Streptococcus pneumoniae ClpL modulates adherence to A549 human lung cells through Rap1/Rac1 activation.

Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens.

Tendon impingement of the extensor digiti minimi: clinical cases series and cadaveric study.

The authors describe two unique clinical cases of closed extensor digiti minimi (EDM) tendon injuries after hyperflexion of the wrist with full finger flexion and one case of chronic tenosynovitis around the EDM tendon. All three cases were thought to be related to the bifurcation of the EDM tendon and synovial septum. Subsequently, variations in EDM tendons were investigated in 49 cadaveric hands with a focus on patterns of tendon bifurcation and their relationships with the surrounding synovial sheath. The EDM tendon was found to be bifurcated in 74% (n = 36) of hands and all of these hands contained a synovial septum. In 9 (25%) hands, the EDM tendon bifurcated proximal to the retinaculum, in 15 (42%), it bifurcated distal to the retinaculum, and in the other 12 hands (33%), the tendon bifurcated at the retinacular level. In 6 of the 15 hands with an infraretinacular bifurcation, the tendon was found to impinge on the synovial septum during passive flexion of the wrist with full finger flexion, and the mean distance between the synovial septum and the bifurcation point in these specimens was 0.6 cm (range, 0.4-0.7 cm), which was differed significantly from hands not showing impingement (P = 0.01). This study shows that distal bifurcation of the EDM tendon may lead to tendon impingement on the septum and suggests that this is a potential etiology of chronic tenosynovitis of the fifth compartment and of acute closed tendon injuries.

Blind subacromial injection from the anterolateral approach: the ballooning sign.

PURPOSE: Our aim was to verify the association of fullness over the skin distal to anterior acromion termed "ballooning" in relation to accuracy of subacromial injection and determine its accuracy in terms of sensitivity, specificity, and predictive value. We hypothesized that a positive ballooning was a sign of an accurately placed injection into the subacromial bursa. METHODS: Data of 136 shoulders with impingement, which received subacromial steroid injections, were evaluated for presence of ballooning signs, pain, motion, and muscle strength. Injections were performed via anterolateral approach, followed by radiographs to locate the contrast. Data were compared between pre- and post-injections as well as between accurate and inaccurate groups to evaluate the correlations between targeting accuracy and immediate outcomes. RESULTS: Ballooning signs were positive in 104 shoulders (76.5%), of which majority were inaccurate (58.7%). The accuracy rate was 49.3% with sensitivity of 64.2%, specificity 11.6% and positive as well as negative predictive values of 41.3% and 25% consecutively. Dispersal rate to the surrounding structures was 86.6% with majority infiltrated the deltoid (29.4%). Significant improvement was noted between pre- and post-injections in all parameters except muscle strength, indicating equal pain relief regardless of locations. CONCLUSION: The ballooning sign is not a reliable indicator for or against subacromial injection. Blind subacromial injections are frequently inaccurate using the anterolateral approach. Nevertheless, immediate improvement of pain, motion, and muscle strength can be expected regardless of location.

Partially purified Curcuma longa inhibits alpha-melanocyte-stimulating hormone-stimulated melanogenesis through extracellular signal-regulated kinase or Akt activation-mediated signalling in B16F10 cells.

Bioassay-guided fractionation of Curcuma longa by solvent partitioning and purification with octadecylsilane open column chromatography yielded a partial purification. The active 80% methanol chromatographic fraction from the ethyl acetate layer [partial purification from C. longa (PPC)] was used to investigate the alpha-melanocyte-stimulating hormone (alpha-MSH)-stimulated melanogenesis signal pathway in B16F10 cells. In cells stimulated alpha-MSH, PPC inhibited cellular melanin contents, tyrosinase activity and expression of melanogenesis-related proteins including microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related proteins (TRP). Melanogenesis-regulating signalling such as mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt was activated by PPC in alpha-MSH-stimulated B16F10 cells. The suppressive activity of PPC on alpha-MSH-induced melanogenesis was abrogated by selective inhibition of MEK/ERK (PD98059) and PI3K (LY294002). MEK/ERK or Akt activation by PPC may contribute to reduced melanin synthesis via MITF and its downstream signal pathway including tyrosinase and TRPs in alpha-MSH-induced melanogenesis.

Naringenin-induced apoptosis is attenuated by Bcl-2 but restored by the small molecule Bcl-2 inhibitor, HA 14-1, in human leukemia U937 cells.

