Functional binding of inner-arm dyneins with demembranated flagella of Chlamydomonas mutants

Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating.     

Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.     

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori

Sericins are soluble silk components encoded in Bombyx mori by three genes, of which Ser1 and Ser3 have been characterized. The Ser1 and Ser3 proteins were shown to appear later in the last larval instar as the major sericins of cocoon silk. These proteins are, however, virtually absent in the highly adhesive silk spun prior to cocoon spinning, when the larvae construct a loose scaffold for cocoon attachment. We show here that the silk-gland lumen of the feeding last instar larvae contains two abundant adhesive proteins of 230 kDa and 120 kDa that were identified as products of the Ser2 gene. We also describe the sequence, exon–intron structure, alternative splicing and deduced translation products of this gene in the Daizo p50 strain of B. mori. Two mRNAs of 5.7 and 3.1 kb are generated by alternative splicing of the largest exon. The predicted mature proteins contain 1740 and 882 amino acid residues. The repetitive amino acid sequence encoded by exons 9a and 9b is apparently responsible for the adhesiveness of Ser2 products. It has a similar periodic arrangement of motifs containing lysine and proline as a highly adhesive protein of the mussel Mytilus edulis.     

αB-crystallin extracellularly suppresses ADP-induced granule secretion from human platelets

αB-crystallin, a low-molecular-weight heat shock protein (HSP), has binding sites on platelets. However, the exact role of αB-crystallin is not clarified. In this study, we investigated the effect of αB-crystallin on platelet granule secretion. αB-crystallin attenuated the adenosine diphosphate (ADP)-induced phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The ADP-stimulated HSP27 phosphorylation was markedly reduced by αB-crystallin. αB-crystallin significantly suppressed the ADP-induced secretions of both platelet-derived growth factor (PDGF)-AB and serotonin. Therefore, our results strongly suggest that αB-crystallin extracellularly suppresses platelet granule secretion by inhibition of HSP27 phosphorylation via p44/p42 MAPK and p38 MAPK.     

Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin

We show that a series of peptides corresponding to individual β-strands in native β-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the β-strands that we considered, βA, were found to promote fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, βI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of βI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.     

Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication in Xenopus Egg Extracts

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.     

Regulation of paracellular Na and Cl conductances by hydrostatic pressure

The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl) has been Investigated. Gp, G_Na and G_Cl were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1-4 cm H₂O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, G_Na and G_Cl in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, G_Na and G_Cl. How the hydrostatic pressure influences Gp, G_Na and G_Cl under an isosmotic condition was also investigated. Gp, G_Na and G_Cl were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, G_Na and G_Cl, while apically applied hydrostatic pressure had little effect on Gp, G_Na or G_Cl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, G_Na and G_Cl.     

Developing a pre-entry weed risk assessment system for use in Japan

We evaluated the applicability of the Australian Weed Risk Assessment (AWRA) system in Japan. Native weeds (n = 117) and introduced plants (n = 142), whose weed status was classified by 20 plant experts, were assessed using a slightly modified version of the AWRA system designed to fit Japanese conditions. A receiver operating characteristic (ROC) curve for the system, when classifying two-thirds of the 259 taxa as weeds or non-weeds, was plotted and the area under the ROC curve was calculated. The area was 0.88 and significantly greater than 0.5. Thus, the validity of the system to classify plants was proven. The best cut-off level for the WRA score using Youden’s index was 10. When taxa whose AWRA scores were greater than 10 were regarded as weeds, the sensitivity and specificity were 0.88 and 0.78, respectively. These values were verified with the remaining one-third of the taxa. From these findings, the modified AWRA system was considered to be effective for use in Japan. However, further studies are required to set the best cut-off level in terms of maximising the benefits gained from using the system. A second screening test associated with the cut-off level also needs to be developed.