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This paper reports on a feasibility study in the detection of nanometer thick lubricated regions on a hard disk by a novel use of a Kelvin probe. A nonvibrating Kelvin probe was constructed and used to measure the voltages between the copper surface of the probe and a hard disk partially-lubricated with a perfluoropolyether. The probe, constructed of a shielded 1.6 mm diameter gold-coated copper wire, was fixed above the hard disk and the probe voltage was obtained as a function of the speed of the disk and lubricant thickness. The probe generates an electrical signal as the interface between the lubricated and unlubricated regions is crossed. Results show that the probe can distinguish between regions on the hard disk that contain 3 and 10 nm thick lubricant films.

Spinal cord infarction (SCI) is a rare but serious complication of endovascular aortic repair (EVAR) for abdominal aortic aneurysms (AAA). It is difficult to predict, prevent, and treat and can cause significant impairment. We describe the case of a patient who experienced paraplegia and thermal pain dysfunction of the lower extremities shortly after EVAR for an infrarenal AAA. Immediately after confirming SCI, we initiated cerebrospinal fluid drainage, administered steroids, naloxone, and free radical scavengers, and maintained high blood pressure. However, the patient's symptoms did not improve sufficiently. Since the possibility of a SCI exists, prompt treatment should be initiated., (@ 2024 The Editorial Committee of Annals of Vascular Diseases.)

The mechanism of bioluminescence in the luminous land snails remains largely unknown. Here, we analyzed basic biochemical properties of the luminous land snail, Quantula weinkauffiana. The luminescence activity was extracted from the light organ located near the mouth using a neutral buffer containing detergent. The reaction of the crude buffer extract was triggered by the addition of only hydrogen peroxide (H 2 O 2 ). These results are inconsistent with the single precedent report on the bioluminescence in the Quantula striata by Shimomura and Haneda in 1986, in which the luminescence of the buffer extract (without detergent) from the light organ was induced by the coaddition of three indispensable components H 2 O 2 , ferrous ion, and 2-mercaptoethanol. Based on the present findings, we suggested that an insoluble photoprotein is involved in the bioluminescence of the luminous land snails and the luminescence reaction is simply triggered by H 2 O 2 ., (© 2024 John Wiley & Sons Ltd.)

A 64-year-old man visited the outpatient department of our hospital for the first time due to bilateral lower limb edema, which he noticed 1 week before the visit. Pain suddenly developed in the left lower limb while the patient was in the waiting room. Nephrotic syndrome was suspected based on blood and urine test results. Acute arterial thromboembolism in the left lower limb associated with hypercoagulation due to nephrotic syndrome was suspected, and a diagnosis was made using computed tomography angiography. Arterial thrombectomy was urgently performed, and the limb was salvaged without sequelae. Based on renal biopsy, minimal change nephrotic syndrome was diagnosed, and the patient underwent remission induction with steroid therapy. Heparin was drip infused and apixaban was orally administered to prevent recurrent thrombosis. Nephrotic syndrome in the acute phase is often complicated by thrombosis. Particularly, arterial thromboembolism requires prompt treatment, and prophylactic anticoagulation therapy needs to be considered., (© 2023. The Author(s) under exclusive licence to The Japan Society of Nephrology.)

The mysterious world of the bioluminescent molluscs in terrestrial ecosystems is mesmerizing, but Quantula striata was previously the only terrestrial mollusc known to be luminescent. Here, we document the new discovery of bioluminescence in four land snails, namely Phuphania crossei, P. globosa, P. carinata, and P. costata. Our observations establish clearly that these four species of Phuphania produce a continuous greenish light from the light-emitting cells located within the mantle and the foot, and that its bright luminescence is intracellular and is not due to any luminous secretion. Although both Quantula and Phuphania can produce a green light, the luminescence patterns are different. The luminescence displayed by Quantula is rhythmical blinking or flashing, while Phuphania glows continuously. In addition, the bioluminescence in Q. weinkauffiana is confirmed, which is similar to that in the related species, Q. striata., (© 2023. Springer Nature Limited.)

