Predatory Odor Exposure as a Potential Paradigm for Studying Emotional Modulation of Memory Consolidation-The Role of the Noradrenergic Transmission in the Basolateral Amygdala.

The pivotal role of the basolateral amygdala (BLA) in the emotional modulation of hippocampal plasticity and memory consolidation is well-established. Specifically, multiple studies have demonstrated that the activation of the noradrenergic (NA) system within the BLA governs these modulatory effects. However, most current evidence has been obtained by direct infusion of synthetic NA or beta-adrenergic agonists. In the present study, we aimed to investigate the effect of endogenous NA release in the BLA, induced by a natural aversive stimulus (coyote urine), on memory consolidation for a low-arousing, hippocampal-dependent task. Our experiments combined a weak object location task (OLT) version with subsequent mild predator odor exposure (POE). To investigate the role of endogenous NA in the BLA in memory modulation, a subset of the animals (Wistar rats) was treated with the non-selective beta-blocker propranolol at the end of the behavioral procedures. Hippocampal tissue was collected 90 min after drug infusion or after the OLT test, which was performed 24 h later. We used the obtained samples to estimate the levels of phosphorylated CREB (pCREB) and activity-regulated cytoskeleton-associated protein (Arc)-two molecular markers of experience-dependent changes in neuronal activity. The result suggests that POE has the potential to become a valuable behavioral paradigm for studying the interaction between BLA and the hippocampus in memory prioritization and selectivity.

Protective Effect of the Novel Melatonin Analogue Containing Donepezil Fragment on Memory Impairment via MT/ERK/CREB Signaling in the Hippocampus in a Rat Model of Pinealectomy and Subsequent Aß1-42 Infusion.

A reduction in melatonin function contributes to the acceleration of Alzheimer's disease (AD), and understanding the molecular processes of melatonin-related signaling is critical for intervention in AD progression. Recently, we synthesized a series of melatonin analogues with donepezil fragments and tested them in silico and in vitro. In this study, one of the most potent compounds, 3c, was evaluated in a rat model of pinealectomy (pin) followed by icvAß1-42 infusion. Melatonin was used as the reference drug. Treatment with melatonin and 3c (10 mg/kg, i.p. for 14 days) had a beneficial effect on memory decline and the concomitant increase in hippocampal Aß1-42 and pTAU in the pin+icvAß1-42 rats. Melatonin supplementation facilitated non-amyloidogenic signaling via non-receptor (histone deacetylase sirtuin 1, SIRT1) and receptor-related signaling (MT/ERK/CREB). The hybrid 3c analogue up-regulated the MT1A and MT2B receptors, pERK and pCREB. Our results strongly support the hypothesis that melatonin-related analogues may become a promising drug candidate for Alzheimer's disease therapy.

Physicochemical, Antioxidant, and Antimicrobial Properties of Three Medicinal Plants from the Western Part of the Rhodope Mountains, Bulgaria.

The present study examined the physicochemical, antioxidant, and antimicrobial properties of three medicinal plants: thyme (Thymus callieri Borbás ex Velen), cotton thistle (Onopordum acanthium L.), and hawthorn fruit (Crataegus monogyna Jacq.) from the Western Rhodope Mountains, Bulgaria. The first stage determined the physicochemical characteristics (moisture, ash, carbohydrates, proteins, and vitamin C) of the three herbs. The second stage investigated four types of extracts (aqueous, oil, methanolic, and ethanolic) of each herb and evaluated their total phenolic content, the presence of phenolic compounds (flavonoids and phenolic acids), their antioxidant activity, and antimicrobial properties. Thyme was characterised by the highest ash, protein, and vitamin C content (6.62%, 11.30%, and 571 mg/100 g, respectively). Hawthorn fruit showed the highest moisture and carbohydrate content (8.50% and 4.20%, respectively). The 70% ethanolic extracts of the three herbs exhibited the highest levels of phenolic compounds and, consequently, pronounced antioxidant activity, compared to the other three types of extracts. The aqueous, oil, methanolic, and ethanolic thyme extracts demonstrated the highest total phenolic content-TPC (27.20 mg GAE/g, 8.20 mg GAE/g, 31.70 mg GAE/g, and 310.00 mg GAE/g, respectively), compared to the extracts of the other two plants. These results were consistent with the highest antioxidant activity of the thyme extracts determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, the oxygen radical absorbance capacity (ORAC) assay, and the hydroxyl radical averting capacity (HORAC) assay (except for the oil extract examined using the DPPH method). The results from the high-performance liquid chromatography (HPLC) analysis revealed that the flavonoid quercetin-3-ß-glucoside had the highest concentration in thyme (374.5 mg/100 g), while myricetin dominated in the cotton thistle (152.3 mg/100 g). The phenolic acid content analysis showed prevalence of rosmaric acid in the thyme (995 mg/100 g), whereas chlorogenic acid was detected in the highest concentration in the cotton thistle and hawthorn fruit (324 mg/100 g and 27.7 mg/100 g, respectively). The aqueous, methanolic, and ethanolic extracts showed moderate to high antibacterial potential but limited antifungal activity. None of the oil extracts inhibited the test microorganisms used in the study.

