Sterically controlled docking of gold nanoparticles on ferritin surface by DNA hybridization.

Novel assemblies of DNA-functionalized gold nanoparticles (DNA-GNPs) have received considerable interest due to their fascinating properties which are desired for various detection applications. In this study, we present innovative GNP assemblies which have a cage-shaped protein ferritin in the center, and discrete GNPs sterically surrounding the central ferritin. These assemblies were constructed by hybridizing DNA-GNP to chemically DNA-modified ferritin, which has a hollow cavity or an iron NP core. Subsequent gel electrophoresis purification and transmission electron microscopy observation showed that ferritin/DNA/GNP assemblies were successfully constructed and can be isolated as independent functional units, which can be used to investigate not only the interaction between the GNPs of complicated GNP clusters but also the interaction between the GNPs and the internalized NP.

A novel type of cell death of lymphocytes induced by a monoclonal antibody without participation of complement.

A monoclonal antibody, RE2, raised by immunizing a rat with cell lysate of a mouse T cell clone, was found to directly kill interleukin 2-dependent T cell clones without participation of serum complement. Fab fragments of RE2 had no cytolytic activity, while the cross-linking of Fab fragments with anti-rat immunoglobulin reconstituted the cytotoxicity. The cytotoxicity was temperature dependent: the antibody could kill target cells at 37 degrees C but not at 0 degrees C. Sodium azide, ethylenediaminetetraacetic acid, and forskolin did not affect the cytolytic activity of RE2, while the treatment of target cells with cytochalasin B and D completely blocked the activity. This suggested that the cell death involves a cytoskeleton-dependent active process. Giant holes on the cell membrane were formed within 5 minutes after the treatment with RE2, as observed by scanning electron microscopy. There was no indication of DNA fragmentation nor swelling of mitochondria during the cytolysis, suggesting that the cell death is neither apoptosis nor typical necrosis. The antibody also killed T cell lymphomas and T and B cell hybridomas only when these cells were preactivated with concanavalin A, lipopolysaccharide, or phorbol myristate acetate. Preactivated peripheral T and B cells were sensitive to the cytotoxicity of RE2, while resting T and B cells were insensitive. These results provide evidence for a novel pathway of cell death of activated lymphocytes by membrane excitation.

Construction of a ferritin dimer by breaking its symmetry.

Ferritin has a mono-dispersed structure and biomineralization properties that allow it to form various kinds of nanoparticles and play an important role in modern nanotechnology. Independent nanoparticles synthesized in ferritin are valuable, but moreover a pair of nanoparticles can bring new properties different from those of the independent nanoparticles. In this study, by breaking ferritin's symmetry, we successfully produced ferritin dimers which provide real protein frameworks for nanoparticle dimer formation. Identical nickel hydro-oxide nanoparticle dimers were produced by simply biomineralizing ferritin dimers. The method presented here can produce multi-functional ferritin dimers with different kinds of nanoparticles.

Site-directed delivery of ferritin-encapsulated gold nanoparticles.

Newly designed porter proteins, which catch gold nanoparticles and deliver the nanoparticles selectively to a silicon dioxide (SiO(2)) surface under the specific conditions were reported. Recombinant apoferritin subunits, each of which has gold-binding peptide and titanium-binding peptide at the C- and N-terminus, respectively, can efficiently encapsulate a gold nanoparticle. The bio-conjugate, a nanogold and surrounding mutant protein subunits, had a property which can deliver itself to the SiO(2) surface through the interaction. In theory, our genetically manipulated apoferritin subunits can encapsulate gold nanoparticles of various sizes, which is a promising property for applications involving surface plasmon resonance.

Infection of human synovial cells by human T cell lymphotropic virus type I. Proliferation and granulocyte/macrophage colony-stimulating factor production by synovial cells.

The present study was performed to clarify the relationship between human T cell lymphotropic virus type I (HTLV-I) infection and chronic inflammatory arthropathy. To determine the ability of HTLV-I to infect synovial cells and the effect on synovial cell proliferation, synovial cells were cocultured with the HTLV-I-producing T cell lines (MT-2 or HCT-1). After coculture with HTLV-I-infected T cells, the synovial cells expressed HTLV-I-specific core antigens, and HTLV-I proviral DNA was detected from the synovial cells by polymerase chain reaction. These cocultured synovial cells with HTLV-I-infected T cells proliferated more actively than the synovial cells cocultured with uninfected T cells. This stimulatory effect of HTLV-I-infected T cells on synovial cell proliferation seems necessary to contact each other. After being cocultured with MT-2 cells, synovial cells proliferated more actively than control cells even after several passages. Furthermore, HTLV-I-infected synovial cells produced significant amounts of granulocyte/macrophage colony-stimulating factor. These results suggest that HTLV-I can infect synovial cells, resulting their active proliferation and may be involved in the pathogenesis of proliferative synovitis similar to that found in rheumatoid arthritis.