The lanternfish is a deep-sea fish with ventral-lateral and head photophores. It uses its ventral-lateral photophores to camouflage its ventral silhouette, a strategy called counterillumination. The bioluminescent reaction of lanternfish involves coelenterazine as a substrate luciferin but the enzyme catalyzing the bioluminescent reaction has not been identified. We report a candidate enzyme of luciferase from lanternfish Diaphus watasei. We purified the luciferase and performed SDS-PAGE analysis resulted in two bands corresponding to the activity, and following mass spectrometry analysis detected three 14-3-3 proteins of which functions is known to exhibit protein-protein interactions. The molecular weights and isoelectric points of the 14-3-3 proteins were almost consistent with the luciferase properties. The addition of two 14-3-3 binding compounds, R18 peptide and fusicoccin, resulted in the inhibition of the luciferase activity. However, the two 14-3-3 recombinant proteins showed very slight luminescence activity. These results suggested that the 14-3-3 proteins are candidate luciferases of D. watasei., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Purposes: The optimal surgical management of renal cell carcinoma with tumor thrombus within the inferior vena cava (IVC) remains to be clarified., Methods: Sixteen consecutive cases were reviewed. Incision, the IVC clamping position, and the venous drainage procedure were modified according to the tumor thrombus extension level: level I or II (below the hepatic vein, n = 8), level III (above the hepatic vein but below the right atrium, n = 5), and level IV (extending into the right atrium, n = 3)., Results: For level I or II, resection could be simply achieved by clamping the IVC below the hepatic vein, without hemodynamic collapse. For level III, clamping the IVC above the hepatic vein and the hepatoduodenal ligament was required. Venous drainage from the lower body (cannulation to distal IVC) and portal system (cannulation to ileocolic vein) were applied. When opening the IVC, the significant backflow was controlled using cardiopulmonary bypass with drop-in suckers. For level IV, median sternotomy, exposure of the right atrium, and cardiopulmonary bypass were mandatory. With the combination of these approaches, the perioperative mortality rate was 0% and the 5-year overall survival rate was 52%., Conclusions: A multidisciplinary surgical approach is essential, especially for level III and IV cases., (© 2021. The Author(s) under exclusive licence to Springer Nature Singapore Pte Ltd.)