Spontaneously hypertensive rats vs. Wistar Kyoto and Wistar rats: An assessment of anxiety, motor activity, memory performance, and seizure susceptibility.

Spontaneously hypertensive rats (SHRs) are widely accepted for modeling essential hypertension and Attention deficit hyperactivity disorder (ADHD). However, data concerning central nervous system changes associated with behavioral responses of this strain and usage of Wistar Kyoto (WKY) rats as controls are confounding. The objective of the present study was to assess the impact of anxiety and motor activity on the cognitive responses of SHRs compared to Wistar and WKY rats. In addition, the role of brain-derived neurotrophic factor (BDNF) in the hippocampus on cognitive behavior and seizure susceptibility in the three strains was evaluated. In Experiment#1, SHR demonstrated impulsive responses in the novelty suppression feeding test accompanied by impaired spatial working and associative memory in the Y maze and object recognition test compared with the Wistar rat but not WKY rats. In addition, the WKY rats exhibited diminished activity compared to Wistar rats in an actimeter. In Experiment#2, the seizure susceptibility was assessed by 3-min electroencephalographic (EEG) recording after two consecutive injections of pentylenetetrazol (PTZ) (20+40 mg/kg). The WKY rats were more vulnerable to rhythmic metrazol activity (RMA) than the Wistar rats. In contrast, Wistar rats were more prone to generalized tonic-clonic seizures (GTCS) than WKY rats and SHRs. Control SHR had lower BDNF expression in the hippocampus compared to Wistar rats. However, while the BDNF levels were elevated in the Wistar and WKY rats after PTZ injection, no change in this signaling molecule was observed in the SHR in the seizure condition. The results suggest Wistar rats as a more appropriate control of SHR than WKY rats for studying memory responses mediated by BDNF in the hippocampus. The higher vulnerability to seizures in Wistar and WKY rats compared to SHR might be linked to PTZ-induced decreased expression of BDNF in the hippocampus.

Value-Added Dietary Fiber Concentrate Obtained as Waste after Protein Isolation from Ethanol-Treated Sunflower Meal.

Deproteinized sunflower meal (DSM) was obtained as waste from ethanol-treated sunflower meal after alkaline extraction of proteins. The study aimed at biochemically and functionally characterizing the material concerning its potential practical application and valuability. The DSM consisted mainly of proteins (19.88%) and dietary fibers (61.06%) the majority of which were insoluble (53.09%). Cellulose (30.87%) and lignin (21.79%) were the most contributing compounds to the total amount of dietary fibers. The DSM contained Fe (133.29 mg/kg), Zn (201.56 mg/kg), and Cu (31.87 mg/kg). The analyses defined the DSM as a fiber concentrate with relatively high thermal stability. The distraction of the material began at 170°Ð¡ with a maximum speed at 277°Ð¡. The highest water absorption capacity (WAC) of the DSM was observed at pH 6 and 7 (approximately 8 g H2O/g sample) under all studied conditions including pH from 3 to 10 and three levels of NaCl concentrations (0.00 M, 0.03 M, and 0.25 M). At pH 7, increasing temperature from 20°C to 60°C increased the WAC of the DSM from 8.13 g H2O/g sample to 9.80 g H2O/g sample, respectively. Further increase in the temperature diminished the WAC of the DSM. At pH 6, the increase in temperature did not influence positively the WAC of the DSM. The study demonstrated the potential of the DSM, a waste obtained from the protein isolation process, as a valuable ingredient/additive in the food industry.