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae.

SPT23 was isolated as a dosage-dependent suppressor of Ty-induced mutations in Saccharomyces cerevisiae. SPT23 shows considerable sequence homology with MGA2, a gene identified as a dosage-dependent suppressor of a snf2-imposed block on STA1 transcription in S. cerevisiae var. diastaticus. Although single mutations in either of these genes have only modest effects on cell growth, spt23 mga2 double mutants are inviable. Unlike SPT23, multicopy expression of a truncated form of MGA2 suppresses a narrow subset of Ty-induced mutations. SPT23/MGA2 and the SNF/SWI genes affect transcription of certain target genes in similar ways. Spt23p appears to be a rate-limiting component required for functional HIS4 expression of his4-912delta, a promoter insertion mutation induced by the Ty1-912 long terminal repeat. Furthermore, both Spt23p and Mga2p can activate transcription when fused to the Gal4p DNA-binding domain, as previously observed with Snf2p and Snf5p. A 50-amino-acid region in the N terminus of the predicted Spt23p protein is necessary and sufficient for the transactivation and necessary for suppression of Ty1-induced mutations and the essential function of Spt23p. Cell fractionation and cytological experiments suggest that Spt23p is associated with the nucleus. Our results suggest that SPT23/MGA2 affects transcription of a subset of genes in yeast, perhaps by changing chromatin accessibility.

Multiple-step method for making exceptionally well-oriented liquid-crystalline sols of macromolecular assemblies.

X-ray fiber diffraction is potentially powerful in solving the atomic structure of filamentous assemblies of macromolecules, as demonstrated for tobacco mosaic virus. However, it requires extremely well-oriented sols to allow for extraction of intensities on closely located layer-lines. A high degree of orientation requires a high filament concentration to restrain the orientational freedom, but orienting concentrated sols is hampered by their high viscosity. Here, we report a systematic method that reproducibly produces extremely good orientation, which involves three steps; liquid crystallization, centrifugation and magnetic orientation. We found that a slow centrifugation can trigger a dynamic self-orientation process to form perfectly homogeneous liquid-crystalline sols, and further centrifugation to concentrate sols followed by magnetic orientation produces exceptionally well-oriented sols. The best-oriented flagellar sol showed a disorientation angle of 0.6 degrees as 1sigma of its Gaussian distribution. The new method has been successfully applied to many other systems, such as tobacco mosaic virus and F-actin.

Preparing well-oriented sols of straight bacterial flagellar filaments for X-ray fiber diffraction.

Well-oriented sols of straight bacterial flagellar filaments have been obtained by preparing reconstituted flagellar filaments with an appropriate length distribution and choosing appropriate solvent conditions. An average filament length of 300 to 500 nm and the use of solvents with very low concentrations of salt has allowed us to prepare highly fluid sols that make flow orientation possible. X-ray fiber diffraction from these sols has shown distinct layer-line reflections to 3.5 A resolution in the meridional direction. Layer-line intensities have been collected by the angular deconvolution method up to 5 A resolution. The possibility of using a magnetic field to further improve the orientation has been explored and a solvent condition that makes flagellar sols sensitive to the magnetic field has been found. General applicability of the method to other systems is also discussed.

Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy.

A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally. However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism. Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament. Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism. In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion. The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences. The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament. This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions.

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.

The structure of the R-type straight flagellar filament of Salmonella at 9 A resolution by electron cryomicroscopy.

