The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.     

Vacuoles in macrophages and reticular cells of regional lymph nodes of the rat after injection of large doses of steroids

The ultrastructure of macrophages and reticular cells of regional lymph nodes of the rat after administration of large doses of cortisone acetate, estrone, progesterone, and cholesterol in aqueous suspensions was investigated. A large number of vacuoles, most of which were surrounded by unit membrane, and lipid droplets not surrounded by unit membrane were observed in the cytoplasm of both macrophages and reticular cells. They were not seen in these cells of control animals and in experimental animals that had received smaller doses of these steroid hormones. After cholesterol injection, many lipid droplets were observed in the cytoplasm of macrophages. These observations suggest that steroids injected in suspension accumulate in macrophages and reticular cells of the regional lymph nodes. Electron-dense material was often present in vacuoles of macrophages but not in those of reticular cells.     

Evaluation of the transpiration rate of lotus using the stem heat-balance method

To reveal the mechanism of transpiration by hydrophytes in the field, it is necessary to evaluate the transpiration rate without the effect of the evaporation from the water surface. In order to test the suitability for evaluating the transpiration rate of lotus (Nelumbo nucifera Gaertn.) leaves in the field, stem heat-balance method was applied and the obtained sap-flow rate was compared with the transpiration rate measured by weighing and with the overall canopy evapotranspiration rate by means of the eddy covariance technique. The transpiration rate estimated with the sap-flow measurements showed good agreement with that obtained from the weighing method. Lotus has many air canals in its petiole to carry oxygen-rich air to the rhizome and methane- and carbon dioxide-rich air back to the atmosphere, but there was little effect of the mass flow of air through these canals on the sap-flow rates. In the field observations, the canopy evapotranspiration rate (0.28 mm h⁻¹ at maximum) was nearly equal to the sum of the transpiration rate from all sunlit leaves (0.30 mm h⁻¹), and the contribution of the transpiration from shaded leaves and evaporation from the water surface was considered to be minor in the seasons when the leaves were fully developed. Evaluation of bulk leaf conductance revealed that the conductance in the leaf boundary layer of lotus could be low (ca. 0.23 mol m⁻² s⁻¹) because of its large leaf area. The low conductance in the leaf boundary layer would increase leaf temperature, which, in turn, would generate air circulation within the plant's ventilation system. Because there was a linear relationship between transpiration rate and the leaf-to-air vapor-pressure deficit, with no apparent maximum, high vapor-pressure deficits (3.4 kPa at maximum) did not appear to cause significant stomatal closure in lotus plants. The stomata of lotus leaves play a role as air inlets to carry oxygen-rich air to the rhizome, so their low sensitivity would help to increase air intake.     

Pepsin, an aspartic protease, converts porcine big endothelin to 21-residue endothelin

Porcine big endothelin (big ET-39) at 1 nM, a concentration with no influence on contractile activity in isolated rat aorta, induced a slow-onset and sustained contraction by the pre-incubation with pepsin. When the incubation mixture of big ET-39 with pepsin was analyzed by high-performance liquid chromatography on an octadecyl silica column, two major products of pepsin hydrolysis were obtained; their amino acid sequences were identical with those of 21-residue endothelin (ET-21) and a C-terminal peptide of big ET-39, big ET (22-39), respectively. On the other hand, no degradation of ET-21 was observed by pepsin treatment. These results indicate that pepsin specifically cleaves a Trp21-Val22 bond in the big ET-39 molecule, producing ET-21 and big ET (22-39). Thus, the possibility that pepsin-like aspartic protease may participate in the conversion of big ET-39 to ET-21 in vivo warrants further attention.     

Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90

The 90-kDa heat shock protein, HSP90, of the mouse has two isoforms, alpha and beta, which are electrophoretically separable. We have investigated the native forms of HSP90 molecules under physiological conditions and determined their isoform compositions. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that HSP90 purified from mouse lymphoma L5178Y cells consists of approximately 40% alpha and 60% beta isoforms. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the purified HSP90 exists predominantly as a dimer, but a considerable amount of monomer was also detected. Western blotting using polyclonal anti-mouse HSP90 antibodies revealed that the native forms of HSP90 in the crude L5178Y cell lysates are also dimer and monomer. The nondenaturing polyacrylamide gel electrophoresis resolved the dimeric forms into two separate bands that were identified as alpha/alpha and beta/beta homodimers by two methods: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. In addition, the results showed that the monomeric form consists mainly of the beta isoform. Both the alpha and beta isoforms were shown to bind equally to actin filaments.     

Evidence for a nitrate-independent function of the nitrate sensor NRT1.1 in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

NRT1.1 is a putative nitrate sensor and is involved in many nitrate-dependent responses. On the other hand, a nitrate-independent function of NRT1.1 has been implied, but the clear-cut evidence is unknown. We found that NRT1.1 mutants showed enhanced tolerance to concentrated ammonium as sole N source in Arabidopsis thaliana. This unique phenotype was not observed in mutants of NLP7, which has been suggested to play a role in the nitrate-dependent signaling pathway. Our real-time PCR analysis, and evidence from a literature survey revealed that several genes relevant to the aliphatic glucosinolate-biosynthetic pathway were regulated via a nitrate-independent signal from NRT1.1. When taken together, the present study strongly suggests the existence of a nitrate-independent function of NRT1.1.     

Characterization of sugar recognition by the toxin complex produced by the Clostridium botulinum serotype C variant strain Yoichi

Clostridium botulinum serotype C strains produce a neurotoxin (BoNT) along with nontoxic proteins, including nontoxic nonhemagglutinin and three hemagglutinin subcomponents, HA-70, HA-33 and HA-17, to form a large toxin complex (L-TC). While L-TCs produced by serotype C strains usually exhibit hemagglutination (HA) activity via HA-33 binding to sialic acid on erythrocytes, serotype C strain Yoichi (C-Yoichi) L-TC exhibited neither HA nor binding activity towards erythrocytes, probably due to a C-terminal truncation of the HA-33 protein. However, here, we demonstrate that C-Yoichi L-TC newly showed full HA and binding activity towards neuraminidase-treated erythrocytes that was completely inhibited in the presence of galactose (Gal) or lactose (Lac). Binding of C-Yoichi L-TC to rat small intestine epithelial cells (IEC-6) treated with neuraminidase was also significantly enhanced compared with untreated IEC-6 cells. Similarly, the HA-33/HA-17 complex isolated from C-Yoichi L-TC also bound to neuraminidase-treated IEC-6 cells. The binding activity of both L-TC and HA-33/HA-17 was inhibited in the presence of Gal or Lac. Additionally, C-Yoichi L-TC adsorbed tightly to a lactose-affinity gel column. These results strongly suggest that the unusual recognition of the Gal moiety on the cells could be due to a variation and/or a truncation in the C-terminal-half of the unique C-Yoichi HA-33 protein.     

Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner

Background

Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state.

Methodology and Results

An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae.

Conclusions

This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis.     

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.