Identification of specifically reduced Th2 cell subsets in allergic rhinitis patients after sublingual immunotherapy.

BACKGROUND: Although Th2 cells are well known to play important roles in allergic diseases including allergic rhinitis (AR), the factors that induce and sustain the pathogenesis of AR remain unclear. The recent development of sublingual immunotherapy (SLIT) is expected to allow changes to the underlying pathogenesis of AR. However, which Th2 cell subsets are important in house dust mite-induced AR (HDM-AR), the influence of SLIT on the pathogenic Th2 cells, and the association of Th2 cell subsets with SLIT efficacy have not been clarified. METHODS: The cytokine production and frequency of HDM-reactive T-cell subsets in peripheral blood mononuclear cells (PBMCs) were evaluated using flow cytometry in 89 HDM-AR patients (placebo [n = 43] and HDM 300 IR [n = 46]) who participated in a placebo-controlled study of SLIT with HDM tablets. All patients provided samples both before treatment as a baseline and at the end of the 52-week study. The PBMCs were stained with CellTrace™ Violet (CTV) before culture with HDM extract, and HDM-reactive T cells were detected as the proliferated cells with diminished CTV. RESULTS: HDM-reactive IL-5+ IL-13+ CD27- CD161+ CD4+ cells and ST2+ CD45RO+ CD4+ cells were observed in the peripheral blood from each patient with HDM-AR; these cells significantly decreased after SLIT in the group treated with active tablets. HDM-reactive ST2+ CD45RO+ CD4+ cells were significantly lower in active-responders. CONCLUSION: Allergen-reactive ST2+ CD45RO+ CD4+ cells or those combined with IL-5+ IL-13+ CD27- CD161+ CD4+ cells may be useful as markers indicating the successful treatment of SLIT. These cells may play a crucial role in the pathogenesis of AR as pathogenic memory Th2 cells.

Intraoperative 5-aminolevulinic acid-induced photodynamic diagnosis of metastatic brain tumors with histopathological analysis.

BACKGROUND: Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is a promising real-time navigation method in the surgical resection of malignant gliomas. In order to determine whether this method is applicable to metastatic brain tumors, we evaluated the usefulness of intraoperative fluorescence patterns and histopathological features in patients with metastatic brain tumors. METHODS: We retrospectively reviewed the cases of 16 patients with metastatic brain tumors who underwent intraoperative 5-ALA fluorescence-guided resection. Patients were given 20 mg/kg of 5-ALA orally 2 h prior to the surgery. High-powered excitation illumination and a low-pass filter (420, 450, or 500 nm) were used to visualize the fluorescence of protoporphyrin IX (PpIX), the 5-ALA metabolite. We evaluated the relationships between the fluorescence and histopathological findings in both tumoral and peritumoral brain tissue. RESULTS: Tumoral PpIX fluorescence was seen in only 5 patients (31%); in the remaining 11 patients (69%), there was no fluorescence in the tumor bulk itself. In 14 patients (86%), vague fluorescence was seen in peritumoral brain tissue, at a thickness of 2-6 mm. The histopathological examination found cancer cell invasion of adjacent brain tissue in 75% of patients (12/16), at a mean ± SD depth of 1.4 ± 1.0 mm (range 0.2-3.4 mm) from the microscopic border of the tumor. There was a moderate correlation between vague fluorescence in adjacent brain tissue and the depth of cancer cell invasion (P = 0.004). CONCLUSION: Peritumoral fluorescence may be a good intraoperative indicator of tumor extent, preceding more complete microscopic gross total resection. TRIAL REGISTRATION: Institutional Review Board of Osaka Medical College No. 42, registered February 17, 1998, and No. 300, registered April 1, 2008. They were retrospectively registered.

ARAP3 inhibits peritoneal dissemination of scirrhous gastric carcinoma cells by regulating cell adhesion and invasion.

