Effects of metabolic rate on thermal responses at different air velocities in −10°C

The effects of exercise intensity on thermoregulatory responses in cold (−10°C) in a 0.2 (still air, NoWi), 1.0 (Wi1), and 5.0 (Wi5) m s⁻¹ wind were studied. Eight young and healthy men, preconditioned in thermoneutral (+20°C) environment for 60 min, walked for 60 min on the treadmill at 2.8 km/h with different combinations of wind and exercise intensity. Exercise level was adjusted by changing the inclination of the treadmill between 0° (lower exercise intensity, metabolic rate 124 W m⁻², LE) and 6° (higher exercise intensity, metabolic rate 195 W m⁻², HE). Due to exercise increased heat production and circulatory adjustments, the rectal temperature (T_re), mean skin temperature (T_sk) and mean body temperature (T_b) were significantly higher at the end of HE in comparison to LE in NoWi and Wi1, and T_re and T_b also in Wi5. T_sk and T_b were significantly decreased by 5.0 m s⁻¹ wind in comparison to NoWi and Wi1. The higher exercise intensity was intense enough to diminish peripheral vasoconstriction and consequently the finger skin temperature was significantly higher at the end of HE in comparison to LE in NoWi and Wi1. Mean heat flux from the skin was unaffected by the exercise intensity. At LE oxygen consumption (V ₂) was significantly higher in Wi5 than NoWi and Wi1. Heart rate was unaffected by the wind speed. The results suggest that, with studied exercise intensities, produced without changes in walking speed, the metabolic rate is not so important that it should be taken into consideration in the calculation of wind chill index.     

Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.     

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20-30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.     

Tight junction proteins in gallbladder epithelium: different expression in acute acalculous and calculous cholecystitis.

There is a paucity of information of tight junction (TJ) proteins in gallbladder epithelium, and disturbances in the structure of these proteins may play a role in the pathogenesis of acute acalculous cholecystitis (AAC) and acute calculous cholecystitis (ACC). Using immunohistochemistry, we investigated the expression of TJ proteins claudin-1, -2, -3, and -4, occludin, zonula occludens (ZO-1), and E-cadherin in 9 normal gallbladders, 30 gallbladders with AAC, and 21 gallbladders with ACC. The number of positive epithelial and endothelial cells and the intensity of the immunoreaction were determined. Membrane-bound and cytoplasmic immunoreactivities were separately assessed. We found that TJ proteins were uniformly expressed in normal gallbladder epithelium, with the exception of claudin-2, which was present in less than half of the cells. In AAC, expression of cytoplasmic occludin and claudin-1 were decreased, as compared with normal gallbladder. In ACC, expression of claudin-2 was increased, and expression of claudin-1, -3, and -4, occludin, and ZO-1 were decreased, as compared with normal gallbladder or AAC. We conclude that there are significant differences in expression of TJ proteins in AAC and ACC, supporting the idea that AAC represents a manifestation of systemic inflammatory disease, whereas ACC is a local inflammatory and often infectious disease.     

Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.     

Phocid seals, seal lice and heartworms: a terrestrial host-parasite system conveyed to the marine environment

Adaptation of pinnipeds to the marine habitat imposed parallel evolutions in their parasites. Ancestral pinnipeds must have harboured sucking lice, which were ancestors of the seal louse Echinophthirius horridus. The seal louse is one of the few insects that successfully adjusted to the marine environment. Adaptations such as keeping an air reservoir and the ability to hold on to and move on the host were necessary, as well as an adjustment of their life cycle to fit the diving habits of their host. E. horridus are confined to the Northern Hemisphere and have been reported from 9 species of northern phocids belonging to 4 genera, including land-locked seal species. The transmission from seal to seal is only possible when animals are hauled-out on land or ice. Lice are rarely found on healthy adult seals, but frequently on weak and young animals. The seal louse is suggested to play an important role as an intermediate host transmitting the heartworm Acanthocheilonema spirocauda among seals. However, the evidence is restricted to a single study where the first 3 larval stages of the heartworm were shown to develop in the louse. The fourth-stage larvae develop in the blood system of seals and eventually transform into the adult stage that matures in the heart. Since all other studies failed to confirm the presence of heartworm larvae in seal lice, other unknown intermediate hosts could be involved in the transmission of the heartworm. Transplacental transmission of microfilariae in seals has been suggested as an additional possibility, but is not likely to be important since the occurrence of heartworms in adult seals is very rare compared with juveniles. Furthermore, there are no findings of the first 3 larval stages in seals. This review shows that the heartworm infects nearly the same species of seals as the seal louse, except for the grey seal Halichoerus grypus, where the heartworm is absent. Prevalence and intensity of infection differ among regions in the Northern Hemisphere. As for seal lice, heartworms mainly infect immature seals, and after infection the prevalence seems to decrease with increasing age of the host.     

Novel Lignan and Stilbenoid Mixture Shows Anticarcinogenic Efficacy in Preclinical PC-3M-luc2 Prostate Cancer Model

Prostate cancer is the most common cancer of men in the Western world, and novel approaches for prostate cancer risk reduction are needed. Plant-derived phenolic compounds attenuate prostate cancer growth in preclinical models by several mechanisms, which is in line with epidemiological findings suggesting that consumption of plant-based diets is associated with low risk of prostate cancer. The objective of this study was to assess the effects of a novel lignan-stilbenoid mixture in PC-3M-luc2 human prostate cancer cells in vitro and in orthotopic xenografts. Lignan and stilbenoid –rich extract was obtained from Scots pine (Pinus sylvestris) knots. Pine knot extract as well as stilbenoids (methyl pinosylvin and pinosylvin), and lignans (matairesinol and nortrachelogenin) present in pine knot extract showed antiproliferative and proapoptotic efficacy at ≥40 μM concentration in vitro. Furthermore, pine knot extract derived stilbenoids enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis already at ≥10 μM concentrations. In orthotopic PC-3M-luc2 xenograft bearing immunocompromized mice, three-week peroral exposure to pine knot extract (52 mg of lignans and stilbenoids per kg of body weight) was well tolerated and showed anti-tumorigenic efficacy, demonstrated by multivariate analysis combining essential markers of tumor growth (i.e. tumor volume, vascularization, and cell proliferation). Methyl pinosylvin, pinosylvin, matairesinol, nortrachelogenin, as well as resveratrol, a metabolite of pinosylvin, were detected in serum at total concentration of 7−73 μM, confirming the bioavailability of pine knot extract derived lignans and stilbenoids. In summary, our data indicates that pine knot extract is a novel and cost-effective source of resveratrol, methyl pinosylvin and other bioactive lignans and stilbenoids. Pine knot extract shows anticarcinogenic efficacy in preclinical prostate cancer model, and our in vitro data suggests that compounds derived from the extract may have potential as novel chemosensitizers to TRAIL. These findings promote further research on health-related applications of wood biochemicals.