High Molecular Weight Protein Fraction Research Articles

Green Synthesis of Copper Oxide Nanoparticles Using Protein Fractions from an Aqueous Extract of Brown Algae Macrocystis pyrifera

Amongst different living organisms studied as potential candidates for the green synthesis of copper nanoparticles, algal biomass is presented as a novel and easy-to-handle method. However, the role of specific biomolecules and their contribution as reductant and capping agents has not yet been described. This contribution reports a green synthesis method to obtain copper oxide nanoparticles (CuO-NPs) using separated protein fractions from an aqueous extract of brown algae Macrocystis pyrifera through size exclusion chromatography (HPLC-SEC). Proteins were detected by a UV/VIS diode array, time-based fraction collection was carried out, and each collected fraction was used to evaluate the synthesis of CuO-NPs. The characterization of CuO-NPs was evaluated by Dynamic Light Scattering (DLS), Z-potential, Fourier Transform Infrared (FTIR), Transmission Electron Microscope (TEM) equipped with Energy Dispersive X-ray Spectroscopy (EDS) detector. Low Molecular Weight (LMW) and High Molecular Weight (HMW) protein fractions were able to synthesize spherical CuO-NPs. TEM images showed that the metallic core present in the observed samples ranged from 2 to 50 nm in diameter, with spherical nanostructures present in all containing protein samples. FTIR measurements showed functional groups from proteins having a pivotal role in the reduction and stabilization of the nanoparticles. The highly negative zeta potential average values from obtained nanoparticles suggest high stability, expanding the range of possible applications. This facile and novel protein-assisted method for the green synthesis of CuO-NPs may also provide a suitable tool to synthesize other nanoparticles that have different application areas.

Proteolysis in kashkaval cheese stored at different temperatures

The aim of the present study was to evaluate the effect of refrigerated storage temperature on the proteolysis in cow's milk Kashkaval cheese. The Kashkaval samples were stored for 12 months at four different temperature regimes - cooled at 4,0 ± 1,0 °C, cooled at 1.0 ± 1.0 °C, superchilled at -7,5 ± 0,5 °C and frozen at -18.0 ± 1.0 °C. The proteolysis in cheese samples was evaluated by determining the non-casein nitrogen (NCN/TN), non-protein nitrogen (NPN/TN) as a percentage of total nitrogen and content of free amino groups. It was found that the storage temperature had a significant impact on the hydrolysis of cheese paracasein. Complete inhibition of the proteolysis was established only in frozen stored Kashkaval. Slight, but statistically significant (P<0,05) increase in the NCN/TN values indicating for retarded proteolysis in samples stored at -7,5 ± 0,5 °C was established. The increasing of the storage temperatures results in increased NCN/TN, NPN/TN values and free amino groups content. The highest proteolysis rate was observed at the refrigeration temperatures of 4.0 ± 1.0 °C. The samples stored at this temperature regime had the highest values of both high molecular weight protein fractions (NCN/TN) and products of deep proteolysis (NPN/TN and free amino groups).

Effect of antibiotics, hormones and anthelmintic on high molecular weight protein fractions in the common carp

We assumed that effect of sulfanilamide, chlortetracycline, nandrolone, and albendazole on the carp fishes depends on substance concentration in the water and associated with changes in the fractional composition of plasma protein. We found that sulfanilamide in concentration of 0.015 mg/dm3 in water practically had no effect, while at concentrations of 0.15 and 0.30 mg/dm3 it changed the protein spectrum of carp blood plasma, reduced the level of proteins with molecular weights of 260 and 140 kDa. Chlortetracycline in the concentration of 1.10 mg/dm3 did not affect the protein content of macromolecular fractions in the blood plasma of fish, and at the doses of 3.15 and 6.30 mg/dm3 it reduced the content of proteins with molecular weights of 450, 340, 260 and 140 kDa. Nandrolone in the concentration of 1.10 mg/dm3 increased the content of individual proteins in carp plasma compared to the control, and in the concentration of 0.10 and 0.50 mg/dm3 it did not affect the fractional composition of proteins with high molecular weight. Albendazole in the concentrations of 0.2, 0.5, and 1.00 mg/dm2 reduced the protein content of macromolecular fractions in carp plasma. Such xenobiotics, like sulfanilamide, chlortetracycline, and albendazole in different concentrations did not affect the total protein content of fish plasma, while the nandrolone increased it. The number of respiratory movements, behavior, body surface, and pathomorphological parameters of the main internal organs in the fish from the experimental groups did not differ from the control. Our results indicated the important role of proteins of high molecular weight fractions in carp blood plasm as these proteins determine the fish adaptation to the water xenobiotics. Keywords: carp, blood plasma, proteins, sulfanilamide, chlortetracycline, nandrolone, albendazole.

Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers

For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater-a non-toxic solution primarily used to stabilize the RNA content of samples-in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins.

Bioluminescent Study of the Distribution of High-Molecular-Weight Protein Fraction of Cellex Daily Preparation in the Brain after Intranasal Administation.

Permeability of the blood-brain barrier for protein fractions 50-100 kDa (PF50-100) of Cellex Daily preparation labeled with fluorescent tracer FITC and non-conjugated FITC were compared after intranasal administration of the preparations to healthy rats. Fluorimetrical analysis of the serum and cerebrospinal fluid samples showed that Cellex Daily PF50-100-FITC administered intranasally penetrated into the blood and cerebrospinal fluid with maximum accumulation in 2 h after administration and persists in the circulation for 24 h probably due to binding with plasma proteins. The differences in the kinetic profile of PF50-100-FITC and free FITC indirectly suggest that the major part of the preparation is not degraded within 24 h and FITC is probably not cleaved from the protein components of the preparation. In vivo fluorescence analysis showed significant fluorescent signal in the olfactory bulbs in 6 h after intranasal administration; hence, the preparation administered via this route can bypass the blood-brain barrier. Scanning laser confocal microscopy of rat brain sections confirmed penetration of the high-molecular weight protein fraction PF50-100-FITC into CNS structures. The most pronounced accumulation of the labeled drug was observed in the olfactory bulb in 6 and 12 h after administration. In contrast to free FITC administered in the control group, significant accumulation of PF50-100-FITC in the olfactory cortex and frontal cortex neurons with functionally active nuclei was observed in 6, 12 and 24 h after intranasal administration.

Characterization of TauC3 antibody and demonstration of its potential to block tau propagation.

The spread of neurofibrillary tangle (NFT) pathology through the human brain is a hallmark of Alzheimer’s disease (AD), which is thought to be caused by the propagation of “seeding” competent soluble misfolded tau. “TauC3”, a C-terminally truncated form of tau that is generated by caspase-3 cleavage at D421, has previously been observed in NFTs and has been implicated in tau toxicity. Here we show that TauC3 is found in the seeding competent high molecular weight (HMW) protein fraction of human AD brain. Using a specific TauC3 antibody, we were able to substantially block the HMW tau seeding activity of human AD brain extracts in an in vitro tau seeding FRET assay. We propose that TauC3 could contribute to the templated tau misfolding that leads to NFT spread in AD brains.

Post-translationally modified human lens crystallin fragments show aggregation in vitro

BackgroundCrystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties. MethodsFour crystallin fragments-containing fractions (Fraction I [∼3.5kDa species], Fraction II [∼3.5–7kDa species], Fraction III [∼7–10kDa species] and Fraction IV [>10–18kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50–70 year old-donors. The crystallin fragments of the Fractions I–IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I–IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins. ResultsCrystallin fragments in Fractions I–IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in Mr from 5.5×105 to 1.0×108Da, with diameters of 10–28nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins. ConclusionThe crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development. General significanceCrystallin fragments play a major role in human cataract development.

Antiedematogenic and antioxidant properties of high molecular weight protein sub-fraction of Calotropis procera latex in rat.

Objectives:The aim was to evaluate the effect of high molecular weight protein fraction of Calotropis procera latex on edema formation and oxidative stress in carrageenan-induced paw inflammation.Methods:A sub-plantar injection of carrageenan was given to induce edema in the hind paw of the rat. The inhibitory effect of high molecular weight protein fraction of C. procera latex was evaluated following intravenous administration (5 and 25 mg/kg body weight) and was compared with that of diclofenac given orally (5 mg/kg). The levels of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) were measured in the inflamed paw tissue at the end of the study.Results:The high molecular weight protein fraction obtained from the latex of C. procera produced a dose-dependent inhibition of edema formation that was accompanied by normalization of levels of oxidative stress markers (GSH and TBARS) and MPO, a marker for neutrophils in the paw tissue.Conclusions:The high molecular weight protein fraction of C. procera latex ameliorates acute inflammation in the paw through its antioxidant effect.

Serum peptidomic biomarkers for pulmonary metastatic melanoma identified by means of a nanopore-based assay

The significant mortality rate associated with metastatic melanoma, which exceeds the number of deaths attributed to the primary tumor, is primarily due to poor diagnosis and increased resistance to systemic therapy. Early detection and treatment of invasive melanoma are therefore crucial to increase survival rates. Low molecular weight proteins and peptides have garnered significant interest as biomarker candidates as they potentially represent a snap shot of pathological condition within the body and, by extension, the organism as a whole. We have developed a nanoporous silica-based platform to segregate the low molecular weight from the high molecular weight protein fraction to aid in the detection of peptides from serum samples using mass spectrometry. The combination of sample treatment with our platform, MALDI-TOF MS and following biostatistical analysis led to the discovery and identification of 27 peptides that are potential biomarkers associated with the development of pulmonary metastatic melanoma. We strongly believe our findings can assist to discover stage-specific peptide signatures and lead to more specific and personalized treatments for patients suffering from pulmonary metastatic melanoma.

