Effect of terminal velocities on macroscopic and microscopic hydrodynamic mixing of stratified suspensions.

We performed numerical experiments to investigate the mixing of stratified suspensions composed of different particle types by gravitational sedimentation. The mixing process is controlled by a dimensionless group Y_{m}∼U_{f}/U_{St1}, where U_{f} is a typical velocity of a macroscopic sedimenting finger and U_{St1} is the Stokes settling velocity of a single spherical particle in the upper suspension. The effects of components of Y_{m}, in particular, terminal velocities of particles, were investigated. For Y_{m}=100, no large difference was observed for the difference of components of Y_{m}, and it was confirmed that the mixing rate is determined by Y_{m}, because macroscopic (vessel-scale) mixing is dominant for large Y_{m}. For Y_{m}=5, macroscopic mixing and microscopic (individual particle-level) mixing due to the particle terminal velocity difference are of the same order, while completely different mixing patterns were observed for positive, zero, and negative terminal velocity differences: macroscopic mixing is promoted by the increase in apparent density due to microscopic mixing, small macroscopic mixing is suppressed by the individual particle settling, and jetting mixing occurs owing to pure liquid layer formation.

Macroscopic and microscopic hydrodynamic mixing of stratified suspensions.

We conducted numerical experiments to investigate the mixing of stratified suspensions containing different types of particles. We used a point-force two-way coupling method. We studied the mixing behavior of stratified suspensions and we discovered two types of mixing: microscopic (individual-particle-level) and macroscopic (vessel-scale) collective mixing. In addition, we examined the vertical mixing speed of the stratified suspension. We used a simple theoretical model to analyze the fingering settling velocity. Then we introduced a nondimensional number representing the difference in collectivities of the upper and lower suspensions while accounting for particle terminal velocities. We discovered that the proposed nondimensional parameter has a negative sign that distinguishes the mixing form of only microscopic individual-particle-level mixing and a positive value that predicts the speed of macroscopic collective mixing of stratified suspensions.

Possibility of non-Fickian mixing at concentration interface between stratified suspensions.

HYPOTHESIS: Relative motion of micro-sized particles suspended in liquid is governed by hydrodynamic effect, in contrast to nano-sized particle suspension in which thermal effect is significant. As a result, the mixing behavior of stratified suspensions with micro-sized particles is totally different from those obeying Fick's diffusion law for nano-sized particles. Such a "non-Fickian" mixing of micro-sized particles is determined not only by the concentration difference but also the physical properties of suspensions. EXPERIMENTS: We conducted an experimental study of gravitational settling of stratified suspensions of micro-sized particles with concentration gradients opposed to gravity. We also performed point-force-type numerical simulations under the same conditions as those in the experiment. Particularly, we focused on the relative motion of particles near the concentration interface, which is an apparent interface between the upper and the lower suspensions having different concentrations. FINDINGS: The experimental and numerical results indicate that, if the number density of particles in suspension is sufficient, the concentration interface seemingly behaves immiscibly and the interface prevents particle mixing. However, a small number of particles cannot maintain the seal of the concentration interface then demonstrates miscible behavior. The mixing mechanism of the suspended particles at the concentration interface is strongly related to the miscible and immiscible characteristics of the interface.

Mouse TMCO5 is localized to the manchette microtubules involved in vesicle transfer in the elongating spermatids.

As a result of a high-throughput in situ hybridization screening for adult mouse testes, we found that the mRNA for Tmco5 is expressed in round and elongating spermatids. Tmco5 belongs to the Tmco (Transmembrane and coiled-coil domains) gene family and has a coiled-coil domain in the N-terminal and a transmembrane domain in the C-terminal region. A monoclonal antibody raised against recombinant TMCO5 revealed that the protein is expressed exclusively in the elongating spermatids of step 9 to 12 and is localized to the manchette, a transiently emerging construction, which predominantly consists of cytoskeleton microtubules and actin filaments. This structure serves in the transport of Golgi-derived non-acrosomal vesicles. Moreover, induced expression of TMCO5 in CHO cells resulted in the co-localization of TMCO5 with ß-tubulin besides the reorganization of the Golgi apparatus. Judging from the results and considering the domain structure of TMCO5, we assume that Tmco5 may have a role in vesicle transport along the manchette.