Among 28 groups of eukaryotes, apart from Metazoa, phosphagen kinase (PKs) is distributed in only a few protist groups, including the Choanomonada with the closest affinity to metazoans. To clarify the origin of metazoan PKs, we performed a database search and focused on 11 sequences of PK homologs from five groups of protists: the Choanomonada, Alveolata, Haptophyta, Stramenopiles, and Cryptophyta. The recombinant enzymes were prepared to determine their substrate specificity. Emiliania (Haptophyta), Anophryoides, Pseudocohnilembus, Vitrella and Chromera (Alveolata), and Monosiga (Choanomonada) all contained a gene for arginine kinase (AK). In contrast, Aphanomyces, Albugo and Ectocarpus (Stramenopiles), and Guillardia (Cryptophyta) possessed a gene for taurocyamine kinase (TK). The Guillardia TK enzyme exhibited rather strong substrate inhibition toward taurocyamine, which was analyzed using the most likely kinetic model. This was the first report of substrate inhibition in a TK. Together with the research results from other groups, the AK, TK, or creatine kinase (CK) activities have been observed sporadically in at least six groups of protists. However, it is not clear the three enzyme activities were emerged early in the evolution and divergence of protist groups, or some of enzyme activities were introduced to the protists by horizontal gene transfer. In addition, we found that seven protist enzymes examined in this study possess a myristoylation signaling sequence at the N-terminus. The amino-acid sequence around the guanidine-specificity region and the key residue at 89 th position of the protist AK and CK were homologous to those of the metazoan enzymes, but those for protist TKs were different indicating that the latter evolved independently., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Opheline kinase (OK) is one of the phosphagen kinases (PKs) restricted to annelids, but the amino acid sequence has not been determined yet. The OK enzyme was isolated in 1966 from the polychaete Ophelia neglecta (Opheliidae) and shown to have somewhat broader activities for the various substrates opheline, lombricine and taurocyamine. To determine the OK sequence, we analyzed the RNA sequencing data for Ophelina sp. and Thoracophelia sp., belonging to Opheliidae. Four PK sequences, namely, taurocyamine kinase (TK), creatine kinase (CK), mitochondrial CK (MiCK) and putative OK, were identified in both species, and the recombinant Ophelina enzymes were expressed in E. coli and purified. Since the substrate opheline was not commercially available, we used the partial activity toward taurocyamine to infer the enzyme specificity. The putative Ophelina OK showed lower activity to taurocyamine with a V max /K m nearly identical to a previously published value for an OK from a related species Ophelia neglecta. Under the same conditions, the true Ophelina TK showed much higher activity. Thus, the putative Ophelina enzyme was determined to be OK. The amino acid sequence alignment indicated that Ophelina and Thoracophelia OKs have five amino acid deletions in the GS region, like those of LKs and AKs, and the guanidino substrate specific residue was Lys, the same as LKs. In the phylogenetic tree constructed from annelid PK amino acid sequences, the OK sequences formed a distinct cluster, and it was placed near the TK and lombricine kinase (LK) clusters. This is the first report of the amino acid sequence for the OK enzyme., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The lantern shark genus Etmopterus contains approximately 40 species of deep-sea bioluminescent cartilaginous fishes. They emit blue light mainly from the ventral body surface. The biological functions of this bioluminescence have been discussed based on the luminescence patterns, but the bioluminescence mechanism remains uncertain. In this study, we detected both coelenterazine and coelenterazine-dependent luciferase activity in the ventral photophore tissue of Etmopterus molleri. The results suggested that bioluminescence in lantern sharks is produced using coelenterazine as the substrate for the luciferin-luciferase reaction, as some luminous bony fishes., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pontodrilus litoralis is a cosmopolitan littoral earthworm known to exhibit bioluminescence. Recently, a congeneric species, Pontodrilus longissimus, from Thailand was described. These species are sympatric, but their burrowing depths on Thai beaches are different. In this study, we examined the in vivo and in vitro bioluminescent properties of P. longissimus and P. litoralis. Mechanical stimulation induced in vivo luminescence in P. litoralis, as reported previously, but not in P. longissimus. In vitro cross-reaction tests between these species revealed the absence of luciferin and luciferase activities in P. longissimus. The coelomic fluid of P. litoralis had strong fluorescence that matched the spectral maximum of its bioluminescence, but the same result was not observed for P. longissimus. These results suggest that P. litoralis has luminescence abilities due to the creation of bioluminescent components (i.e., luciferin, luciferase, and light emitters). The presence of both luminous and nonluminous species in a single genus is likely widespread, but only a few examples have been confirmed. Our findings provide insight into the possible functions of bioluminescence in earthworms, such as avoiding predation by littoral earwigs.

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Purpose: Postoperative spinal cord injury is a devastating complication after aortic arch replacement. The purpose of this study was to determine the predictors of this complication., Methods: A group of 254 consecutive patients undergoing aortic arch replacement via median sternotomy, with (n = 78) or without (n = 176) extended replacement of the upper descending aorta, were included in a risk analysis. The frozen elephant trunk technique was used in 46 patients. The patients' atherothrombotic lesions (extensive intimal thickening of > 4 mm) were identified from computed tomography images., Results: Complete paraplegia (n = 7) and incomplete paraparesis (n = 4) occurred immediately after the operation (permanent spinal cord injury rate, 1.97%; transient spinal cord injury rate, 2.36%). A multivariable logistic regression analysis identified the use of the frozen elephant trunk technique (odds ratio 36.3), previous repair of thoracoabdominal aorta or descending aorta (odds ratio 29.4), proximal atherothrombotic aorta (odds ratio 9.6), chronic obstructive lung disease (odds ratio 7.1) and old age (odds ratio 1.1) as predictors of spinal cord injury (p < 0.0001, area under curve 0.93)., Conclusions: Spinal cord injury occurs with a non-negligible incidence following aortic arch replacement. The full objective assessment of the morphology of the whole aorta and the recognition of the risk factors are mandatory.

