Feasibility of 2nd generation STS retinal prosthesis in dogs.

We developed a 2(nd) generation suprachoroidal transretinal stimulation (STS) system with a 49 channel electrode array and implanted in 2 dogs. One month after surgery, all electrodes were functioning and the ocular fundus was normal in both dogs. The results indicate the 2(nd) generation STS retinal prosthesis is feasible and can be considered for clinical use.

[Primary arterial switch operation for complete transposition of the great arteries (type I) of a neonate weighing 1,378 g].

A 2-day-old female baby, delivered by emergent cesarean section at 35 weeks of gestational age with a birth weight of 1,378 g, was referred to our institute for intensive care of heart failure. By echocardiography and cardiac catheterization, the patient was diagnosed with isolated complete transposition of the great arteries. Primary arterial switch operation was performed at 13 days of age. No technical difficulty arose, imposed by the small size of cardiovascular structure. On the 5th postoperative day, surgical repair of intestinal perforation was performed. Convalescence thereafter was uneventful. She returned home on the 64th postoperative day with the body weight of 2,310 g. We conclude that primary arterial switch operation can be a feasible surgical option even in a neonate with very low birth weight.

Color-coding reveals tandem repeats in the Escherichia coli genome.

Genomic DNA contains a wide variety of repetitive sequences. In Escherichia coli, there have been several classes of repetitive sequences reported, some of which cluster as tandem repeats. We propose a novel method for analyzing symbolic sequences by two-dimensional pattern formation with color-coding. We applied this method for searching tandem repeats in the E. coli genome and found approximately 50 repeats with periods longer than 30 bases. The longest repeat has a period of 1267 bases.

A new purification method for overproduced proteins sensitive to endogenous proteases.

Proteolysisis a major problem in purification of overproduced proteins for structural studies. We developed a new method to avoid proteolysis of the products even in cases where popular protease inhibitors do not work effectively. When we cloned FlgF, a flagellar rod protein, from Salmonella typhimurium and overproduced it in Escherichia coli, FlgF was highly susceptible to cleavage by endogenous proteases after cell disruption even in the presence of various protease inhibitors. However, FlgF was not digested when the cells were disrupted in the presence of urea, which allowed us to develop the following new purification procedure. After cell disruption in the presence of urea and removal of the cell debris, the supernatant was passed through tandem-connected cation- and anion-exchange columns. Proteases were trapped in the cation-exchange column, and protease-free FlgF was eluted from the disconnected anion-exchange column. This gave a stable full-length product suitable for crystallization trials. The key procedures are cell disruption in the presence of urea and linked ion-exchange chromatography to quickly remove proteases as well as urea. This fast and simple method can be applied to purification of other overproduced proteins that are very sensitive to proteolysis.

Assembly characteristics of flagellar cap protein HAP2 of Salmonella: decamer and pentamer in the pH-sensitive equilibrium.

The cap of the bacterial flagellum is an oligomeric assembly of HAP2 protein (also called FliD), tightly attached to the tip of the flagellar filament. Flagellar growth does not occur in fliD-deficient mutants because flagellin monomers transported through the central channel of the flagellum leak out without polymerizing at the distal end. The structure of the cap complex is not known yet. An in vitro assembly of HAP2 proteins was found to have a pentagonal shape, while its molecular mass corresponded roughly to that of a dodecamer. To characterize the structure and assembly behavior of the complex formed in vitro in more detail, the stoichiometry of the complex and the association equilibrium have been studied. Crosslinking experiments now clearly show that the HAP2 complex is decameric. The assembly equilibrium is mainly between the monomer and decamer with a minor population of intermediate oligomers involved, and is highly dependent on the solution pH as well as the salt concentration: the fraction of the decamer sharply rises as the pH decreases from 8.5 to 8.0; the physiological concentration of salt partially suppresses the decamer formation. A preferential crosslinking within a pentameric unit together with a bipolar feature of the complex particle observed by electron microscopy suggests that the decamer is a bipolar pair of pentamers. Because of the polar nature of the filament cap structure, the pentamer is suggested to be the cap complex with its decamer forming surface involved in interactions with the filament.

A structural feature in the central channel of the bacterial flagellar FliF ring complex is implicated in type III protein export.

The FliF ring complex, which consists of the M-S ring and a proximal portion of the rod of the flagellar basal body, is the base structure for the bacterial flagellar assembly. The FliF ring is also thought to be part of the export apparatus for flagellar proteins from its amino acid sequence homology to proteins involved in type III protein export systems. We established a new purification procedure for the FliF ring particles and carried out electron microscopic image analyses in their two distinct forms: well-dispersed single particles in the presence of salt and ordered monolayer arrays of hexagonal packing formed in the absence of salt. In both cases, the axial projection maps showed a common feature, a pair of concentric rings: the inner ring corresponds to the proximal rod; the outer ring represents the thick, edge portion of the M-S ring. However, the central channel of the FliF ring, the putative pathway for the flagellar protein export, appeared to show distinct structural features in the two forms. This suggests that a domain of FliF partially occupies the central channel to be involved in the export and gate mechanism, and the domain changes its conformation depending on the ionic strength.

Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer.

Many transmembrane receptors are oligomeric proteins. Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both. The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands. Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA. In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate. These results suggest that heterodimers with only one intact cytoplasmic domain are functional. The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.

Self-assembly of the filament capping protein, FliD, of bacterial flagella into an annular structure.

A bacterial flagellum has a cap structure at the tip of the external filament. The cap is composed of the FliD protein (Mr, 49 x 10(3)), and plays an essential role in the polymerization of the filament protein, flagellin, which is believed to be transported through a central channel in the flagellum. A fliD-deficient mutant becomes non-motile because it lacks flagellar filaments and leaks flagellin monomer out into the medium. We have constructed a FliD-overproducing plasmid and purified the protein. The purified FliD at high concentration formed a large complex (Mr, ca. 600 x 10(3)) under physiological conditions. The complex was found by electron microscopy to be ring shaped. Image analysis revealed that the complex consisted of five substructures arranged in a pentagonal shape. Its outer diameter, approximately 10 nm, was about the same as that of the cap at the tip of the wild-type flagella. When the annular structure was added to the culture medium of a Salmonella fliD mutant, almost all of the cells became able to swim. Overall, about ten molecules of FliD self-assemble into an annular structure in vitro, forming the functional capping structure by incorporating flagellin at the tip of the flagellar filament in vivo.

Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images.

The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy. In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes of Salmonella typhimurium overproduced in the cytoplasmic membrane of Escherichia coli. The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm. On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible. We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm. It was identified as the C ring, since it was located at the base of the polyhook. Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photogrammetry, and a three-dimensional model of the total basal structure is presented.

Radial mass analysis of the flagellar filament of Salmonella: implications for the subunit folding.

X-ray fiber diffraction patterns of the R-type straight flagellar filament of Salmonella typhimurium SJW1655 strain showed layer-lines with an axial spacing of 1/437 A-1, which could be resolved only due to very small disorientation angles (< 2 degrees) of the filaments in oriented sol specimens. Although the equatorial layer-line was situated between the relatively strong first layer-lines right above and below it, these small disorientation angles and a new method of two-dimensional angular deconvolution allowed us to determine the equatorial layer-line intensities quite accurately. The equatorial data were phased by using the amplitude difference between the native flagellar filament and its heavy atom derivatives. One of the heavy-atom derivatives was prepared by introducing a cysteine residue by site-directed mutagenesis and applying a mercury compound. From the equatorial structure factors, the radial density distribution of the filament was calculated at 11 A resolution. A prominent feature was two pairs of high density peaks at radii of around 25 and 45 A and a deep density trough between them, which corresponds to the concentric double tubular structure in the core region that has been found in the density map recently deduced by helical image reconstruction from electron micrographs of frozen hydrated filaments. The molecular masses were estimated for four radial segments that correspond to the morphological domains identified in the map of helical image reconstruction. Then the domains were assigned to sequence positions by correlating the estimated masses with those of proteolytic fragments of flagellin. The assignment is consistent with the distributions of secondary structures and in particular alpha-helical coiled-coils that were predicted from the sequence. It also helps to understand how the polymerization behaviour is affected by truncation of the disordered terminal regions of flagellin and why mutations in a specific region are responsible for changes in the polymorphic shape of the filament.

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.).

Plants were regenerated from adventitious buds and somatic embryos (R0) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R1) of R0 plants. Among 5,618 R1 seeds harvested from 23 R0 plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R2 seeds harvested from 2 R1 plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R1 seeds harvested from 50 R0 plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R2 seeds harvested from 17 R1 plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R3 seeds were collected from these R2 plants following self-pollination. Forty-five of the 47 lines (R3) originated from 10 R0 plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.

Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium.

The length of flagellar hooks isolated from wild-type and mutant cells with various hook lengths were measured on electron micrographs. The length of the wild-type hook showed a narrow distribution with a peak (+/- standard deviation) at 55.0 +/- 5.9 nm, whereas fliK mutants (so-called polyhook mutants) showed a broad distribution of hook lengths ranging from 40 to 900 nm, strongly indicating that FliK is involved in hook length determination. Among pseudorevertants isolated from such polyhook mutants, fliK intragenic suppressors gave rise to polyhook filaments. However, intergenic suppressors mapping to flhB also gave rise to hooks of abnormal length, albeit they were much shorter than polyhooks. Furthermore, double mutations of flhB and flgK (the structural gene for hook-associated protein 1; HAP1) resulted in polyhooks, suggesting another way in which hook length can be affected. The roles of FliK, FlhB, and HAP1 in hook length determination are discussed.

Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM.

CheY is the response regulator of bacterial chemotaxis. Previously, we showed that CheY binds to the flagellar switch protein FliM and that this binding is increased upon phosphorylation of CheY [Welch, M., Oosawa, K., Aizawa, S.-I., & Eisenbach, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8787-8791]. Here, we demonstrate that it is the phosphorylated conformation of CheY, rather than the phosphate group itself, that is recognized and bound by FliM. We found that subsequent to the phosphorylation of CheY, Mg2+ was not required for the binding of CheY to FliM. However, phosphorylation of CheY did cause a change in the coordination properties of Mg2+ in the acid pocket of the protein. This change in the coordination of Mg2+ required the presence of the absolutely conserved residue Lys109. When Lys109 was substituted by arginine, the resulting CheY protein was unable to adopt an active conformation upon phosphorylation, and the protein was not bound by FliM. Surprisingly, the CheY13DK mutant protein, which is active in vivo but cannot be phosphorylated in vitro, exhibited only a low level of FliM binding activity, suggesting that its ability to cause clockwise rotation in the cell is not due to a constitutively high level of FliM binding. On the basis of these findings, we propose a mechanism for CheY activation by phosphorylation.

Overproduction of the bacterial flagellar switch proteins and their interactions with the MS ring complex in vitro.

The flagellar switch proteins (FliG, FliM, and FliN) of Salmonella typhimurium were overproduced in Escherichia coli and partially purified in soluble form. They were mixed with purified MS ring complexes (which consist of subunits of FliF protein) to examine their interactions in vitro. The degree of interaction was estimated by ultracentrifugation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the band density on the gel, we estimated that FliG bound to the MS ring complex at an approximately 1:1 molar ratio (FliG:FliF), whereas FliM did so only at a 1:5 molar ratio (FliM:FliF). FliN did not bind to the MS ring complex by itself or in the presence of the other switch proteins. A possible configuration of the switch proteins is discussed.

Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium.

The proximal end of the flagellar basal body consists of a structure called the MS ring complex: two (M and S) rings with different thicknesses closely apposed and a rod extending from the center of the S ring. It has been shown that the MS ring complex consists of multiple copies of single protein FliF (molecular mass 61 kDa). We analyzed the domains of FliF to elucidate how a single protein can be used to construct a complicated particle with several distinct sub-structures. Tryptic digestion of the MS ring complex gave rise to a structure which lacked most of the M ring portion by electron microscopy and showed a major band at 25 kDa by SDS/gel electrophoresis. Amino acid sequence analysis of this band showed that both terminal regions of FliF have been digested, leaving a semi-stable peptide starting from Phe120 and ending at around 400. In addition, we constructed a truncated fliF gene which encodes a FliF lacking 103 amino acid residues from the C terminus. Amplification of the truncated FliF gave rise to a ring complex lacking the rim of the M ring. From these results we assign both terminal regions of FliF to the M ring. Possible domain structures of FliF corresponding to the S ring and the rod are also discussed.

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.).

The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.

Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria.

Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three proteins (FliG, FliM, and FliN) and located at the base of the flagellar motor. The mechanism of function of the switch is unknown. Here we demonstrate that the diffusible clockwise-signal molecule, the CheY protein, binds to the switch, that the primary docking site is FliM, that the extent of CheY binding to FliM is dependent upon the phosphorylation level of CheY, and that it is unaffected by the other two switch proteins. This study provides a biochemical demonstration of binding of a signal molecule to the bacterial switch and demonstrates directly that phosphorylation regulates the activity of this molecule.

Activin A: serum levels and immunohistochemical brain localization in rats given diets deficient in L-lysine or protein.

When a L-lysine (Lys)-deficient diet is given to rats, Lys in plasma and brain declines and rats will then select a Lys solution from among other L-amino acids (AAs). The recording of single-unit activity in the lateral hypothalamic area of these rats suggested that neural plasticity occurred, specifically responding to the deficient nutrient, Lys, centrally and during ingestion of AA. Possible neurotrophic factors in serum from rats with or without deficiency of either protein or Lys was assayed by Hydra japonica. An increase in serum inhibin and activin A was observed in rats fed a Lys-sufficient and nonprotein diet, respectively. However, serum activin A-like activity was severely suppressed under Lys deficiency. Additionally, the immunohistochemical distribution of activin A in the brain was found in the nucleus tractus solitarius, the area postrema, and the arcuate nucleus. These facts indicate that ingestion of Lys-deficient or nonprotein diet caused a change in serum levels of activin A as a possible neurotrophic factor. This release may elicit plasticity in the sensitivity of neurons to deficient AA in the nuclei that could selectively drive ingestive behavior for its particular AA (e.g., Lys) to maintain AA homeostasis.