Retort beef aroma that gives preferable properties to canned beef products and its aroma components.

The objective of this study is to identify the properties and responsible compounds for the aromatic roast odor (retort beef aroma) that commonly occurs in canned beef products and could contribute to their palatability. The optimal temperature for generating retort beef aroma was 121°C. An untrained panel evaluated both uncured corned beef and canned yamato-ni beef and found that they had an aroma that was significantly (P < 0.01) similar to the odor of 121°C-heated beef than 100°C-heated beef. The panel also noted that the aroma of 121°C-heated beef tended to be (P < 0.1) preferable than that of 100°C-heated beef. These results suggest that retort beef aroma is one constituent of palatability in canned beef. GC-MS (gas chromatography-mass spectrometry) analysis of the volatile fraction obtained from 100°C- and 121°C-heated beef showed that the amounts of pyrazine, 2-methylpyrazine and diacetyl were higher in the 121°C-heated beef than in the 100°C-heated beef. GC-sniffing revealed that the odor quality of pyrazines was similar to that of retort beef aroma. Therefore, pyrazines were suggested to be a candidate responsible for the retort beef aroma. Analysis of commercial uncured corned beef and cured corned beef confirmed the presence of pyrazine, 2-methylpyrazine and 2,6-dimethylpyrazine.

Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers.

YAP and TAZ oncoproteins confer malignancy and drug resistance to various cancer types. We screened for small molecules that inhibit the nuclear localization of YAP/TAZ. Dasatinib, statins and pazopanib inhibited the nuclear localization and target gene expression of YAP and TAZ. All three drugs induced phosphorylation of YAP and TAZ, and pazopanib induced proteasomal degradation of YAP/TAZ. The sensitivities to these drugs are correlated with dependence on YAP/TAZ in breast cancer cell lines. Combinations of these compounds with each other or with other anti-cancer drugs efficiently reduced cell proliferation of YAP/TAZ-dependent breast cancer cells. These results suggest that these drugs can be therapeutics and chemosensitizers for YAP/TAZ-dependent breast cancers.

Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2ß stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode.

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2ß bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2ß-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2ß-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2ß mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2ß and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.

X-ray snapshot of the mechanism of inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives.

The mechanism of action of a general class of mechanism-based inhibitors of serine proteases, including human neutrophil elastase (HNE), has been elucidated by determining the X-ray crystal structure of an enzyme-inhibitor complex. The captured intermediate indicates that processing of inhibitor by the enzyme generates an N-sulfonyl imine functionality that is tethered to Ser195, in accordance with the postulated mechanism of action of this class of inhibitors. The identity of the HNE-N-sulfonyl imine species was further corroborated using electrospray ionization mass spectrometry.

Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection.

Eukaryotic initiation factor (eIF) 1 is a small protein (12 kDa) governing fidelity in translation initiation. It is recruited to the 40 S subunit in a multifactor complex with Met-tRNA(i)(Met), eIF2, eIF3, and eIF5 and binds near the P-site. eIF1 release in response to start codon recognition is an important signal to produce an 80 S initiation complex. Although the ribosome-binding face of eIF1 was identified, interfaces to other preinitiation complex components and their relevance to eIF1 function have not been determined. Exploiting the solution structure of yeast eIF1, here we locate the binding site for eIF5 in its N-terminal tail and at a basic/hydrophobic surface area termed KH, distinct from the ribosome-binding face. Genetic and biochemical studies indicate that the eIF1 N-terminal tail plays a stimulatory role in cooperative multifactor assembly. A mutation altering the basic part of eIF1-KH is lethal and shows a dominant phenotype indicating relaxed start codon selection. Cheung et al. recently demonstrated that the alteration of hydrophobic residues of eIF1 disrupts a critical link to the preinitiation complex that suppresses eIF1 release before start codon selection (Cheung, Y.-N., Maag, D., Mitchell, S. F., Fekete, C. A., Algire, M. A., Takacs, J. E., Shirokikh, N., Pestova, T., Lorsch, J. R., and Hinnebusch, A. (2007) Genes Dev. 21, 1217-1230 ). Interestingly, eIF1-KH includes the altered hydrophobic residues. Thus, eIF5 is an excellent candidate for the direct partner of eIF1-KH that mediates the critical link. The direct interaction at eIF1-KH also places eIF5 near the decoding site of the 40 S subunit.