During the analysis of phosphotyrosine-containing proteins in scirrhous gastric carcinoma cell lines, we observed an unusual expression of Arf-GAP with Rho-GAP domain, ankyrin repeat and PH domain 3 (ARAP3), a multimodular signaling protein that is a substrate of Src family kinases. Unlike other phosphotyrosine proteins, such as CUB domain-containing protein 1 (CDCP1) and Homo sapiens chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa), which are overexpressed and hyperphosphorylated in scirrhous gastric carcinoma cell lines, ARAP3 was underexpressed in cancerous human gastric tissues. In this study, we found that overexpression of ARAP3 in the scirrhous gastric carcinoma cell lines significantly reduced peritoneal dissemination. In vitro studies also showed that ARAP3 regulated cell attachment to the extracellular matrix, as well as invasive activities. These effects were suppressed by mutations in the Rho-GTPase-activating protein (GAP) domain or in the C-terminal two tyrosine residues that are phosphorylated by Src. Thus, the expression and phosphorylation state of ARAP3 may affect the invasiveness of cancer by modulating cell adhesion and motility. Our results suggest that ARAP3 is a unique Src substrate that suppresses peritoneal dissemination of scirrhous gastric carcinoma cells.

Overview of studies comparing human normal cartilage with minimal and advanced osteoarthritic cartilage.

A major area under study in the osteoarthritis (OA) research field is the characterization of specific molecular and biochemical changes that distinguish advanced diseased cartilage from less involved or normal tissue. This information is important to better define the pathogenic mechanisms that are operating during OA progression and to identify disease-specific markers. This review describes recent studies that have addressed changes in chondrocyte gene expression, proliferation, and apoptosis in "experimental" (more advanced OA cartilage) versus "control" (less involved or non-OA cartilage). Included is a comprehensive listing of recently published studies in this area with general findings. The review also includes a discussion of study design and the strengths and weaknesses of the various approaches. In addition, specific strategies to deal with some of the important issues are discussed. One particular model utilizing minimal and advanced OA cartilage obtained from the same patient is described in more detail.

A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila.

Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musculature, largely utilizing the Paxillin LIM domains.

An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization.

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein beta-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.

The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

Superconductor-insulator transition in a two-dimensional array of resistively shunted small josephson junctions

We have fabricated two-dimensional (2D) small-Josephson-junction arrays of which each Al-AlOx-Al junction is shunted by a Cr resistor. The arrays with large junction resistance and large charging energy show a transition from insulating to superconducting behavior when the shunt resistance is lowered below a critical value, which is close to 2R(Q) ( R(Q) identical withh/4e(2) = 6.45 kOmega). The measured phase diagram is consistent with theories of quantum-fluctuation-driven and dissipation-driven phase transitions in the 2D Josephson-junction array with Ohmic shunt resistors.

Poly(allylamine) beads as selective sorbent for preconcentration of formaldehyde and acetaldehyde in high-performance liquid chromatographic analysis.

Formaldehyde and acetaldehyde in water were determined by preconcentration with poly(allylamine) beads, derivatization with 2,4-dinitrophenylhydrazine (DPH) and analysis by HPLC. Poly(allylamine) beads (0.5 g) were used to adsorb formaldehyde and acetaldehyde at 1.2-150 microg l(-1) and 3.5-220 microg l(-1) from water (1 l). The concentration factor is 50 fold. The aldehydes were eluted and derivatized with 2 mM DPH in 0.5 M H2SO4 (10 ml). The time of analysis was 1 h. The detection limits (S/N=3) for formaldehyde and acetaldehyde were 0.6 and 2 microg l(-1), respectively.

Relationship between EMG signals and force in human vastus lateralis muscle using multiple bipolar wire electrodes.

This paper describes the relationship between knee extension force and EMG signals detected by multiple bipolar wire electrodes inserted into the human vastus lateralis muscle under isometric conditions. Six healthy male volunteers participated in this study. Eight pairs of bipolar wire electrodes were inserted into the right vastus lateralis muscle and the EMG data were simultaneously detected and analyzed. The EMG raw data and individual force-IEMG relations were influenced by the location of the electrode inserted into the muscle. The force and IEMG relationship averaged across subjects detected from the eight electrodes, however, showed almost the same linear correlation in spite of different electrode locations. No linear correlation was observed between MdF and the knee extension force. This result suggests that, if all of the muscle fibers participate in the same action at the same time, the averaged normalized IEMG from any places using wire electrodes could reflect the total activities of that muscle even if the muscle is large.

