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The aim of our study was to improve the texture quality of salted Alaska pollock (Theragra chalcogramma) roe product using microbial transglutaminase (MTGase). Thus, the effect of 0–1.0% MTGase on the physical properties of the salted roe was assessed. Endogenous TGase activity in the roe, which contributes to improving its texture, decreased at −20 °C during frozen storage. Mechanical properties of the salted roe, such as roe firmness and egg breaking strength, were improved using MTGase, the optimum concentration of which was 0.5%. Protein polymerizations using SDS-PAGE and decreasing of protein solubilities in SDS–urea solution were observed in the salted roe with MTGase. Therefore, the texture of salted Alaska pollock roe can be significantly enhanced by adding 0.5% MTGase because of catalyzing protein crosslinking. Additionally, this study most likely indicates that MTGase can be used to improve the texture of aquatic processed foods at low temperatures, especially 5 °C.

HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z’ ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.

Soil Cd pollution is a serious environmental issue associated with human activities. However, the factors determining exogenous Cd dynamics in the soil profile in a complex environment are not well understood. Based on regional observations from 169 soil profiles across the Chengdu Plain, this study explored the key factors controlling Cd accumulation in the soil profile under actual field conditions. Results showed that total soil Cd contents decreased from 0.377 to 0.196 mg kg−1 with increasing soil depth. The effects of phosphate fertilizer rates, road density and precipitation on the difference in total soil Cd content were only observed in topsoil, while agricultural land-use type and topography had no impact. In contrast, significant differences in the total soil Cd content among different parent material types were found in the 0–20, 40–60 and 60–100 cm soil depths. One sample t-tests showed that significant Cd accumulation occurred in the whole soil profile in soils formed from Q4 (Quaternary Holocene) grey alluvium, while soils formed from Q3 (Quaternary Pleistocene) old alluvium and Q4 grey-brown alluvium showed significant Cd accumulation only in the 0–40 cm soil layers. In the topsoil, acid soluble Cd accounted for the largest proportion of the total Cd in soils formed from Q4 grey alluvium, reducible Cd was the main fraction in soils formed from Q4 grey-brown alluvium, while reducible Cd and residual Cd contributed the largest proportion of the total soil Cd in soils formed from Q3 old alluvium. The above results indicated that parent material was the decisive factor determining the magnitudes and depths of exogenous Cd accumulation in the soil profile due to its impacts on the Cd fraction distributions. These findings suggested that the parent material-induced Cd fraction distributions and accumulation should be considered for effectively exploring targeted remediation strategies for Cd pollution.

The quality of seasoned products of Alaska pollack (Theragra chalcogramma) roe declined dramatically during frozen storage.This research investigated the effects of processing sequence and frozen storage period on seasoned Alaska pollack roe product quality. In addition, the relationship between mechanical properties and protein composition of ovary membrane and eggshell was discussed.The seasoned roe product made by the Seasoning-Frozen storage process showed higher mechanical property values, better surface color and more stable protein composition.The results clarified that this process allowed endogenous transglutaminase to act before it had been denatured and effectively prevented protein degradation during frozen storage.The retardation of protein degradation was probably attributed to cryoprotective effects from components in seasoning solution., 公開日: 2022-12-07

Salt-reduced Alaska pollack roe benefits public health by decreasing NaCl intake; however, it has a poor texture with low breaking strength. This study addresses the feasibility of NaCl reduction in salted roe products, with focusing on the improvement of breaking strength using CaCl2. Salted roe products were prepared by immersing Alaska pollack roe in either NaCl solutions (3.5, 7.0, 15.0, 20.0, and 25.0%) or 7.0% NaCl solutions with added CaCl2 (0.0, 0.5, 1.0, 2.0, and 3.0%). Breaking strength, moisture and salt contents, eggshell protein composition of the salted roe products, as well as total endogenous transglutaminase (TGase) activity in various NaCl and CaCl2 concentrations were analyzed. CaCl2 addition enhanced eggshell protein crosslinking and breaking strength of the salt-reduced roe products. An acyl transfer reaction catalyzed by calcium-dependent TGase may be responsible for the eggshell protein crosslinking and improved texture. Thus, we successfully developed a salt-reduced Alaska roe product using CaCl2.

HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.

Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology. RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function, and catalytic activity similar to proteins. However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinch away from RNA nanotechnology. Here we report the construction of stable RNA nanoparticles resistant to RNase digestion. The 2'-F (2'-fluoro) RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing the phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase-resistant, biologically active, and stable RNA for application in nanotechnology.

HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg 21 . The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg 21 in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg 21 plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a ✓ outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg2+, Ca2+, and Mn2+. Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer → tetramer → hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that phi29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23-97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggests that segment 23-97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1-22 and 98-120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization.

Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation. Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid. We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions. Mutation in only one loop results in inactive pRNAs. However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules. The stoichiometry of pRNA for the packaging of one viral DNA genome is six. Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels. These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.

Background Regulated autoprocessing of HIV Gag-Pol precursor is required for the production of mature and fully active protease. We previously reported that H69E mutation in a pseudo wild type protease sequence significantly (>20-fold) impedes protease maturation in an in vitro autoprocessing assay and in transfected mammalian cells. Results Interestingly, H69E mutation in the context of a laboratory adapted NL4-3 protease showed only moderate inhibition (~4-fold) on protease maturation. There are six point mutations (Q7K, L33I, N37S, L63I, C67A, and C95A) between the NL4-3 and the pseudo wild type proteases suggesting that the H69E effect is influenced by other residues. Mutagenesis analyses identified C95 as the primary determinant that dampened the inhibitory effect of H69E. L63 and C67 also demonstrated rescue effect to a less extent. However, the rescue was completely abolished when H69 was replaced by aspartic acid in the NL4-3 backbone. Charge substitutions of surface residues (E21, D30, E34, E35, and F99) to neutral or positively charged amino acids failed to restore protease autoprocessing in the context of H69E mutation. Conclusions Taken together, we suggest that residue 69 along with other amino acids such as C95 plus L63 and C67 to a less extent modulate precursor structures for the regulation of protease autoprocessing in the infected cell.

Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S -transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli . Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PR H69E ) folds into active enzymes, and it does so with an apparent K d (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PR H69E and suggesting a folding defect in the precursor.

Objective evaluation of commercial spicy pollock roes was conducted. Ovary weight, ovary length, ovary membrane thickness, egg diameter, pH, color parameters, chemical composition, mechanical properties and protein composition were included in the evaluation items. The measurements of size, pH, color parameters, chemical composition and free amino acid may be not suitable for assessing the quality of commercial spicy pollock roe in the current industry, as they do not differ much between high-priced and low-priced products. However, we found that most of products showed significant differences in ovary hardness between them. Therefore, we propose the measurements of mechanical properties such as ovary hardness as objective quality assessing methods. Keywords: Alaska pollock roe ,Chemical composition, Ovary hardness

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer → tetramer → hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75–81 of the left stem were found to be oriented toward the head loop and proximate to bases 26–31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45–71 and 82–91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.

The metal complexes, cis-Rh(phen)2Cl2+ and its more hydrophobic analog cis-Rh(phen)(phi)Cl2+, have been shown to photocross-link the 120-base 29-encoded pRNA. Primer extension on the cis-Rh(phen)(phi)Cl2+-photocross-linked RNA revealed that guanines are responsible for the interstrand cross-links.

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.