The supercoiled forms of the flagellar filaments are thought to be constructed from a mixture of two distinct subunit conformations arranged in a regular manner. We analyzed the structure of one of the two straight flagellar filaments, each of which is built up with all its subunits in one of the two conformations. The filament we studied was isolated from the strain SJW1655 of Salmonella typhimurium and had a right-handed helical symmetry. With recent advancements in electron cryomicroscopy, such as a liquid helium temperature stage for frozen hydrated specimens and a stable field emission source, and also by averaging high resolution data with a proper correction of the contrast transfer function, the density distribution map of this straight flagellar filament was generated in far more detail than before by including data up to 9 A resolution. The structure shows a densely packed core region from about 15 to 55 A in radius, where a pair of concentric tubular features of high density is present without well-defined subunit boundaries, and an outer part from 55 to 115 A, where the subunits are mostly well separated from each other. The outer tube in the core region, from 35 to 55 A in radius, contains many rod-like features with near-axial orientation and closest lateral distances of around 10 A, which are most likely to represent the alpha-helical bundles that were predicted in our previous report. In the inner tube, from 15 to 30 A in radius, the rod-like features are less clear. Between the inner and outer tubes are the short spoke-like densities, which are radially tilted and are connecting the two tubes. The outer part, from 55 to 115 A, contains an axially elongated column density and a slewed projection with a narrow neck region. When compared with the other straight filament having left-handed helical symmetry, this outer part does not show any significant changes in orientation, suggesting that the switch in the subunit conformation and packing involved in the polymorphic transitions is quite subtle and only occurs within the core region. Reassignment of each structural domain to the amino acid sequence is suggested, based on the volume of each domain, which was determined rather precisely by a proper correction of the contrast transfer function for both amplitudes and phases.

HTLV-I induced myeloneuropathy in WKAH rats: apoptosis and local activation of the HTLV-I pX and TNF-alpha genes implicated in the pathogenesis.

To investigate the pathogenesis of HTLV-I associated diseases, we established a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in WKAH rats. In the spinal cords of WKAH rats carrying HTLV-I, chronological histopathology revealed the occurrence of apoptotic cell death starting at 9 months after the infection, followed by demyelination, macrophage infiltration, and the activation of astrocytes starting at 12, 15 and 20 months, respectively. Apoptosis of the Schwann cells was also observed in the peripheral nerves of these rats. By RT-PCR, pX mRNA of HTLV-I was selectively expressed in the diseased spinal cords and peripheral nerves, but not in the unaffected cerebra, cerebella, even though provirus DNAs were consistently identified in these tissues. Among several cytokines examined, mRNA expression and production of TNF-alpha were frequently detected in the spinal cord and the cerebrospinal fluid. The collective evidence suggests that the selective activation of HTLV-I, in particular Tax expression, and/or the production of TNF-alpha in target spinal cord and peripheral nerves are causally related to apoptotic death of the oligodendrocytes and Schwann cells, a major pathogenetic pathway of HTLV-I induced myeloneuropathy in the WKAH rat.

Models of HTLV-I-induced diseases. Infectious transmission of HTLV-I in inbred rats and HTVL-I env-pX transgenic rats.

To examine the pathogenic roles of HTLV-I in HTLV-I-induced diseases, we developed two models; namely HTLV-I carrier rats and HTLV-I env-pX transgenic rats. Among life long HTLV-I carriers in seven rat strains, only WKAH rats with the RT1k haplotype developed chronic progressive myeloneuropathy, resembling HAM/TSP clinically and histologically in humans, designated as HAM rat disease and after long incubation periods. Apoptosis of myelin forming cells, oligodendrocytes and Schwann cells associated with HTLV-I infection appears to be the primary cause of HAM rat disease. Local activation of the pX gene and TNF alpha gene was evident in these rats. WKAH rats transgenic for HTLV-I env-pX gene were established and at age 5 weeks, swelling of the bilateral ankle joints began to develop and histological features of the affected joints resembled findings in cases of rheumatoid arthritis (RA): high-titers of rheumatoid factors were present in these rats. A series of vascular collagen diseases such as polyarteritis nodosa-like angiitis, polymyositis, myocarditis, and Sjögren's syndrome-like sialodenitis together with RA were present, even in one individual animal. These transgenic rats as well as HAM rats appear to be suitable animal models for elucidating pathogenic mechanisms implicated in HTLV-I-induced diseases and also various demyelinating vascular collagen diseases of unknown etiology.

Further analysis of the control of voluntary saccadic eye movements in schizophrenic patients.