Metabolic footprint in young, middle-aged and elderly rats with melatonin deficit.

The pineal gland is suggested to be an essential area involved in the programming of fertility, growth, aging, and death of mammals via the released hormone melatonin.The present study aimed to ascertain the effect of melatonin deficit on several physiological and metabolic parameters, closely associated with the aging process, at certain stages of ontogenesis. Sham and rats with pinealectomy, operated at ages 3, 14, and 18-months, respectively, were tested two months later. Sham rats demonstrated an age-related decline of muscle strength, exercise endurance, motor activity, food intake, calorimetric parameters, and impaired lipid profile. Pinealectomy reduced the maximal time to exhaustion and body weight gain while diminished motor activity, food intake, O2 consumption, CO2 production, and energy expenditure during the Dark phase in the youngest rat group. In addition, melatonin deficit elevated arterial blood pressure (systolic, diastolic, and mean arterial pressure) and increased serum glucose and triglyceride level in 3-month-old rats while decreased the liver enzyme activity in 14-month-old rats. In conclusion, the present study brought new insights confirming the complex impact of melatonin deficit on important physiological, metabolic and biochemical markers related to aging and demonstrated for the first time that the lack of melatonin hormone is harmful in young adult rats.

Multifunctionality of Rapeseed Meal Protein Isolates Prepared by Sequential Isoelectric Precipitation.

Rapeseed meal is a by-product of the oil-producing industry with a currently underestimated application. Two protein isolates, PI2.5-8.5 or PI10.5-2.5, were obtained from industrial rapeseed meal after treatment with an aqueous ethanol solution. The alkaline-extracted proteins were sequentially precipitated by two different modes, from pH 10.5 to 2.5, and vice versa, from 2.5 to 8.5, with a step of 1 pH unit. The preparation approach influenced both the functional and antioxidant properties of the isolates. The PI10.5-2.5 exhibited higher water and oil absorption capacities than PI2.5-8.5, reaching 2.68 g H2O/g sample and 2.36 g oil/g sample, respectively. The emulsion stability of the PI2.5-8.5, evaluated after heating at 80 °C, was either 100% or close to 100% for all pH values studied (from 2 to 10), except for pH 6 where it reached 93.87%. For the PI10.5-2.5, decreases in the emulsion stability were observed at pH 8 (85.71%) and pH 10 (53.15%). In the entire concentration range, the PI10.5-2.5 exhibited a higher scavenging ability on 2,2-diphenyl-1-picryl hydrazyl (DPPH) and hydroxyl radicals than PI2.5-8.5 as evaluated by DPPH and 2-deoxyribose assays, respectively. At the highest concentration studied, 1.0%, the neutralization of DPPH radicals by PI10.5-2 reached half of that exhibited by synthetic antioxidant butylhydroxytoluene (82.65%). At the same concentration, the inhibition of hydroxyl radicals by PI10.5-2 (71.25%) was close to that achieved by mannitol (75.62%), which was used as a positive control. Established antioxidant capacities add value to the protein isolates that can thus be used as both emulsifiers and antioxidants.

Comparative analysis of morphological and molecular approaches integrated into the study of the dinoflagellate biodiversity within the recently deposited Black Sea sediments - benefits and drawbacks.

One of the assets, assigned to the phytoplankton resting stages, is that of serving as the "memory" of the aquatic ecosystems and preserved biodiversity in the course of time. However, an accurate cyst identification proves to be a more difficult and extremely challenging process, even today. In order to gain a better taxonomic coverage of cyst assemblages in the Black Sea, an integrated approach of the classical morphological identification with metabarcoding methods (MySeq sequencing of V7-V9 regions of the 18S rDNA) was applied on thirteen surface sediment samples collected from different sites. A total number of 112 dinoflagellate taxa was detected at the species level and ascribed to 51 genera. In general, it is the molecular analysis that yields a higher number of taxa as compared to those obtained through the morphological taxonomy (66 taxa based on the DNA sequences versus 56 morphologically-identified taxa). Besides, it should be pointed out that the integrated dataset includes 14 potentially toxic dinoflagellate species. Discerned, subsequently, was a good dataset consistency for ten species, followed by some discrepancies as to a number of taxa, identified with one of the methods only, due to specific methodological biases. On the whole, it could be concluded that the combination of morphological and molecular methods is likely to increase the potential for a more reliable taxonomic assessment of phytoplankton diversity in marine sediments which, in turn, proves conclusively the utmost importance of the integrated approach.