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λ max 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Coronary artery spasm after coronary artery bypass grafting(CABG) is rare and often fatal. There is no established treatment for perioperative coronary spasm because of the rare and not fully understood complications. We report a 67-year-old male, who experienced perioperative spasm of native coronary arteries and the left thoracic artery graft following CABG. Several transcatheter intracoronary injections of isosorbide sulfate failed to relieve the spasm completely. After 5 days' circulatory support with intra-aortic balloon pumping, the cardiac function improved and the patient recovered. Echocardiography performed before discharge showed decreased left ventricular systolic function with apical akinesis. Postoperative coronary angiography revealed 4 of the 5 grafts were patent.

Paramecium tetraurelia expresses four types of arginine kinase (AK1-AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis-Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3-15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R 2 , AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V max SES  = 30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V max ES  = 241.7. The dissociation constant for the SES complex (K i SES : 0.34 mM) was two times smaller than that of the ES complex (K s ES : 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V max SES . The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K i SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V max ES and V max SES values for the mutants decreased by one-third, except for the V max SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.

The cDNA sequence of arginine kinase (AK) from the precious coral Corallium rubrum was assembled from transcriptome sequence data, and the deduced amino acid sequence of 364 residues was shown to conserve the structural features characteristic of AK. Based on the amino acid sequence, the DNA coding C. rubrum AK was synthesized by overlap extension PCR to prepare the recombinant enzyme. The following kinetic parameters were determined for the C. rubrum enzyme: K a Arg (0.10 mM), K ia Arg (0.79 mM), K a ATP (0.23 mM), K ia ATP (2.16 mM), and k cat (74.3 s -1 ). These are comparable with the kinetic parameters of other AKs. However, phylogenetic analysis suggested that the C. rubrum AK sequence has a distinct origin from that of other known cnidarian AKs with unusual two-domain structure. Using oligomers designed from the sequence of C. rubrum AK, the coding region of genomic DNA of another coral Paracorallium japonicum AK was successfully amplified. Although the nucleotide sequences differed between the two AKs at 14 positions in the coding region, all involved synonymous substitutions, giving the identical amino acid sequence. The P. japonicum AK gene contained one intron at a unique position compared with other cnidarian AK genes. Together with the observations from phylogenetic analysis, the comparison of exon/intron organization supports the idea that two distinct AK gene lineages are present in cnidarians. The difference in the nucleotide sequence between the coding regions of C. rubrum and P. japonicum AKs was 1.28%, which is twice that (0.54%) of mitochondrial DNA, is consistent with the general observation that the mitochondrial genome evolves slower than the nuclear one in cnidarians.

A 43-year-old woman was diagnosed with aortitis associated with mild aortic regurgitation (AR) at the age of 25, which advanced to a severe grade requiring surgical treatment at age 28. Dilation of the sinotubular junction (STJ) was treated with ascending aortic replacement (Hemashield Gold 24 mm), which reduced AR from severe to mild. The patient conceived 9 years later and delivered a baby by cesarean section at 38 weeks of gestation. By appropriate control of inflammation with steroid, neither deterioration of the aortic valve nor cardiac function has been noted during the 15 years of follow-up after surgery.