Naringenin, a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth and there is increasing interest in its therapeutic applications. In this study, the effect of ectopic Bcl-2 expression on naringenin-induced apoptosis was investigated. We found that Bcl-2 overexpression markedly protected human leukemia U937 cells from time- and dose-dependent induction of apoptosis by naringenin, as did caspase-3 and caspase-9 inhibitors. Additionally, Bcl-2 overexpression attenuated naringenin-induced Bax translocation and cytosolic release of cytochrome c. Our results also indicated that co-administration of HA14-1 and naringenin increased apoptosis in Bcl-2 overexpressing U937 cells by restoring mitochondrial dysfunction and activation of caspase-9 and caspase-3, as well as by cleavage of poly (ADP-ribose) polymerase. Taken together, these observations indicate that Bcl-2 confers apoptosis resistance to naringenin by inhibiting a mitochondrial amplification step in U937 cells.

Dichloromethane fraction of Cimicifuga heracleifolia decreases the level of melanin synthesis by activating the ERK or AKT signaling pathway in B16F10 cells.

Cimicifuga rhizoma has long been used in traditional Korean medicine. In particular, a Cimicifuga heracleifolia extract (CHE) was reported to inhibit the formation of glutamate and the glutamate dehydrogenase activity in cultured rat islet. Glutamate activates melanogenesis by activating tyrosinase. Accordingly, it was hypothesized that a CHE might inhibit the melanogenesis-related signal pathways including the inhibition of microphthalmia-associated transcription factor (MITF)-tyrosinase signaling and/or the activation of extracellular signal-regulated kinase (ERK)-Akt signaling. The results showed that CHE inhibits the cellular melanin contents, tyrosinase activity and expression of melanogenesis-related proteins including MITF, tyrosinase and tyrosinase-related protein (TRP)s in alpha-melanocyte-stimulating hormone-stimulated B16 cells. Moreover, CHE phosphorylates MEK, ERK1/2 and Akt, which are melanogenesis inhibitory proteins. The data suggest that CHE inhibits melanogenesis signaling by both inhibiting the tyrosinase directly and activating the MEK-ERK or Akt signal pathways-mediated suppression of MITF and its downstream signal pathway, including tyrosinase and TRPs. Therefore, C. heracleifolia would be a useful therapeutic agent for treating hyperpigmentation and an effective component in whitening and/or lightening cosmetics.

Effects of mycelial culture of Phellinus linteus on ethanol-induced gastric ulcer in rats.

The gastroprotective effects of a mycelial culture of Phellinus linteus (MCPL) were evaluated by determining the ulcer index, gastric mucus content, histopathological observation and histochemical properties of mucin in an ethanol-induced ulcer model of rats. Preadministration with MCPL at doses of 20 and 60 mg/kg, showed a significant decrease of bleeding and ulcer index and alleviated the histopathological changes induced by ethanol such as hemorrhage and necrosis. Ethanol treatment decreased the gastric adhesion mucus content, but a higher level of gastric mucus persisted after preadministration of MCPL. As for the histochemical properties of mucins, marked changes were observed in both the surface and gland mucous cells in ethanol-treated rats, but these changes were detected only in the surface mucous cells in rat preadministered with MCPL. Using conventional methods for mucins, ethanol-treated rats revealed a decrease of neutral and acid mucin in the surface epithelium and mucous neck cells compared with normal rats. A marked decrease of BSL-1 by lectin histochemistry was also revealed in the ethanol-treated rats. But the MCPL preadministered rats showed similar stainabilities and lectin affinity patterns for mucins as the normal rats. These results indicate that pretreatment with MCPL provided protection of the gastric mucosa from ethanol-induced injury by maintaining the mucus barrier in rats.

Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells.

Water-soluble proteoglycan (WSPG) of Agaricus blazei Murill has been known to stimulate the non-specific complements and humoral immune functions to act as polyclonal activators of B cells and to inhibit tumor growth and metastasis. However, little is known about its immunomodulating effects on murine bone marrow (BM)-derived dendritic cells (DC). In the present study, we examined the maturation process of murine BM-DC. BM cells were cultured in the presence of IL-4 and GM-CSF and the generated immature DC were stimulated with WSPG or LPS (WSPG-DC and LPS-DC, respectively) for 24 h. WSPG significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) II, as did LPS. IL-12p70 production in WSPG-DC was also identical to that in LPS-DC. The antigen-uptake capacity of WSPG-DC was determined by FITC-labeled dextran uptake. WSPG-DC lost dextran uptake capacity comparable to LPS-DC. The antigen-presenting capacity of WSPG-DC as analyzed by allogeneic T cell proliferation was significantly increased in comparison with immature DC, was identical to LPS-DC, and induced higher levels of IL-2 in responding T cells. These results indicate the immunomodulatory properties of WSPG, which might be therapeutically useful in the control of cancers and immunodeficient diseases through the up-regulation of DC maturation.