Enhanced Solubility of Rapeseed Meal Protein Isolates Prepared by Sequential Isoelectric Precipitation.

The solubility of plant protein isolates is a key determinant of their potential application. Two protein isolates (PI) from ethanol-treated industrial rapeseed meal, PI10.5-2.5 and PI2.5-8.5, were prepared by sequential isoelectric precipitation of alkali-extracted proteins (pH 12) starting from pH 10.5 to 2.5 or from pH 2.5 to 8.5, respectively. Biochemical analyses revealed that PI2.5-8.5 contained a higher amount of crude protein (72.84%) than PI10.5-2.5 (68.67%). In the same protein isolate, the level of total phenols (0.71%) was almost two-fold higher than that in PI10.5-2.5 (0.42%). No glucosinolates were established in both protein isolates. SDS-PAGE analysis demonstrated that PI10.5-2.5 contained 10 to 15 kDa protein fractions in a relatively higher amount, while PI2.5-8.5 was enriched in 18 to 29 kDa protein fractions. PI10.5-2.5 exhibited high solubility, varying from 41.74% at pH 4.5 to 65.13% at pH 6.5, while PI2.5-8.5 was almost two-fold less soluble under the same conditions. Up to pH 5.5, the addition of NaCl at 0.03 and 0.25 M diminished the solubility of PI2.5-8.5, while the solubility of PI10.5-2.5 was increased. The supplementation of PI10.5-2.5 with 0.25 M NaCl enhanced the protein solubility to 56.11% at pH 4.5 and 94.26% at pH 6.5. The addition of 0.03 M NaCl also increased the solubility of this protein isolate but to a lower extent. Overall, the approach for sequential precipitation of proteins influenced the biochemical characteristics, protein fractional profile and solubility of prepared protein isolates.

Stability of sunflower and rapeseed oil-in-water emulsions supplemented with ethanol-treated rapeseed meal protein isolate.

A protein isolate (ERPI) was prepared from ethanol-treated rapeseed meal and used as a stabilizing agent in sunflower and rapeseed oil-in-water emulsions. The aim of the current study was to explore the influence of protein and oil concentrations on initial stability of sunflower and rapeseed oil-in-water emulsions by evaluating Gibbs free energy (ΔG) and particle size distribution. The 7-day dynamics of emulsion stability was investigated by turbidity measurement as well. A 32 factorial design was applied to assess the significance of oil (5%, 10% and 15% w/w) and ERPI protein (0.25%, 0.5% and 1.0% w/w) addition on stability of the emulsions. The results demonstrated that the increase of oil concentrations from 5 to 15% positively influenced the initial stability of sunflower and rapeseed oil-in-water emulsions. In both oil types, ERPI protein supplementation at all levels resulted in significant differences in the stability of 5% and 10% oil emulsions but did not alter the initial stability of the emulsions prepared with either 15% sunflower or rapeseed oil. With a few exceptions, there was a good agreement between Gibbs free energy data and microstructural profiles of the emulsions. Overall, emulsions with all sunflower oil concentrations and 1.0% ERPI protein exhibited better initial and a 7-day stability dynamics compared to all rapeseed oil-based emulsions. The study demonstrated the potential of ethanol-treated rapeseed meal protein isolate to serve as an emulsifying agent in sunflower and rapeseed oil containing emulsions.

Foaming properties of acid-soluble protein-rich ingredient obtained from industrial rapeseed meal.

The use of the rapeseed meal as a source for preparation of protein-rich ingredients for the food industry is an alternative to the current limited application as a feed additive. The aim of this study was to evaluate foaming properties of an acid-soluble protein-rich ingredient (ASP) obtained from industrial rapeseed meal as a co-product of a protein isolate. Foam capacity and stability over a period of 60 min were evaluated by using volumetric and image analyzing methods. The influence of NaCl at two boundary concentrations (0.03 and 0.25 M) was studied over a pH range from 2 to 10. The ASP exhibited high foamability (> 90%), not influenced by pH or salt addition. In contrast, foam stability, measured over a 60 min period, was pH and NaCl dependent. By the end of the observation period, the addition of 0.25 M NaCl reduced the foam volume by more than 70% at all pH values. After 30 min at pH values 4, 6 and 8, which are the most common for food products, the foams without NaCl retained 51, 38 and 41% of the initial foam volume, respectively. The results were in agreement with image analysis observations where microstructure of the foams with NaCl was more heterogeneous than that of the foams without salt addition. The high foamability and relatively high foam stability at pH from 4 to 8 without NaCl addition shows that ASP could be a potential alternative to plant proteins currently used as foaming agents in the food industry.

