursts. This improvement has been attributed to phonemic restoration, a top-down repair mechanism that helps intelligibility of degraded speech in daily life. Two hypotheses were investigated using perceptual learning of interrupted speech. If different cognitive processes played a role in restoring interrupted speech with and without filler noise, the two forms of speech would be learned at different rates and with different perceived mental effort. If the restoration benefit were an artificial outcome of using the ecologically invalid stimulus of speech with silent gaps, this benefit would diminish with training. Two groups of normal-hearing listeners were trained, one with interrupted sentences with the filler noise, and the other without. Feedback was provided with the auditory playback of the unprocessed and processed sentences, as well as the visual display of the sentence text. Training increased the overall performance significantly, however restoration benefit did not diminish. The increase in intelligibility and the decrease in perceived mental effort were relatively similar between the groups, implying similar cognitive mechanisms for the restoration of the two types of interruptions. Training effects were generalizable, as both groups improved their performance also with the other form of speech than that they were trained with, and retainable. Due to null results and relatively small number of participants (10 per group), further research is needed to more confidently draw conclusions. Nevertheless, training with interrupted speech seems to be effective, stimulating participants to more actively and efficiently use the top-down restoration. This finding further implies the potential of this training approach as a rehabilitative tool for hearing-impaired/elderly populations.     

Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I,II and IX with some hydroxylic compounds

A series of hydroxylic compounds (1–10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068–4003, 0.012–9.9 and 0.025–115?μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the K_I values were calculated.     

A New Type of Anatolian Propolis: Evaluation of Its Chemical Composition,Activity Profile and Botanical Origin

This study was undertaken to analyze the phenolic profiles of 19 propolis samples from Turkey by using a high‐performance thin‐layer chromatographic (HPTLC) method in order to identify their plant origins. Furthermore, their antioxidant and antimicrobial activity profiles were comparatively evaluated. For the appraisal of antioxidant potential, total phenolic (TPC) and total flavonoid contents (TFC) of propolis samples were firstly determined and then their effects on free radicals were evaluated by FRAP, ABTS^.+, CUPRAC, DPPH^. and HPTLC‐DPPH^. methods. Antimicrobial activity of propolis samples against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 11229) and Candida albicans ATCC 10231 were determined by disc diffusion and broth dilution methods. HPTLC fingerprinting analyses revealed that O‐type (botanical origin from Populus nigra L.) was the primarily available propolis type in Turkey. Moreover, 3‐O‐methylquercetin (3MQ) rich propolis was identified as a new propolis type for the first time. Principal component analysis (PCA) indicated that 3MQ‐type propolis differs from the O‐type. Antioxidant activity studies showed that O‐type of propolis possesses higher antioxidant effect than the other tested propolis types. Quercetin, caffeic acid, caffeic acid phenethyl ester (CAPE) and galangin were determined to contribute significantly to the antioxidant potential of O‐type propolis among others. Propolis extracts exerted moderate antimicrobial activity against the tested microorganisms with MIC values between the ranges of 128–512 μg/mL.     

Physical and functional interactions of Galphaq with Rho and its exchange factors

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.