Many schizophrenic patients reveal abnormalities in the antisaccade task. To better understand the nature of these abnormalities, in the present study we have assigned to schizophrenics the no-saccade task (subjects were required to remain fixated without being disturbed by a reflexive saccade) and memory-saccade task (subjects were required to look at a remembered target) in addition to the antisaccade and saccade tasks used previously. Many schizophrenics revealed higher error rates in the no-saccade task, and latencies of saccades to a memorized target were significantly longer than controls in the memory-saccade task. Peak velocities of saccades of large amplitudes in the memory-saccade and antisaccade tasks (but not in the saccade task) were significantly slower and durations of such saccades were longer than normal controls despite the similarity between the distributions of amplitudes of such saccades between the patients and controls. These results suggest that many schizophrenics have difficulty suppressing reflexive saccades and initiating and executing appropriate volitional saccades when the goal for the movements is known but not visible.

Voluntary control of saccadic eye movement in patients with frontal cortical lesions and parkinsonian patients in comparison with that in schizophrenics.

To investigate whether the abnormalities of antisaccades in schizophrenics could be explained by a dysfunction of the frontal cortex, we examined 10 patients with frontal cortical lesions and 22 patients with idiopathic Parkinson's disease with mild symptoms (Yahr I-II) using the same tasks, and compared the results with those obtained in schizophrenics. The frontal patients with lesions covering the frontal eye field and prefrontal cortex showed more errors, longer latencies, and lower peak velocities in the antisaccade task, despite giving normal results in the visually guided saccade task. This was similar to the results observed in schizophrenics. Parkinsonian patients did not consistently show a significant difference in the antisaccade task. These results indicate specific abnormalities of antisaccades in schizophrenics and patients with frontal cortical lesions but not consistently in Parkinsonian patients. This suggests that the abnormalities of antisaccades in schizophrenics might be explained by a frontal cortical dysfunction.

B-HT 920, a dopamine D2 agonist, in the treatment of negative symptoms of chronic schizophrenia.

A prospective, nonblind 8-week trial of talipexole dihydrochloride (B-HT 920), a dopamine D2 agonist, was conducted in 15 schizophrenic patients with predominantly negative symptoms. B-HT 920 was initiated at 0.15 mg/day and then adjusted at 0.15-2.4 mg/day on the basis of clinical response and side effects. Dosage of concurrent neuroleptics was fixed at least 3 weeks prior to the trial and was unchanged throughout the study period. In addition to clinical assessment, levels of plasma homovanillic acid (pHVA), a potential index of central dopamine turnover, were measured. There was a small but significant (p < 0.01, Wilcoxon test) reduction in total scores of the Scale for the Assessment of Negative Symptoms or in a cluster score of three negative items (Emotional Withdrawal, Blunted Affect, and Psychomotor Retardation) of the Brief Psychiatric Rating Scale (BPRS). No change was observed in cluster scores of positive items of BPRS. There was a weak negative correlation between pHVA levels and the cluster scores of negative items of BPRS both at weeks 0 and 8 of the trial. The clinical results suggest that activation of D2 receptors was related to partial amelioration of the negative symptoms. The clinical and biochemical findings are consistent with a hypothesis that decreased dopaminergic activity may be related to the etiology of negative symptoms of schizophrenia.

Disturbances of voluntary control of saccadic eye movements in schizophrenic patients.

To study whether or not schizophrenic patients have disturbances in voluntary control of saccades, we examined visually elicited saccade and antisaccade tasks in 10 normal control subjects and 12 schizophrenic patients. The latencies of saccades in the schizophrenic patients were not significantly different from those of normal controls. However, 6 of the 12 schizophrenics showed significant abnormalities in the antisaccade task; 6 made more errors and 3 of them showed longer latencies than normal controls. Five of these 6 patients revealed an atrophy of the frontal cortex on computed tomography (CT) scans. These results indicate that many schizophrenics show difficulties in voluntary control of saccades, suggesting a dysfunction of the frontal cortex.

Repeated naloxone administration in schizophrenia: a phase II World Health Organization Study.

In the context of a previous WHO collaborative study, six research centers reported that naloxone (0.3 mg/kg) produced significant improvement in symptomatology in neuroleptic-treated patients. In the current Phase II WHO study, repeated (4 days) naloxone (0.3 mg/kg) administration was performed in schizophrenic patients (n = 43) from five WHO collaborating centers using a double-blind, placebo-controlled design. Both naloxone and placebo administrations were associated with significant reductions in symptoms. Naloxone, however, was not superior to placebo. These data are discussed in relation to endorphin hypotheses of schizophrenia.