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We assembled a phosphagen kinase gene from the Expressed Sequence Tags database of Myzostoma cirriferum, a basal member of annelids. The assembled gene sequence was synthesized using an overlap extension polymerase chain reaction method and was expressed in Escherichia coli. The recombinant enzyme (355 residues) exhibited monomeric behavior on a gel filtration column and showed strong activity only for l-arginine. Thus, the enzyme was identified as arginine kinase (AK). The two-substrate kinetic parameters were obtained and compared with other AKs. Phylogenetic analysis of amino acid sequences of phosphagen kinases indicated that the Myzostoma AK gene lineage differed from that of the polychaete Sabellastarte spectabilis AK, which is a dimer of creatine kinase (CK) origin. It is likely that the Myzostoma AK gene lineage was lost at an early stage of annelid evolution and that Sabellastarte AK evolved secondarily from the CK gene. This work contributes to our understanding of the evolution of phosphagen kinases of annelids with marked diversity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Two arginine kinase cDNAs (AK1 and AK2) were isolated from the marine feather star Tropiometra afra macrodiscus, and the gene structure (exon/intron organization) of AK1 was determined. The cDNA-derived amino acid sequences and the exon/intron organization of the Tropiometra AK1 gene were homologous to those of a human creatine kinase (CK) as well as the AK of the sea cucumber Stichopus. Phylogenetic analysis also supports the close relationship between human CKs and echinoderm AKs, indicating that the latter AKs evolved from an ancestral CK gene. We observed that the Tropiometra AK1 gene has a novel C-terminal extension (approximately 50 amino acid residues) encoded by a unique exon. Moreover, a typical prenylation signal sequence (CSLL) was found at the C-terminal end of this extension, suggesting that AK1 is anchored to a membrane. AK2 had no such C-terminal extension. This is the first finding of a prenylation signal in metazoan phosphagen kinases. Recombinant Tropiometra AK1 and AK2 enzymes were successfully expressed in Escherichia coli, and their kinetic constants were determined. Both enzymes showed activity comparable to that of typical invertebrate AKs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) mediate specific membrane fusion between transport vesicles or organelles and target membranes. VAM3/SYP22 and PEP12/SYP21 are Qa-SNAREs that act in the vacuolar transport pathway of Arabidopsis thaliana, and are localized predominantly on the vacuolar membrane and the pre-vacuolar compartment (PVC), respectively. Previous studies have shown that loss-of-function mutants of VAM3/SYP22 or PEP12/SYP21 showed male gametophytic lethality, suggesting that VAM3/SYP22 and PEP12/SYP21 possess different, non-redundant functions. We have re-evaluated the effects of mutations in these genes using T-DNA insertion mutants in the Columbia accession. We found that a mutation in VAM3/SYP22 (vam3-1) caused pleiotropic abnormalities, including semi-dwarfism and wavy leaves. In contrast, a loss-of-function mutant of PEP12/SYP21 (pep12) showed no apparent abnormal phenotype. We also found that the double vam3-1 pep12 mutant had severely reduced fertilization competence, although male and female gametophytes (vam3-1(-) pep12(-) ) maintained the ability to fertilize. Moreover, promoter swapping analysis revealed that expression of a GFP-PEP12/SYP21 fusion under the control of the VAM3/SYP22 promoter suppressed all phenotypes of the vam3-1 mutant. These results indicate that the functions of VAM3/SYP22 and PEP12/SYP21 were redundant and interchangeable., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Plants can sense the direction of gravity and change the growth orientation of their organs. The molecular mechanisms of gravity sensing and signal transduction during gravitropism are not well known. We have isolated several shoot gravitropism (sgr) mutants of Arabidopsis. The sgr3-1 mutant exhibits a reduced gravitropic response in the inflorescence stems. In the inflorescence stems of Arabidopsis, gravity is sensed in endodermal cells that contain sedimentable amyloplasts. In sgr3-1, some amyloplasts in the endodermis failed to sediment in the direction of gravity. SGR3 encodes a syntaxin, AtVAM3, which had previously been cloned as a homologue of yeast Vam3p. AtVAM3 is localized to the prevacuolar compartment and vacuole and is suggested to function in vesicle transport to the vacuole. We have also cloned another soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), ZIG/AtVTI11, a mutation that causes abnormal gravitropism. This mutant displayed an abnormal distribution of amyloplasts in the endodermal cells similar to that in sgr3-1. Endodermis-specific expression of SGR3 and ZIG by using the SCR promoter could complement the abnormal shoot gravitropism of each mutant. Protein-protein interaction between AtVAM3 and AtVTI11 in the endodermal cells was detected immunologically. The sgr3-1 mutation appeared to reduce the affinity of AtVAM3 for AtVTI11 or SYP5. These results suggest that vesicle transport to the prevacuolar compartment/vacuole in the endodermal cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11, plays an important role in shoot gravitropism.

We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes. To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system. Among the candidates thus obtained, many clones encoded the C-terminal half of an L. edodes 14-3-3 homologue (designated cip3). Southern blot analysis indicated that L. edodes contains only one 14-3-3 gene. Overexpression of the L. edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L. edodes and S. pombe. The interaction between L. edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation. Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S. pombe. The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L. edodes and S. pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function.