Thapsigargin induces microglial transformation from amoeboid- to ramified- type in vivo

Yagi R, Tanaka S, Koike T. 1999. Thapsigargin induces microglial transformation from amoeboid- to ramified- type in vivo. Glia 28:49-52. The article referenced above was published as an Original Article instead of a Short Communication. The publisher regrets this error.

Reduction in subluxation and improved muscle function of the hemiplegic shoulder joint after therapeutic electrical stimulation.

Seventeen hemiplegic patients with chronic shoulder subluxation secondary to a cerebrovascular accident (CVA) were divided into three groups, two of which were subjected to 6 weeks of therapeutic electrical stimulation (TES) for 15 minutes twice a day, in order to assess the effectiveness of the treatment in reducing subluxation, and in improving shoulder abduction function. The third group was used as a control (C group). After 6 weeks of electrical stimulation of the supraspinatus (S group) and deltoid (D group), a significant (p<0.05) reduction in subluxation was observed in both groups when compared to the C group. The maximal force of shoulder abduction showed a tendency to increase in the S group (p<0.10). A significant increase in maximal force was also observed in the D group. In most of the TES-treated muscles, the interference pattern of EMG at maximum voluntary contraction increased. The amplitude of the EMG activity of the stimulated muscle also increased. Thus, we concluded that electrical stimulation therapy of the supraspinatus and the deltoid muscle is an effective treatment modality for shoulder subluxation and shoulder abduction function in hemiplegic patients.

Structure-activity relationship of HIV-1 protease inhibitors containing alpha-hydroxy-beta-amino acids. Detailed study of P1 site.

The structure-activity relationship of HIV-1 protease (HIV-1 PR) inhibitors containing alpha-hydroxy-beta-amino acids is discussed. We demonstrated that substituent groups on the P1 aromatic rings of the inhibitors exert significant influence on their biological activity. Inhibitors bearing an alkyl or a fluorine atom at the meta and para position on their P1 benzene ring were found to be good inhibitors. We also discovered that the substitution positions of the P2 benzamides were crucial for good antiviral potency. In this study, inhibitor 48 was the most potent [IC90 (CEM/HIV-1 IIIB) 27 nM] and showed good pharmacokinetics in rats.

Thapsigargin induces microglial transformation from amoeboid to ramified type in vitro.

Microglia generally display amoeboid morphology under prevalent culture conditions. We found that cultured microglia derived from rat cerebral cortex undergo a morphological transformation from amoeboid to process-bearing microglia upon treatment with thapsigargin (TG), a specific Ca2+-ATPase inhibitor of endoplasmic reticulum. Microglial transformation was further enhanced by exposure of amoeboid microglia to serum-free (N2) medium containing TG (TG/N2 treatment). TG/N2-treated microglia showed a marked reduction in the activity of phagocytosis and showed down-regulated expression of MRF-1 or F4/80, which are markers for activated microglia. Thus, both morphological and physiological criteria suggest that TG promotes the ramification of amoeboid microglia in vitro. This method would be helpful in characterization of ramified microglia in vitro.

A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator.

A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein-protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c-Jun, AP-2, NF-E2, C/EBPalpha and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. We show here that mutation of the PY motif in the subregion of the activation domain of the DNA-binding subunit of PEBP2, PEBP2alpha, abolishes its transactivation function. Using yeast two-hybrid screening, we demonstrate that Yes-associated protein (YAP) binds to the PY motif of PEBP2alpha through its WW domain. The C-terminal region of YAP fused to the DNA-binding domain of GAL4 showed transactivation as strong as that of GAL4-VP16. Exogenously expressed YAP conferred transcription-stimulating activity on the PY motif fused to the GAL4 DNA-binding domain as well as to native PEBP2alpha. The osteocalcin promoter was stimulated by exogenous PEBP2alphaA and a dominant negative form of YAP strongly inhibited this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes.

