Aspects of milk protein catabolism by lactobacilli

Lactobacillus plantarum and subspecies of Lactobacillus casei were isolated from good quality mature Cheddar cheese and characterized with respect to metabolic functions that would allow their use in cheesemaking. In this way microbiological control of the maturation process with particular emphasis on protein catabolism was achieved. The lactobacilli isolated were selected for low growth rates (and acid production) in milk, and low proteinase activity to allow for their addition in high numbers to cheesemilk together with the normal starter flora (group N streptococci). The growth and acid production of the starter bacteria were unaffected by the presence of the lactobacilli during cheese manufacture and it was found that the added lactobacilli were able to grow and function under the conditions prevalent in Cheddar cheese during maturation. It was also demonstrated that the lactobacilli could be grown in an artificial medium to high numbers under controlled conditions and could be harvested for the preparation of cell concentrates, a necessary characteristic for commercialization. The lactobacilli also metabolized citrate, a potential problem in cheese maturation associated with C02 production but this did not adversely affect the maturation process under the conditions used. Compared to the group N streptococci the non-starter lactobacilli possessed a proteinase system that had a higher temperature optimum and was less affected by heat and sodium chloride. They also possessed a more active peptidase system although both the lactobacilli and the starter organisms possessed a similar range of peptidases. Non-starter lactobacilli were added to normal cheese and cheese made with proteinase negative starter. The added organisms did not adversely affect manufacturing parameters and did not metabolize citrate or lead to the formation of biogenic amines. However protein catabolism rates, particularly with respect to peptide degradation, were increased, as was flavour development and intensity. It was observed that the body and texture of the cheeses was unaffected by the treatment. By controlling both the starter and non-starter microflora in the cheeses a practical system for favourably influencing cheese maturation was possible. The investigation has demonstrated that carefully selected and characterized non-starter lactobacilli can be incorporated into Cheddar cheese manufacture in order to influence flavour development during maturation. Moreover the organisms can be added to the vat stage of manufacture without causing problems to the manufacturing process. This approach is a simple cost effective means of improving the cost of Cheddar cheese production and provides an unique opportunity to improve and control quality of all Cheddar cheese produced.

Catabolism attack and anabolism defense: a novel attack and traceback mechanism in opportunistic networks

Security is a major challenge in Opportunistic Networks (OppNets) because of its characteristics, such as open medium, dynamic topology, no centralized management and absent clear lines of defense.A packet dropping attack is one of the major security threats in OppNets since neither source nodes nor destination nodes have the knowledge of where or when the packet will be dropped. In this paper, we present a novel attack and traceback mechanism against a special type of packet dropping where the malicious node drops one or more packets and then injects new fake packets instead. We call this novel attack a Catabolism Attack and we call our novel traceback mechanism against this attack Anabolism Defense. Our novel detection and traceback mechanism is very powerful and has very high accuracy. Each node can detect and then traceback the malicious nodes based on a solid and powerful idea that is, hash chain techniques. In our defense techniques we have two stages. The first stage is to detect the attack, and the second stage is to find the malicious nodes. Simulation results show this robust mechanism achieves a very high accuracy and detection rate.

Effect of AICAR treatment on gycogen metabolism in skeletal muscle

AMP-activated protein kinase (AMPK) is proposed to stimulate fat and carbohydrate catabolism to maintain cellular energy status. Recent studies demonstrate that pharmacologic activation of AMPK and mutations in the enzyme are associated with elevated muscle glycogen content in vivo. Our purpose was to determine the mechanism for increased muscle glycogen associated with AMPK activity in vivo. AMPK activity and glycogen metabolism were studied in red and white gastrocnemius muscles from rats treated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in vivo, and also in muscles incubated with AICAR in vitro. In vivo AICAR treatment reduced blood glucose and increased blood lactate compared with basal values. AICAR increased muscle α2 AMPK activity, glycogen, and glucose-6-phosphate concentrations. Glycogen synthase activity was increased in the red gastrocnemius but was decreased in the white gastrocnemius. Glycogen phosphorylase activity increased in both muscles, with an inhibition initially observed in the red gastrocnemius. In vitro incubation with AICAR activated α2 AMPK but had no effect on either glycogen synthase or glycogen phosphorylase. These results suggest that AICAR treatment does not promote glycogen accumulation in skeletal muscle in vivo by altering glycogen synthase and glycogen phosphorylase. Rather, the increased glycogen is due to the well-known effects of AICAR to increase glucose uptake.