Degradation of an Old Human Protein

Long-lived proteins exist in a number of tissues in the human body; however, little is known about the reactions involved in their degradation over time. Lens proteins, which do not turn over, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS-crystallin, were studied. By teenage years, insoluble intact γS-crystallin was detected, indicative of protein denaturation. This was not the only change, however, because blots revealed evidence of significant cross-linking as well as cleavage of γS-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS-crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.

Role of steaming and Toasting on the Odor, Protein Characteristics of Chickpea (Cicer arietinum L.) Flour, and Product Quality

Proteins play an important role in imparting functional attributes like texture and shape, which determine the sensory quality of the foods. Boondi, a deep fried product from chickpea (Cicer arietinum L.) flour dispersion, is a popular snack food in India. Chickpea dhal (splits) or flour was subjected to various processing conditions like steaming and toasting, to determine their effect on the chickpea flour protein characteristics and on the product quality. Dhal and flour subjected to different heat treatments showed differences in their odor profiles. The SDS-PAGE of sodium phosphate buffer extracts of steamed dhal or flour showed that the high molecular weight (HMW) proteins of 66 to 100 kDa that were present in the untreated dhal were found to be absent in steamed dhal extracts. However, SDS buffer extracts on SDS-PAGE of these steamed samples did not show any difference between untreated and thermally treated dhal samples. Phosphate buffer extracts of the thermally treated flours were subjected to gel filtration chromatography and the results indicated that the HMW protein fraction content decreased significantly in the treated dhal or flour samples compared to control. Boondi prepared from the thermally treated dhal samples resulted in the loss of spherical shape of boondi. Thus, the results indicate that thermal treatment of chickpea dhal and flour influence changes in protein characteristics, the sensory profile and quality of boondi.

Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate–PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356-5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.

Humoral immune response to antigens of Porphyromonas gingivalis ATCC 33277 in chronic periodontitis

Introduction:Periodontitis is a chronic disease that results from an interaction of a mixed bacterial challenge and the host response.Objective:The purposes of this study were to evaluate the IgG serum levels to Porphyromonas gingivalis antigens by ELISA in individuals with different periodontal conditions correlated with clinical parameters, and to analyze the immunoreactivity profiles by Western blotting.Methods:Serum IgG levels against the cell sonicate antigen from P. gingivalis ATCC 33277 of 28 patients with chronic periodontitis (CP), 10 patients with gingivitis (G) and 21 periodontally healthy individuals (H) were measured by ELISA and Western immunoblotting.Results:In the CP group, sera reactivity by ELISA was significantly higher than in the G and H groups (Kruskal-Wallis p<0.001; Dunnet t3 p= 0.001 and Dunnet t3 p= 0.0001). There was no statistically significant difference between G and HP reactivity (Dunnett t3 p=0.617). Among individuals with chronic periodontitis, the IgG-anti-P. gingivalis serum levels were positively correlated with percentage of clinical attachment level =5mm (rs = + 0.375, p<0.05) and a negative correlation was found between IgG-anti-P. gingivalis levels and percentage of probing pocket depth 0-3mm (rs = - 0. 411, p< 0.05). The analysis of sera immunoreactivity profiles to sonicate antigen by Western blotting showed differences between the sera of CP, G and H group individuals. The serum from CP frequently reacted with high molecular weight (103 kDa, 86 kDa, 72 kDa, 60 kDa, 58 kDa, 52 kDa) protein fractions.Conclusions:Serum levels of IgG anti-P. gingivalis distinguished individuals with chronic periodontitis, gingivitis and healthy periodontium. There was a correlation between clinical parameters and serum IgG levels against P. gingivalis. There was a difference in the recognition profile of protein fractions among the studied groups and some bands were more specific

Urinary pheromones promote ERK/Akt phosphorylation, regeneration and survival of vomeronasal (V2R) neurons

The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Galpha(o) and V2R, whereas V1R and Galpha(i) immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Galpha(o). Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.

