Proteolytic activity amongst selected Saprolegnia species

Saprolegia ssp. effectively utilized the protein casein as a sole source of carbon, nitrogen and sulphur, indicating considerable proteolytic activity. In the presence of a more simple carbon source such as glucose, which was readily assimilated, catabolite repression was not observed and casein exploitation was enhanced. Free proteinase activity was not detected by a number of methods, irrespective of culture conditions. However, clearing by mycelia of skimmed milk agar or agar amended with bacteria demonstrated a close association between proteinases and hyphae, suggestive of natural immobilization of proteinases. Casein breakdown was accompanied by release of individual amino acids and ammonia. The latter, indicative of amino acid assimilation and metabolism, was also associated with an increase in pH of culture medium. Single amino acids did not support growth of Saprolegnia but in combination with other amino acids, methionine encouraged greatest biomass production. Certain groupings of amino acids affected growth in a manner which departed from that expected, as assessed by multifactorial analysis of variance, and either enhanced or reduced growth.

Attenuation of muscle atrophy by an N-terminal peptide of the receptor for proteolysis-inducing factor (PIF)

Background: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. Methods: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C2C12 murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. Results: The process resulted in almost a 23?000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. Conclusion: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation.

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E214k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB. © 2005 Elsevier Inc. All rights reserved.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

American Alligator Digestion Rate of Blue Crabs and Its Implications for Stomach Contents Analysis

Stomach contents analysis (SCA) provides a snap-shot observation of a consumer's diet. Interpretation of SCA data can be complicated by many factors, including variation in gastric residence times and digestion rates among prey taxa. Although some SCA methods are reported to efficiently remove all stomach contents, the effectiveness of these techniques has rarely been tested for large irregular shaped prey with hard exoskeletons. We used a controlled feeding trial to estimate gastric residency time and decomposition rate of a large crustacean prey item, the Blue Crab (Callinectes sapidus), which is consumed by American Alligators (Alligator mississippiensis), an abundant apex predator in coastal habitats of the southeastern United States. The decomposition rate of C. sapidus in the stomachs of A. mississippiensis followed a predictable pattern, and some crab pieces remained in stomachs for at least 14 days. We also found that certain portions of C. sapidus were prone to becoming caught within the stomach or esophagus, meaning not all crab parts are consistently recovered using gastric lavage techniques. However, because the state of decomposition of crabs was predictable, it is possible to estimate time since consumption for crabs recovered from wild alligators. This information, coupled with a detailed understanding of crab distributions and alligator movement tactics could help elucidate patterns of cross-ecosystem foraging by the American Alligator in coastal habitats.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.

Assessment of genotoxicity of aflatoxin M1 and B1 contaminated milks after in vitro human digestion

Introduction - Milk is considered a complete food from the nutritional point of view. Milk can be exposed to various types of contamination, such as mycotoxins. These metabolites are naturally occurring toxic compounds produced by fungi. Several studies on milk samples have reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1), due to the high incidence in samples intended for human consumption, carcinogenicity proven AFB1 and resistance of the contaminants to the process of digestion, making those available for intestinal absorption. Considering these aspects, the objective of this study was to evaluate the genotoxicity of milk samples contaminated by AFB1 and AFM1 before and after the action of lactic acid bacteria using Caco-2 intestinal human cells.

Co-digestion of terrestrial plant biomass with marine macro-algae for biogas production

This paper investigates factors affecting anaerobic degradation of marine macro-algae (or seaweed), when used as a co-substrate with terrestrial plant biomass for the production of biogas. Using Laminaria digitata, a brown marine seaweed species and green peas, results showed that when only 2% of feedstock of a reactor treating the green peas at an organic loading rate (OLR) of 2.67 kg VS.m3.day-1 was replaced with the seaweed, methane production was disrupted, whilst acidogenesis, seemed to be less adversely affected, resulting in excessive volatile acids accumulation. Reactor stability was difficult to achieve thereafter. The experiment was repeated with a lower initial OLR of green peas of 0.70 kg VS.m3.day-1 before the addition of the seaweed. Although similar symptoms as in first trial were observed, process stability was restored through the control of OLR and alkalinity. These measures led to an increase in overall OLR of 1.25 kg VS.m3.day-1 comprising of 35% seaweed. This study has shown that certain seaweed constituents are more inhibitory to the methanogens even at trace concentrations than to the other anaerobic digestion microbial groups. Appropriate adaptation strategy, involving initial low proportion of the seaweed relative to the total OLR, and overall low OLR, is necessary to ensure effective adaptation of the microorganisms to the inhibitory constituents of seaweed. Where there is seasonal availability of seaweed, the results of this study suggest that a fresh adaptation or start-up strategy must be implemented during each cycle of seaweed availability in order to ensure sustainable process stability.

Thermodynamic efficiency of carbon capture and utilisation in anaerobic batch digestion process

Carbon capture and storage (CCS) in the oil and water industries is becoming common and a significant consumer of energy typically requiring 150–450 °C and or several hundred bar pressure [1] particularly in geological deposition. A biological carbon capture and conversion has been considered in conventional anaerobic digestion processes. The process has been utilised in biological mixed culture, where acetoclastic bacteria and hydrogenophilic methanogens play a major key role in the utilisation of carbon dioxide. However, the bio catalytic microorganisms, hydrogenophilic methanogens are reported to be unstable with acetoclastic bacteria. In this work the biochemical thermodynamic efficiency was investigated for the stabilisation of the microbial process in carbon capture and utilisation. The authors observed that a thermodynamic efficiency of biological carbon capture and utilisation (BCCU) had 32% of overall reduction in yield of carbon dioxide with complimentary increase of 30% in yield of methane, while the process was overall endothermic. Total consumption of energy (≈0.33 MJ l−1) was estimated for the carbonate solubility (0.1 mol l−1) in batched BCCU. This has a major influence on microbial composition in the bioreactor. This thermodynamic study is an essential tool to aid the understanding of the interactions between operating parameters and the mixed microbial culture.

Anaerobic digestion process for biogas and biomolecules production: microflora identification and characterization

The anaerobic process was efficient in organic matter removal. During the process, an interesting compound as quercetin was produced inside of reactor. Phylogenetic analysis showed the presence of phylotypes affiliated with gamma-Proteobacteria, Choroflexi, and Bacteroidetes. Archaea were represented by phylotypes belonging to the genus Methanosarcina and Methanosaeta.

Bioactive compounds through anaerobic digestion of heterotrophic microalgae residues

Several important biomolecules are available into anaerobically digested effluents that were obtained from the biodiesel production process using heterotrophically grown microalga Chlorella protothecoides. Defatted microalgae residues and crude glycerol may undergo anaerobic digestion, separately and in admixture, providing methane/hydrogen and a digestate exploitable for agriculture applications. Furthermore, industrial interesting bioactive compounds such as polyphenols provided with antioxidant activity can be obtained. Anaerobic process offers a promising chance and can be advantageously combined with algae lipid-extraction techniques in order to make it more sustainable.