Change in nutritional status modulates the abundance of critical pre-initiation intermediate complexes during translation initiation in vivo.

In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation.

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.

The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G.

Eukaryotic translation initiation factor (eIF) 5 is crucial for the assembly of the eukaryotic preinitiation complex. This activity is mediated by the ability of its C-terminal HEAT domain to interact with eIF1, eIF2, and eIF3 in the multifactor complex and with eIF4G in the 48S complex. However, the binding sites for these factors on eIF5-C-terminal domain (CTD) have not been known. Here we present a homology model for eIF5-CTD based on the HEAT domain of eIF2Bepsilon. We show that the binding site for eIF2beta is located in a surface area containing aromatic and acidic residues (aromatic/acidic boxes), that the binding sites for eIF1 and eIF3c are located in a conserved surface region of basic residues, and that eIF4G binds eIF5-CTD at an interface overlapping with the acidic area. Mutations in these distinct eIF5 surface areas impair GCN4 translational control by disrupting preinitiation complex interactions. These results indicate that the eIF5 HEAT domain is a critical nucleation core for preinitiation complex assembly and function.

Eukaryotic translation initiation factor 5 is critical for integrity of the scanning preinitiation complex and accurate control of GCN4 translation.

The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal pre-initiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for pre-initiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts- phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd- phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNA(i)Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn- phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the post-assembly process in the 48S complex, likely during the scanning process.

Physical association of eukaryotic initiation factor (eIF) 5 carboxyl-terminal domain with the lysine-rich eIF2beta segment strongly enhances its binding to eIF3.

The carboxyl-terminal domain (CTD) of eukaryotic initiation factor (eIF) 5 interacts with eIF1, eIF2beta, and eIF3c, thereby mediating formation of the multifactor complex (MFC), an important intermediate for the 43 S preinitiation complex assembly. Here we demonstrate in vitro formation of a nearly stoichiometric quaternary complex containing eIF1 and the minimal segments of eIF2beta, eIF3c, and eIF5. In vivo, overexpression of eIF2 and tRNA(Met)(i) suppresses the temperature-sensitive phenotype of tif5-7A altering eIF5-CTD by increasing interaction of the mutant eIF5 with eIF2 by mass action and restoring its defective interaction with eIF3. By contrast, overexpression of eIF1 exacerbated the tif5-7A phenotype because eIF1 forms unusual inhibitory complexes with a hyperstoichiometric amount of eIF1. Formation of such complexes leads to increased GCN4 translation, independent of eIF2 phosphorylation (general control derepressed or Gcd(-) phenotype). We also provide biochemical evidence indicating that the association of eIF5-CTD with eIF2beta strongly enhances its binding to eIF3c. Our results suggest strongly that MFC formation is an ordered event involving specific enhancement of eIF5-CTD binding to eIF3 on its binding to eIF2beta. We propose that the primary function of eIF5-CTD is to serve as an assembly guide by rapidly promoting stoichiometric MFC assembly with the aid of eIF2 while excluding formation of nonfunctional complexes.

Efficient incorporation of eukaryotic initiation factor 1 into the multifactor complex is critical for formation of functional ribosomal preinitiation complexes in vivo.

Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation. It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents. Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2beta but not that with eIF3c. Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo. C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of eIF2.GTP.Met-tRNA(i)(Met) ternary complex binding to the ribosome. Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation. Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions. We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex.

Nascent-peptide-mediated ribosome stalling at a stop codon induces mRNA cleavage resulting in nonstop mRNA that is recognized by tmRNA.