Ability of conventional and nutritionally-modified whey protein concentrates to stabilize oil-in-water emulsions

Abstract The ability of a modified whey protein concentrate (MWPC), which contains relatively high proportions of phospholipid and high molecular weight protein fractions, to form and stabilize 10 wt% corn oil-in-water emulsions (pH 7.0, 5 mM phosphate buffer) was compared with that of a conventional whey protein concentrate (CWPC). The MWPC stabilized emulsions required less protein to prepare stable emulsions with monomodal particle size distributions and small mean droplet diameters (d43 ≈ 0.3 μm at [WPC] ⩾ 0.5 wt%) than CWPC stabilized emulsions (d43 ≈ 0.4 μm at [WPC] ⩾ 0.9 wt%) under similar homogenization conditions (5 passes at 5000 psi). In addition, the emulsions stabilized by 0.9 wt% MWPC were more stable to high salt concentration (NaCl ⩽ 200 mM), thermal processing (30–90 °C for 30 min) and pH (3, 6 and 7) than those stabilized by the same concentration of CWPC, which was attributed to polymeric steric repulsion rather than electrostatic repulsion. This study has important implications for the wide application of WPC as a natural emulsifier in food products.

Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates

Porcine haemoglobin hydrolysates were prepared through hydrolysis by Alcalase followed by Flavourzyme, and their protein compositions were analyzed using Sephadex G-50 gel filtration chromatography. The antioxidant activities, including reducing power, ferrous ion chelating ability, and DPPH radical scavenging activity, of the hydrolysates were evaluated. The results showed that the hydrolysates of haemoglobin exhibited low reducing powers, but high ferrous ion chelating abilities and DPPH radical scavenging activities. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 h and followed by 1% Flavourzyme for 6 h, had the highest ferrous ion chelating ability of 63.54% at a concentration of 5.0 mg/mL. The hydrolysate, obtained through hydrolysis by 2% Alcalase for 4 hrs, had the highest DPPH radical scavenging activity of 41.94% at a concentration of 5.0 mg/mL. According to the results of protein composition analysis, we divided the hydrolysates into three groups, including high molecular weight (MW) group (Group I), medium MW group (Group II), and low MW group (Group III). The reducing power and ferrous ion chelating ability of the hydrolysates were significantly and positively correlated to the relative amount of Group I, and negatively correlated to the relative amount of Group III. This study revealed that the antioxidant activities of porcine haemoglobin hydrolysates were dependent on their protein compositions. The high MW protein fraction (Group I) was responsible for the high reducing power and ferrous ion chelating ability of the hydrolysate.

Effects of storage in slurry ice on the microbial, chemical and sensory quality and on the shelf life of farmed turbot ( Psetta maxima)

The application of slurry ice, a binary mixture of small spherical ice crystals surrounded by seawater at subzero temperature, is a potentially new preservation method for farmed turbot ( Psetta maxima), a flat fish species of increasing commercial interest. Comparative biochemical, microbiological and sensory analyses were carried on turbot specimens stored in either slurry ice or flake ice for up to 40 days. The results obtained in the sensory analysis correlated well with the observed chemical and microbial changes. Storage of turbot in slurry ice resulted in a slowing-down of the nucleotide degradation pathway and lipid oxidation mechanisms. A good stabilisation of the high molecular weight protein fraction of turbot muscle was also achieved as a consequence of storage in slurry ice. A slower production of both trimethylamine and total volatile bases was also observed. Likewise, low levels of total aerobes, anaerobes, coliforms, and proteolytic bacteria were attained. The application of slurry ice to farmed turbot is advisable to achieve better quality maintenance during storage and distribution.

The Microglia-activating Potential of Thrombin: THE PROTEASE IS NOT INVOLVED IN THE INDUCTION OF PROINFLAMMATORY CYTOKINES AND CHEMOKINES

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.

Male urinary chemosignals differentially affect aggressive behavior in male mice.

Chemical signals modulate aggressive behavior in mice. For example, male urinary cues enhance aggression against other adults: a resident mouse attacks a male but not a castrated intruder, unless it is anointed with male urine. Our purpose was to understand whether molecules excreted with urine also act as aggression triggers in a different context. Therefore, the effect of urine, or molecules purified from urine, voided by different animals (males or females), was tested on the aggression of male mice against pups. Latency to the first attack, percentage of pups receiving the first attack, and percentage of attacked pups after 5 and 15 min were recorded. At variance with intermale aggression, male urinary chemosignals sprayed on pups reduced infanticide, while female urine did not. Male urine also delayed infanticide when compared to female urine. Pups anointed with low molecular weight dialyzed urine and with the high molecular weight protein fraction were attacked later than controls. Pups anointed with Major Urinary Proteins (MUPs) also were attacked later. The volatiles retained by MUPs act in the same way as adult male urine. MUPs and their ligands did not modify biting of food items. The results show that mice do not perceive male chemosignals as compulsory aggression triggers but rather can consistently and differentially shape their behavior in response to the same molecules according to different contextual events.