A serial comparison of arthroscopic repairs for partial- and full-thickness rotator cuff tears.

PURPOSE: To compare the outcome of arthroscopic rotator cuff repair and subacromial decompression in partial-thickness rotator cuff tears (PTRCT) with those in full-thickness rotator cuff tears (FTRCT). TYPE OF STUDY: Prospective serial follow-up study. METHODS: Of 46 consecutive patients who were treated with arthroscopic rotator cuff repair, 42 patients who were followed-up serially for 2 years were enrolled as study subjects. The average age of the patients at the time of the operation was 55 years, and the mean duration of follow-up was 34 months. The subjects included 22 cases of PTRCT and 20 cases of FTRCT. RESULTS: At the final follow-up, the PTRCT group showed changes in scores from 7.2 to 0.9 for average pain and from 34 to 91 for the shoulder functional evaluation score of the American Shoulder and Elbow Society (ASES score). The FTRCT group showed changes in scores from 7.6 to 1.2 for pain and from 29 to 88 for the ASES score. There were no significant differences between the 2 groups ( P >.05). The average range of shoulder motion was significantly improved in both groups at the final follow-up versus their preoperative values. Evaluation at the final follow-up showed that 93% of the total subjects showed good or excellent results, and 95% showed satisfactory results with regard to pain reduction and functional outcomes. The 2 fair results were the result of acromioclavicular arthritis. CONCLUSIONS: It may be anticipated that arthroscopic rotator cuff repair and subacromial decompression will give satisfactory postoperative outcomes in both PTRCT and FTRCT in terms of pain relief and functional recovery. However, careful preoperative examination of the acromioclavicular joint is critical to avoid procedural failure. LEVEL OF EVIDENCE: Level IV.

Purification of a fibrinolytic enzyme (myulchikinase) from pickled anchovy and its cytotoxicity to the tumor cell lines.

A fibrinolytic enzyme, myulchikinase, from a Korean seasoning ingredient, myul-chi-jeot-gal, has been purified to electrophoretic homogeneity. The molecular mass of the myulchikinase was estimated to about 28 kDa by SDS-PAGE and gel filtration. Amino acid sequence of the NH2-terminal of myulchikinase showed significant homology with other fibrinolytic enzymes including trypsin from starfish, katsuwokinase, and rat pancreatic elastase II. The purified myulchikinase hydrolyzed various synthetic substrates with different substrate specificity and cytotoxic to the tumor cell lines.

A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins.

We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies.

Amphetamine increases phosphorylation of extracellular signal-regulated kinase and transcription factors in the rat striatum via group I metabotropic glutamate receptors.

Amphetamine is an indirect dopamine receptor agonist and increases glutamate release in the striatum. Activation of group I metabotropic glutamate receptors (mGluRs) upregulates cAMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1 and 2 (ERK1/2) in the striatum in vivo. In the present study the role of mGluRs in the regulation of ERK1/2 pathways leading to CREB and Elk-1 phosphorylation by amphetamine was investigated using immunohistochemistry and Western blot in the rat dorsal striatum. Acute administration of amphetamine (5 mg/kg, i.p.) caused increases in phosphorylated (p)CREB, pElk-1, and pERK1/2 immunoreactivity. Intrastriatal blockade of group I mGluRs with N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) significantly attenuated amphetamine-induced pCREB, pElk-1, pERK1/2, and Fos immunoreactivity in both medial and lateral areas of the striatum. Systemic injection of an mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 10 mg/kg, i.p.), also blocked the amphetamine induction of these phosphoproteins. In contrast, intrastriatal blockade of group II/III mGluRs with (RS)-alpha-methylserine-o-phosphate monophenyl ester (MSOPPE; 25 nmol) did not affect amphetamine-induced increases in all the four markers. Similarly, intrastriatal dantrolene (2 or 20 nmol) that blocks intracellular Ca(2+) release from ryanodine-sensitive stores did not affect amphetamine effects. Injection of PHCCC, MPEP, MSOPPE, or dantrolene alone did not alter basal levels of the three phosphoproteins and Fos. These data suggest that acute amphetamine is able to facilitate the phosphorylation of CREB, Elk-1, and ERK1/2 signaling proteins and Fos gene expression via a group I mGluR-dependent mechanism in the dorsal striatum.

BAP, a mammalian BiP-associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP.

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae.

Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17965.9+/-5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56-83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted alpha-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic alpha-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240-340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.