Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter

CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli. Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity. By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified. Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA. At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity. Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V.     

Proline synthesis and redox regulation: Differential functions of pyrroline-5-carboxylate reductase in human lymphoblastoid cell lines

Pyrroline-5-carboxylate (P5C) reductase not only catalyzes the final step in proline biosynthesis but also mediates redox regulation. We examined these dual functions in human lymphoblastoid cell lines (REH, LHN₁₃) one of which (REH) is deficient in proline synthesis. In spite of the deficiency in proline synthesis in REH, the PC-mediated redox effect was relatively intact. The dissociation of these functions was due to qualitative as well as quantitative differences in the REH enzyme relative to its utilization of NADPH and NADH.     

Kappaphycus malesianus sp. nov.: a new species of Kappaphycus (Gigartinales,Rhodophyta) from Southeast Asia

A new species, Kappaphycus malesianus, is established as a new member of the genus Kappaphycus. Locally known as the “Aring-aring” variety by farmers in Malaysia and the Philippines, this variety has been commercially cultivated, often together with Kappaphycus alvarezii due to the similarities in morphology. Despite also producing kappa-carrageenan, the lower biomass of the K. malesianus when mixed with K. alvarezii ultimately affects the carrageenan yield. Morphological observations, on both wild and cultivated plants, coupled with molecular data have shown K. malesianus to be genetically distinct from its Kappaphycus congeners. The present study describes the morphology and anatomy of this new species as supported by DNA data, with additional morphological features for distinguishing between commercial Kappaphycus cultivars.     

Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors

Global demand for seaweed resources has increased due to their emergent use as sources of biopharmaceuticals, nutraceuticals and biofuels. These high-valued products make possible the use of micropropagation techniques that may be more costly than conventional mariculture. This study reports the successful tissue culture of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott) Abbott, Zhang and Xia. Callus induction of K. alvarezii was successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was observed in solidified Provasoli’s enriched seawater medium. Different culture conditions such as agar concentration, growth hormones, nutrients, irradiance and enrichment media were investigated to determine the suitable conditions for explant culture of G. changii. Proliferations of adventitious shoots were induced under the most suitable culture conditions. G. changii explants were successfully cultured in airlift photo-bioreactors, with no decrease in the carbohydrate content in the G. changii explants. This micropropagation technique can provide a useful alternative system for seedling production of economically important seaweeds.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting 'curing' of the adhesive; some force curves also contained a 'fingerprint' of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative 'tanning' mechanism in barnacle cyprid cement.     

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca²⁺-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.     

Effects of irradiance and salinity on the growth of carpospore-derived tetrasporophytes of Gracilaria edulis and Gracilaria tenuistipitata var liui (Rhodophyta)

Gracilaria edulis and Gracilaria tenuistipitata var liui are agarophytes with high commercial value which are currently cultivated in countries like India and Thailand. They have great potential for mariculture in Malaysia. Experiments were carried out to study carpospore germination and determine the effects of irradiance and salinity on the growth of these two species. Both species showed the Dumontia type of carpospore development. Both species showed increased daily growth rate (% day^?1) with increasing irradiance and tolerance for a wide range of salinity with a preference for low salinity. G. edulis grew best at 100 μmol photons m^?2 s^?1 and 15 psu while G. tenuistipitata var liui grew best at 60–130 μmol photons m^?2 s^?1 and 15 psu. The highest growth rate obtained for G. edulis and G. tenuistipitata var liui was 13.57 and 19.7 % day^?1 respectively. tenuistipitata var liui. ANOVA showed that both irradiance and salinity have significant effect on the growth of both species (P?<?0.05). The results showed that G. tenuistipitata var liui is a good candidate for mass cultivation in Malaysian brackish waters. Besides, this study also showed the feasibility of using spore culture to provide stocks for sustainable farming of Gracilaria.