Pepsin-Assisted Transglutaminase Modification of Functional Properties of a Protein Isolate Obtained from Industrial Sunflower Meal.

The utilization of industrial sunflower meal to produce protein-rich products for the food industry is an alternative approach for better and more efficient use of this agricultural by-product. Sunflower meal proteins possess specific functional properties, which however need improvement to broaden their potential as supplements for delivering high--quality products for human nutrition. The aim of the study is to evaluate the combined influence of low-degree pepsin hydrolysis and transglutaminase (TG) modification on industrial sunflower meal protein isolate functionality at pH=2 to 10. Three TG-modified pepsin hydrolysates with the degree of hydrolysis of 0.48, 0.71 and 1.72% were produced and named TG-PH1, TG-PH2 and TG-PH3, respectively. All three TG-modified pepsin hydrolysates exhibited improved solubility at pH between 3.5 and 5.5 as the highest was observed of TG-PH3 at protein isoelectric point (pI=4.5). Sunflower meal protein isolate and TG-modified sunflower meal protein isolate had greater solubility than the three TG-modified hydrolysates at pH<3 and >7. Significant improvement of foam making capacity (p<0.05) was achieved with all three TG-modified pepsin hydrolysates in the entire pH area studied. Pepsin hydrolysis of the protein isolate with the three degrees of hydrolysis did not improve foam stability. Improved thermal stability was observed with TG-PH3 up to 80 °C compared to the protein isolate (pH=7). At 90 °C, TG modification of the protein isolate alone resulted in the highest thermal stability. Pepsin hydrolysis followed by a treatment with TG could be used to produce sunflower protein isolates with improved solubility, foam making capacity and thermal stability for use in the food industry.

Applicability of massively parallel sequencing on monitoring harmful algae at Varna Bay in the Black Sea.

In this study the plankton diversity in 13 environmental samples from Varna Bay (in the western Black Sea) was analyzed using massively parallel sequencing (MPS). This preliminary study was undertaken to assess the potential of this technology for future implementation in monitoring programs in the Black Sea. Amplicon sequences of the 18S rRNA gene (V4-5 regions) were obtained using the Illumina MiSeq 250PE platform. A total of 1137 operational taxonomic units (OTUs) were obtained among which 242 OTUs with >0.990 BLAST top hit similarity (21.3% of all detected OTUs) closely related to sequences belonging to -protists. A large portion (175 OTUs=72.3%) was identified at the species levels, including species typical for the Bulgarian Black Sea plankton community, as well as many that haven't been reported earlier in the Bulgarian Black Sea coast (124 OTUs=51.2%). Dinoflagellates were represented by the highest species number (77 OTUs comprising 31.8% of protist species), with dominant genera Gyrodinium and Heterocapsa. The present survey revealed the presence of 12 species listed as harmful, some of which have been previously overlooked, such as Cochlodinium polykrikoides, Karenia bicuneiformis, and Karlodinium veneficum. Species identification was possible for 10.3-36.0% of the detected OTUs in the six major supergroups. The frequency in Rhizaria was significantly lower than that in other major groups (p<0.05-0.01), implying difficulties in the classification from morphology-based observations. The metagenetic data had an insufficient resolution of the 18S rRNA gene for species identification in many genera. These issues may hamper the implementation of MPS-based surveys for plankton monitoring, especially for detecting harmful algal blooms (HAB). The sequencing technology is steadily improving and it is expected that sequence length and quality issues will be resolved in the near future. The ongoing efforts to register taxonomic information and quality controls in the international nucleotide sequence databases (INSDs) will be essential for improving taxonomic identification power.

Preparation of stereoregular isotactic poly(mandelic acid) through organocatalytic ring-opening polymerization of a cyclic O-carboxyanhydride.