Isolation of an Antioxidative Substance Produced by Aspergillus repens.

The acidic fraction of an extract of the culture liquid of Aspergillus repens MA0197 showed strong antioxidative activity when tested by the ferric thiocyanate and TBA methods. Chromatographic purification of this acidic fraction gave an active substance identified as Neoechinulin A. This compound showed higher antioxidative activity than α-tocopherol and could be expected to act as an antioxidant in Katsuobushi.

Analysis of voluntary finger movements during hand tasks by a motion analyzer.

Finger movements have primarily been classified by the final position of the hand and finger during deliberate hand activities, rather than as a description of the movement process. In addition, as of yet there have been no reports based upon objective data from the analysis of the motion of three finger joints during movement, and no reports exist that describe the relationship of the three joints' motion during these movements. This paper describes the relationship of the three finger joints during simple finger movements and hand tasks using measurements and analysis from a two-dimensional motion analyzer. Two prehensile movements were examined in 15 healthy volunteers: pure finger extension from finger flex position in different wrist positions (dorsi-flexion position, neutral position and palmar-flexion position of the wrist joint) and the grasping of discs of different diameter (10, 11, 12 and 13 cm). In the sequence of pure finger extension, where the grasping task was not requested, results showed that the movement was started from the proximal joint and extended to the distal joint of the finger, and full finger extension accomplished from distal to proximal, one after another, in any wrist position in most subjects. With the grasping of a disc, however, joint movement was initiated from distal to proximal and the final motion for grasping was carried to completion from the proximal to distal joints of the finger in most subjects. In addition, it was recognized that the proportion of the angular change in each of the three joints was different, as were the time duration of the joint motion and the pattern of the angular change. From these results, it is suggested that deliberate activities of the finger and sophisticated joint movements provided delicate adjustments to fit the fingers to the size of the object, as compared to the simple finger extension movement.

Rotational action of the supraspinatus muscle on the shoulder joint.

The shoulder joint allows three-dimensional movement. In order to analyze the function of the muscles which act on the shoulder joint, three-dimensional movements, including rotation, must be considered. Among muscles participating in the shoulder joint movement, the supraspinatus muscle is known to have abduction and stabilization effects on the shoulder joint. However, the rotational function of the supraspinatus muscle has not been identified, because few studies have been reported on it. This study investigates the rotating function of the supraspinatus muscle using electrical stimulation, magnetic resonance imaging (MRI) and anatomical examination. Electrical stimulation was applied selectively to the supraspinatus muscle of healthy subjects using percutaneous wire electrodes. The electrical stimulation was given at different positions of the shoulder joint. It was found that the electrically induced rotational movements changed their direction depending on the position of the shoulder joint. When the humerus was relatively in internal rotation, internal rotation resulted. When it was in external rotation, external rotation occurred. Regarding the abduction angle of the shoulder joint, external rotation was induced with an increase in the abduction angle, whereas internal rotation occurred when the abduction angle was decreased. By the dissection of cadavers and MRI examination, it was indicated that the relation between the running direction of the supraspinatus muscle and the center of rotation of the humeral head was dependent on the position of the shoulder joint. Those findings supported the results of electrical stimulation of the supraspinatus muscle at various shoulder positions. These results indicate that the bi-directional rotating function of the supraspinatus muscle is characterized by an anatomical relationship between the running direction of the supraspinatus muscle and the center of rotation of the humeral head.

Design and anti-HIV-1 activity of hammerhead and hairpin ribozymes containing a stable loop.

Three ribozymes, a hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the U5 region in the long terminal repeat (LTR) of HIV-1 RNA. The t1/2 values of chemically synthesized substrates mediated by three ribozymes were measured. The transformed CEM cells possessing these three ribozyme-encoding genes were challenged with a HIV-1IIIB strain, and two of these three ribozymes, HR112 and RZ119, were shown to possess strong anti-HIV-1 activity.