Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis,Autophagy, and CLN3 Protein Function

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) Ca²⁺ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca²⁺-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca²⁺ chelation. Interrogation of intracellular Ca²⁺ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca²⁺ pools and in store-operated Ca²⁺ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca²⁺ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.     

Phenoloxidase activity of intact and chemically modified functional unit RvH1: a from molluscan Rapana venosa hemocyanin

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0mM SDS, and RvH1-a shows both, dopamine (K(M)=6.53mM, k(cat)/K(M)=1.29) and L-Dopa (K(M)=2.0mM, k(cat)/K(M)=2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K(M) value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.     

Lactoferrin stimulates erythrocyte Na+/K+ -adenosine triphosphatase: effect of some modulators of membrane phosphorylation

We studied the effect of some modulators of signal transduction on the erythrocyte Na+/ K+-ATPase. Go6976 and Go6983 (protein kinase C inhibitors) showed a stimulatory effect and calyculin A (protein phosphatase inhibitor) exerted an inhibitory effect on the Na pump activity. Some of the tested modulators of cell-signaling [protein phosphatase(s), phosphodiesterase, calmodulin and some protein kinases] interfered with the lactoferrin (Lf) stimulatory effect on the sodium pump. Lf itself was able to modulate the effect of some agents upon the pump activity. Moreover, an additive effect of stimulation was found when Lf and some agents were used simultaneously. The summarized results showed that: (i) Lf upregulates the Na+/K+-ATPase in erythrocytes and facilitates the K+ influx into the erythrocytes; (ii) the effect of pump stimulation is mediated by phosphorylation processes. These results suggest a potential opportunity for using Lf alone or together with other agents as a stimulator of the erythrocyte Na+/K+-ATPase.     

Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells.     

The effect of meat consumption on body odor attractiveness

Axillary body odor is individually specific and potentially a rich source of information about its producer. Odor individuality partly results from genetic individuality, but the influence of ecological factors such as eating habits are another main source of odor variability. However, we know very little about how particular dietary components shape our body odor. Here we tested the effect of red meat consumption on body odor attractiveness. We used a balanced within-subject experimental design. Seventeen male odor donors were on "meat" or "nonmeat" diet for 2 weeks wearing axillary pads to collect body odor during the final 24 h of the diet. Fresh odor samples were assessed for their pleasantness, attractiveness, masculinity, and intensity by 30 women not using hormonal contraceptives. We repeated the same procedure a month later with the same odor donors, each on the opposite diet than before. Results of repeated measures analysis of variance showed that the odor of donors when on the nonmeat diet was judged as significantly more attractive, more pleasant, and less intense. This suggests that red meat consumption has a negative impact on perceived body odor hedonicity.     

Guanylins--agents with natriuretic effect

Guanylins and uroguanylins are natriuretic peptides with different effects in many of tissues. In context with guanylins, the intestine-renal axis is presented. The overproduction of guanylin or uroguanylin leads to secondary diarrhea with stimulation of Cl(-) secretion. A diet high in salt lead especially to increased guanylin and uroguanylin secretion. Interesting applications with guanylins measurement could to be in hypertension diagnosis, monitoring of heart dysfunction treatment, intensive care etc.     

o-Diphenol oxidase activity of molluscan hemocyanins

Diphenoloxidase activities of two molluscan hemocyanins, isolated from the marine snails Rapana venosa and garden snails Helix vulgaris were studied using o-diphenol and L-Dopa as substrates. The dimers of H. vulgaris Hc show both, diphenol (K(m)=2.86 mM and K(cat)=4.48) and L-Dopa activity due to a more open active sites of the enzyme and better access of the substrates. The K(m) value of molluscan H. vulgaris Hc is very close to those of Helix pomatia and Sepia officinalis Hcs, but several times higher compared to those of Rapana and Octopus Hcs. Also HvH has a very high enzyme activity compared with other molluscan Hcs. Kinetic measurements with native RvH and both structural subunits, RvH1 and RvH2, show that RvH and only one structural subunit, RvH2, exhibited only o-diphenol activity, but no L-Dopa oxidizing activity.     

Arylamidase activity of neutral proteinase from Saccharomonospora canescens. Comparison with other Zn-proteinases that exhibit the same activity

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S(1), S(2), S(3), S(4) subsites shows that variations in all positions P(1) to P(4) in the substrate structure affect the catalytic efficiency. The importance of P(4)-S(4) and P(1)-S(1) interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases-astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn(2+) and a tyrosyl residue as a fifth ligand to the Zn(2+). It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.