Poly(mandelic acid) (PMA) is an aryl analogue of poly(lactic acid) (PLA) and a biodegradable analogue of polystyrene. The preparation of stereoregular PMA was realized using a pyridine/mandelic acid adduct (Py⋅MA) as an organocatalyst for the ring-opening polymerization (ROP) of the cyclic O-carboxyanhydride (manOCA). Polymers with a narrow polydispersity index and excellent molecular-weight control were prepared at ambient temperature. These highly isotactic chiral polymers exhibit an enhancement of the glass-transition temperature (T(g)) of 15 °C compared to the racemic polymer, suggesting potential future application as high-performance commodity and biomedical materials.

Divergence with gene flow between Ponto-Caspian refugia in an anadromous cyprinid Rutilus frisii revealed by multiple gene phylogeography.

The Black and Caspian Seas have experienced alternating periods of isolation and interconnection over many Milankovitch climate oscillations and most recently became separated when the meltwater overflow from the Caspian Sea ceased at the end of the last glaciation. Climate-induced habitat changes have indisputably had profound impacts on distribution and demography of aquatic species, yet uncertainties remain about the relative roles of isolation and dispersal in the response of species shared between the Black and Caspian Sea basins. We examined these issues using phylogeographical analysis of an anadromous cyprinid fish Rutilus frisii. Bayesian coalescence analyses of sequence variation at two nuclear and one mitochondrial genes suggest that the Black and Caspian Seas supported separate populations of R. frisii during the last glaciation. Parameter estimates from the fitted isolation-with-migration model showed that their separation was not complete, however, and that the two populations continued to exchange genes in both directions. These analyses also suggested that majority of migrations occurred during the Pleistocene, showing that the variation shared between the Black and Caspian Seas is the result of ancient dispersal along the temporary natural connections between the basins, rather than of incomplete lineage sorting or recent human-mediated dispersal. Gene flow between the refugial populations was therefore an important source of genetic variation, and we suggest that it facilitated the evolutionary response of the populations to changing climate.

Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4.

Chlamydophila pneumoniae, an obligately intracellular Gram-negative bacterium and a common causative agent of respiratory tract infections, has been implicated in the induction and progression of atherosclerosis and coronary artery disease. In this study, the signalling mechanism of C. pneumoniae in human fibroblasts, a prominent cell population in chronic inflammation and persistent infection, contributing to plaque formation, was investigated. C. pneumoniae elementary bodies were demonstrated to up-regulate the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in human fibroblasts. The effect was independent of the chlamydial lipopolysaccharide and was likely to be mediated by a heat-labile chlamydial protein. Furthermore, an anti-Toll-like receptor 4 (TLR4) antibody was shown to abolish C. pneumoniae-induced cell activation, whereas an anti-TLR2 antibody had no effect, indicating the role of TLR4 in p44/p42 MAPK activation. Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 and phosphodiesterase 4 (PDE 4) inhibitor Rolipram enhanced C. pneumoniae-induced MAPK phosphorylation and attenuated C. pneumoniae infectivity in vitro. Together the results indicate that C. pneumoniae triggers rapid TLR4-mediated p44/p42 MAPK activation in human fibroblasts and chemical enhancement of MAPK phosphorylation modulates in vitro infection at the molecular level.

Biochemical-genetic comparison of Cobitis simplicispina from Turkey, and C. taenia, C. albicoloris and C. elongata from Bulgarian freshwaters.

Taxonomic investigations of 4 species of the genus Cobitis: C. simplicispina Hanko 1925, C. taenia Linnaeus 1758, C. albicoloris Chichkoff, 1932 (= C. peshevi Sivkov & Dobrovolov, 1984) and C. elongata (Heckel & Kner 1858) from Turkey and Bulgarian freshwaters were carried out using biochemical-genetic methods. The analyses of one general muscle protein (PROT) and six enzyme systems including esterase (EST), lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malic enzyme (MEP), malate dehydrogenase (MDH) and superoxide dismutase (SOD) revealed electrophoretic differences between the species examined. Fractions controlled by the LDH-B locus of C. simplicispina gave completely different electrophoretic mobility from those of the other Cobitis species. Nei's genetic distance (D(Nei)) between C. simplicispina and C. albicoloris, C. taenia and C. elongata was estimated as 1.39, 1.16 and 1.27, respectively. C. simplicispina might have diverged from the other three species 5.8 - 6.95 million years ago.