Body composition changes, metabolic fuel use and energy expenditure during extended fasting in subantarctic fur seal (Arctocephalus tropicalis) pups at Amsterdam Island

The fasting metabolism of 71- to 235-d-old subantarctic fur seal (Arctocephalus tropicalis) pups from Amsterdam Island, southern Indian Ocean, was investigated during the long foraging trips of their mothers. Body lipid reserves were proportionally greater in female than male pups and higher in postmoult (37%) than premoult (10%) animals. The mass-specific rate of mass loss did not differ between the sexes but was lower than observed in other species. Daily mass loss was estimated to 56% fat, 10% protein, and 34% water. The rate of protein catabolism (15 g d⁻¹) was negatively related to the size of initial lipid stores and accounted for 9% (±1%) of total energy expenditure. However, body composition changes during the fast were not equal between the sexes, with females relying more on protein catabolism than males (11% and 5% of total energy expenditure, respectively). Energy expenditure (270 kJ kg⁻¹ d⁻¹) and metabolic water production (11.5 mL kg⁻¹ d⁻¹) rates are the lowest reported for an otariid species. These results suggest that subantarctic fur seal pups greatly reduce activity levels to lower energy expenditure in addition to adopting protein-sparing metabolic pathways in order to survive the extreme fasts they must endure on Amsterdam Island.

Fasting metbabolism in Antarctic fur seal (Artocephalus gazella) pups

The metabolism of 52–73-day old Antarctic fur seal pups from Bird Island, South Georgia, was investigated during fasting periods of normal duration while their mothers were at sea foraging. Body mass decreased exponentially with pups losing 3.5–3.8% of body mass per day. Resting metabolic rate also decreased exponentially from 172–197 ml (O2)·min⁻¹ at the beginning of the fast and scaled to M_b^0.74 at 2.3 times the level predicted for adult terrestrial mammals of similar size. While there was no significant sex difference in RMR, female pups had significantly higher (F₁,₁₈=6.614, P<0.019) mass-specific RMR than male pups throughout the fasting period. Fasting FMR was also significantly (t₁₅=2.37, P<0.035) greater in females (823 kJ·kg⁻¹·d⁻¹) than males (686 kJ·kg⁻¹·d⁻¹). Average protein turnover during the study period was 19.3 g·d⁻¹ and contributed to 5.4% of total energy expenditure, indicating the adoption of a protein-sparing strategy with a reliance on primarily lipid catabolism for metabolic energy. This is supported by observed decreases in plasma BUN, U/C, glucose and triglyceride concentrations, and an increase in β-HBA concentration, indicating that Antarctic fur seals pups adopt this strategy within 2–3 days of fasting. Mean RQ also decreased from 0.77 to 0.72 within 3 days of fasting, further supporting a rapid commencement of protein-sparing. However, RQ gradually increased thereafter to 0.77, suggesting a resumption of protein catabolism which was not substantiated by changes in plasma metabolites. Female pups had higher TBL (%) than males for any given mass, which is consistent with previous findings in this and other fur seal species, and suggests sex differences in metabolic fuel use. The observed changes in plasma metabolites and protein turnover, however, do not support this.

The effect of estrogen on Akt signalling and protein synthesis in C2C12 mouse skeletal myotubes

The maintenance of skeletal muscle mass is a critical component of health in both chronic wasting diseases and aging. A considerable amount of progress has been made in the understanding of the signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Akt is seen as a key molecular protein involved in the maintenance of skeletal muscle mass as it has the dual ability to positively influence protein syntheses and negatively regulate protein degradation in its active state (Glass, 2003). Potential mechanisms which may assist with maintaining skeletal muscle mass are the estrogen hormones. Estrogens increase the proliferation of mouse and rat myoblasts and can also attenuate immobilization-induced skeletal muscle atrophy in rats in vivo (Kahlert et al., 1997). No studies have investigated the effect of estrogens on the activation of skeletal muscle hypertrophy and atrophy signalling pathways. Estrogens may contribute to maintaining skeletal muscle mass via their activation of the Akt signalling pathways. Therefore, the aims of the present study were to determine if treatment of C2C12 myotubes with either 17β-estrodiol or estrone increases the activity of Akt and its downstream anabolic signalling proteins, GSK, p70s6k and 4E-BP1 and decreases its catabolic stimulating targets, FOXO, atrogin-1 and MuRF-1. A secondary aim was to determine if this was associated with an increased rate of protein synthesis.

C2C12 myotubes were incubated at 37°C in serum free DMEM without phenol red containing 10 000 units/ml penicillin, 10 000 μg/ml streptomycin, and 250μg/ml amphotericin B for 24h. Myotubes were then stimulated with 17-β estradiol (10nM) for 24h. Phosphorylated and total proteins for Akt, p70S6k, GSK3β, 4E-BP1, FOXO and atrogin-1 were measured using western blotting techniques. Atrogin-1 and MuRF1 mRNA levels were measured using real time-PCR. Protein synthesis rates were measured by incorporation of [3H]-tyrosine into the myotubes during the last hour of treatment.

