Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca(2+) concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca(2+)-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.     

Antioxidant properties of some different molecular weight chitosans

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6–CT7) with that of low Mr chitosans (CT1–CT5) showed that low Mr chitosans (CT1–CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1–CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the ‘TPAC’ test. These results suggest that low Mr chitosans (CT1–CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.     

Salinity affects the photoacclimation of <Emphasis Type="Italic">Chlamydomonas raudensis</Emphasis> Ettl UWO241

Chlamydomonas raudensis Ettl UWO241, a natural variant of C. raudensis, is deficient in state transitions. Its habitat, the deepest layer of Lake Bonney in Antarctica, features low irradiance, low temperature, and high salinity. Although psychrophily and low-light acclimation of this green alga has been described, very little information is available on the effect of salinity. Here, we demonstrate that this psychrophile is halotolerant, not halophilic, and it shows energy redistribution between photosystem I and II based on energy spillover under low-salt conditions. Furthermore, we revealed that C. raudensis exhibits higher non-photochemical quenching in comparison with the mesophile Chlamydomonas reinhardtii, when grown with low-salt, which is due to the lower proton conductivity across the thylakoid membrane. Significance of the C. raudensis UWO241 traits found in the low salinity culture are implicated with their natural habitats, including the high salinity and extremely stable light environments.     

Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas'' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection.     

Age-related differences in cutaneous warm sensation thresholds of human males in thermoneutral and cool environments

The purpose of this study was to investigate age-related differences in cutaneous temperature thresholds for warm thermal sensitivity in a thermoneutral (28 °C) and in a cool environment (22 °C). Peripheral warm thresholds were measured on nine body regions (cheek, chest, abdomen, upper arm, forearm, hand, thigh, shin, and foot) using a thermal stimulator in 12 young (22±1 years) and 13 elderly male subjects (67±3 years). The results showed that: (1) mean skin temperature did not differ by age in both environments; (2) the cutaneous warm thresholds for the hand, shin, and foot were significantly higher for the elderly than for the young in both environments (p<0.01), whereas the remaining body parts showed no age difference; (3) the most insensitive region for elderly males was the shin for both environments (p<0.01), while for young there was no statistical significant difference with T_a 28 °C; (4) the shin of the elderly was seven and nine times less sensitive to warmth when compared to those of the cheek at T_a 28 and 22 °C, respectively; and (5) warm thresholds were 3-4 °C greater at T_a 22 °C than at 28 °C, only for the elderly males' shin and foot (p<0.05), while for young the difference between T_a 22 and 28 °C was not statistically significant. The results indicate that age-related differences in cutaneous warm perception appear to be non-uniform over the body and significant on extremities; there is a greater bluntness of warm sensitivity in the cool environment for elderly males.