Recent studies have established that tmRNA-mediated protein tagging occurs at stop codons depending on the C-terminal amino acid sequence of the nascent polypeptide immediately adjacent to those codons. We investigate here how the trans-translation at a stop codon occurs by using model crp genes encoding variants of cAMP receptor protein (CRP). We demonstrate that a truncated crp mRNA is efficiently produced along with a normal transcript from the model gene where tmRNA-mediated protein tagging occurs. The truncated crp mRNA was not detected in the presence of tmRNA, indicating that its degradation was facilitated by tmRNA. The major 3'-ends of the truncated crp mRNA in cells unable to express tmRNA were mapped at and near the stop codon. When RNA derived from the model crp-crr fusion gene was analyzed, crr mRNA was detected as a downstream cleavage product along with the upstream crp mRNA. These results are compatible with the hypothesis that ribosome stalling caused by the tagging-provoking sequences leads to endonucleolytic cleavage of mRNA around the stop codon, resulting in nonstop mRNA. In addition, the data are consistent with the view that mRNA cleavage is the cause of trans-translation at stop codons. Neither the bacterial toxin RelE nor the known major endoribonucleases are required for this cleavage, indicating that either other endoribonuclease(s) or the ribosome itself would be responsible for the mRNA cleavage in response to ribosome stalling caused by the particular nascent peptides.

Comparison of the sequences of Turbo and Sulculus indoleamine dioxygenase-like myoglobin genes.

Some archaeogastropodic molluscs, including Sulculus and Turbo, contain an unusual approximately 40 kDa myoglobin in their buccal masses. This myoglobin can bind oxygen reversibly, but has a lower oxygen affinity than vertebrate and invertebrate myoglobins. Amino acid sequences clearly show that Sulculus and Turbo myoglobins evolved not from the globin gene but from the gene for indoleamine dioxygenase (IDO), a tryptophan-degrading enzyme. The Turbo myoglobin gene has been determined to consist of 14 exons and 13 introns. Compared with the known Sulculus IDO-like myoglobin gene, all splice junctions except two are conserved exactly between the two genes. The exon/intron organization of these myoglobin genes is also highly homologous with human IDO (ten exon/nine intron structure); splice junctions of six introns were exactly conserved among the three genes, suggesting that these introns have been conserved for at least 600 million years. To look for putative IDO genes in Turbo or Sulculus, we re-examined the genomic DNA fragments amplified by PCR in full detail, and found intron 2 in two distinct Sulculus fragments (A and B). Fragment A with a 576 bp intron corresponded exactly to the myoglobin gene of Sulculus. On the other hand, fragment B, containing a 239 bp intron, differed significantly from fragment A in nucleotide and translated amino acid sequences. Detailed sequence comparison suggests that fragment B may be derived from a putative IDO gene of Sulculus.

SsrA-mediated trans-translation plays a role in mRNA quality control by facilitating degradation of truncated mRNAs.

An important unsolved question regarding the bacterial SsrA system is the fate of target mRNAs replaced by SsrA RNA during trans-translation. The aim of the present study is to address the potential role of SsrA system in mRNA quality control, focusing on truncated mRNAs that are expected to arise from 3'-to-5' exonucleolytic attack. We found that significant amounts of truncated mRNAs and polypeptides were produced from genes lacking a rho-independent terminator in SsrA-deficient cells. These truncated mRNAs, hence truncated polypeptides, were no longer observed in the presence of SsrA RNA. The data indicate that the SsrA system facilitates degradation of "nonstop" mRNAs by presumably removing the stalled ribosomes. Furthermore, analysis of affinity-purified proteins indicated that truncated polypeptides could be produced even from a gene with an intact rho-independent terminator, although less efficiently, implying that C-terminally truncated proteins and 3'-truncated mRNA may be produced from virtually all protein-coding genes. We conclude that the SsrA system not only promotes the degradation of incomplete polypeptides but also minimizes the synthesis of incomplete polypeptides by facilitating the degradation of truncated mRNAs that are produced in cells.

Bacterial SsrA system plays a role in coping with unwanted translational readthrough caused by suppressor tRNAs.

BACKGROUND: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C-terminus of the nascent polypeptide when a ribosome is stalled at the 3' end of an mRNA lacking a stop codon. This process, called trans-translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP-dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans-translation in Escherichia coli. RESULTS: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Rho-independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3' end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA. CONCLUSION: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.