Compared to control myotubes, treatment with 17β-estradiol increased the ratio of phosphorylated to total protein contents for Akt, GSK-3β and P70s6k by, 1.62, 1.53 and 2.2 fold, respectively (n=6 per group; p < 0.05). There was, however, no difference in the ratios of phosphorylated to total 4E-BP1 or Foxo3a or Atrogin-1 and MuRF1 mRNA. Protein synthesis rates remained unchanged.

This study demonstrates that in C2C12 mouse myotubes, 17β-estradiol treatment increases the phosphorylation of the hypertrophy signalling protein, Akt, and its downstream hypertrophy signalling targets, GSK-3β and P70s6k; no associated changes in protein synthesis were observed. Future studies should investigate the ability of 17β-estradiol to activate these proteins in a model of myotube catabolism and to determine if protein degradation is attenuated.

Fish oil replacement in finfish nutrition

Unsustainable fishing practices have placed a heavy emphasis on aquaculture to meet the global shortfalls in the supply of fish and seafood, which are commonly accepted as the primary source of health-promoting essential omega-3 (n-3 highly unsaturated fatty acids). However, dietary fish oil is required for the production of omega-3-rich farmed fish and this commodity, in a vicious circle, is at present derived solely from wild fisheries. Decreasing global availability coupled with the highly variable price of this resource has forced the aquaculture industry to investigate the possibilities of alternative dietary lipid sources. This review attempts to compile all principal information available regarding the effects of fish oil replacement for the diets of farmed finfish, analysing the findings using a comparative approach among different cultured fish species. The review initially focuses on the present situation with regard to the production, availability and main nutritional characteristics of fish oil and the principal alternative lipid sources (such as vegetable oils and animal fats). Following this, the effects of fish oil replacement in finfish nutrition on feed quality, fish performance, feed efficiency, fish lipid metabolism, final eating quality and related economic aspects are presented and discussed.

Physiological response to extreme fasting in subantarctic fur seal (Arctocephalus tropicalis) pups : metabolic rates, energy reserve utilization, and water fluxes

Physiological response to extreme fasting in subantarctic fur seal (Arctocephalus tropicalis) pups: metabolic rates, energy reserve utilization, and water fluxes. Am J Physiol Regul Integr Comp Physiol 297: R1582–R1592, 2009. First published September 23, 2009; doi:10.1152/ajpregu.90857.2008.— Surviving prolonged fasting requires various metabolic adaptations, such as energy and protein sparing, notably when animals are simultaneously engaged in energy-demanding processes such as growth. Due to the intermittent pattern of maternal attendance, subantarctic fur seal pups have to repeatedly endure exceptionally long fasting episodes throughout the 10-mo rearing period while preparing for nutritional independence. Their metabolic responses to natural prolonged fasting (33.4 ± 3.3 days) were investigated at 7 mo of age. Within 4–6 fasting days, pups shifted into a stage of metabolic economy characterized by a minimal rate of body mass loss (0.7%/day) and decreased resting metabolic rate  (5.9 ± 0.1 ml O₂ ·kg^-1·day^-1) that was only 10% above the level predicted for adult terrestrial mammals. Field metabolic rate (289 ± 10 kJ·kg^-1 ·day^-1) and water influx (7.9 ± 0.9 ml·kg^-1 ·day^-1) were also among the lowest reported for any young otariid, suggesting minimized energy allocation to behavioral activity and thermoregulation. Furthermore, lean tissue degradation was dramatically reduced. High initial adiposity (>48%) and predominant reliance on lipid catabolism likely contributed to the exceptional degree of protein sparing attained. Blood chemistry supported these findings and suggested utilization of alternative fuels, such as β-hydroxybutyrate and de novo synthesized glucose from fat-released glycerol. Regardless of sex and body condition, pups tended to adopt a convergent strategy of extreme energy and lean body mass conservation that appears highly adaptive for it allows some tissue growth during the repeated episodes of prolonged fasting they experience throughout their development.

The nutritional selectivity of a siderophore-catabolizing bacterium

The ability of a siderophore-catabolizing bacterium to assimilate ferric ion was examined. While the bacterium utilizes the siderophore deferrioxamine B (DFB) as a carbon source, it was incapable of using the ferricion analogue (ferrioxamine B) as an iron source. It did, however, assimilate the ferric ion of the chelator ferric nitrilotriacetic acid and of the siderophore ferrirhodotorulic acid (ferriRA). Neither ferriRA nor its deferrated analog (RA), however, were capable of functioning as carbon sources for the bacterium. The microbe thus employs a nutritional selectivity with respect to these two siderophores. That is, it does not use the siderophore it employs as a carbon source (DFB) as an iron source nor does the siderophore utilized as an iron source, i.e. ferriRA, nor its deferrated analog (RA), serve as carbon sources for the organism.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

n-3 LC-PUFA deposition efficiency and appetite regulating hormones are modulated by the dietary lipid source during rainbow trout grow-out and finishing periods

Largely attributable to concerns surrounding sustainability, the utilisation of omega-3 long-chain polyunsaturated fatty acid-rich (n-3 LC-PUFA) fish oils in aquafeeds for farmed fish species is an increasingly concerning issue. Therefore, strategies to maximise the deposition efficiency of these key health beneficial fatty acids are being investigated. The present study examined the effects of four vegetable-based dietary lipid sources (linseed, olive, palm and sunflower oil) on the deposition efficiency of n-3 LC-PUFA and the circulating blood plasma concentrations of the appetite-regulating hormones, leptin and ghrelin, during the grow-out and finishing phases in rainbow trout culture. Minimal detrimental effects were noted in fish performance; however, major modifications were apparent in tissue fatty acid compositions, which generally reflected that of the diet. These modifications diminished somewhat following the fish oil finishing phase, but longer-lasting effects remained evident. The fatty acid composition of the alternative oils was demonstrated to have a modulatory effect on the deposition efficiency of n-3 LC-PUFA and on the key endocrine hormones involved in appetite regulation, growth and feed intake during both the grow-out and finishing phases. In particular, n-6 PUFA (sunflower oil diet) appeared to ‘spare’ the catabolism of n-3 LC-PUFA and, as such, resulted in the highest retention of these fatty acids, ultimately highlighting new nutritional approaches to maximise the maintenance of the qualitative benefits of fish oils when they are used in feeds for aquaculture species.

Flight costs and fuel composition of a bird migrating in a wind tunnel

We studied the energy and protein balance of a Thrush Nightingale Luscinia luscinia, a small long-distance migrant, during repeated 12-hr long flights in a wind tunnel and during subsequent two-day fueling periods. From the energy budgets we estimated the power requirements for migratory flight in this 26 g bird at 1.91 Watts. This is low compared to flight cost estimates in birds of similar mass and with similar wing shape. This suggests that power requirements for migratory flight are lower than the power requirements for nonmigratory flight. From excreta production during flight, and nitrogen and energy balance during subsequent fueling, the dry protein proportion of stores was estimated to be around 10%. A net catabolism of protein during migratory flight along with that of fat may reflect a physiologically inevitable process, a means of providing extra water to counteract dehydration, a production of uric acid for anti-oxidative purposes, and adaptive changes in the size of flight muscles and digestive organs in the exercising animal.

What is the most effective way of increasing the bioavailability of dietary long chain omega-3 fatty acids-daily vs. weekly administration of fish oil?

The recommendations on the intake of long chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA) vary from eating oily fish ("once to twice per week") to consuming specified daily amounts of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ("250-500 mg per day"). It is not known if there is a difference in the uptake/bioavailability between regular daily consumption of supplementsvs. consuming fish once or twice per week. In this study, the bioavailability of a daily dose of n-3 LC-PUFA (Constant treatment), representing supplements, vs. a large weekly dose of n-3 LC-PUFA (Spike treatment), representing consuming once or twice per week, was assessed. Six-week old healthy male Sprague-Dawley rats were fed either a Constant treatment, a Spike treatment or Control treatment (no n-3 LC-PUFA), for six weeks. The whole body, tissues and faeces were analysed for fatty acid content. The results showed that the major metabolic fate of the n-3 LC-PUFA (EPA+docosapentaenoic acid (DPA) + DHA) was towards catabolism (β-oxidation) accounting for over 70% of total dietary intake, whereas deposition accounted less than 25% of total dietary intake. It was found that significantly more n-3 LC-PUFA were β-oxidised when originating from the Constant treatment (84% of dose), compared with the Spike treatment (75% of dose). Conversely, it was found that significantly more n-3 LC-PUFA were deposited when originating from the Spike treatment (23% of dose), than from the Constant treatment (15% of dose). These unexpected findings show that a large dose of n-3 LC-PUFA once per week is more effective in increasing whole body n-3 LC-PUFA content in rats compared with a smaller dose delivered daily.

Sarcopenia and the common mental disorders: a potential regulatory role of skeletal muscle on brain function?

While it is understood that body composition impacts on physical conditions, such as diabetes and cardiovascular disease, it is only now apparent that body composition might play a role in the genesis of common mental disorders, depression and anxiety. Sarcopenia occurs in ageing and comprises a progressive decline in muscle mass, strength and function, leading to frailty, decreased independence and poorer quality of life. This review presents an emerging body of evidence to support the hypothesis that shared pathophysiological pathways for sarcopenia and the common mental disorders constitute links between skeletal muscle and brain function. Contracting skeletal muscle secretes neurotrophic factors that are known to play a role in mood and anxiety, and have the dual role of nourishing neuronal growth and differentiation, while protecting the size and number of motor units in skeletal muscle. Furthermore, skeletal muscle activity has important immune and redox effects that impact behaviour and reduce muscle catabolism.