Characteristics of meso-particles formed in coagulation process causing irreversible membrane fouling in the coagulation-microfiltration water treatment.

In coagulation-membrane filtration water treatment processes, it is still difficult to determine the optimal coagulation condition to minimize irreversible membrane fouling. In microfiltration (MF), meso-particles (i.e., 20 nm-0.5 µm) are thought to play an important role in irreversible membrane fouling, especially their characteristics of particle number (PN) and zeta potential (ZP). In this study, a new nanoparticle tracker combined a high-output violet laser with a microscope was developed to identify the physicochemical characteristics of these microscopic and widely dispersed meso-particles. The effects of pH and coagulant dose on ZP and PN of micro-particles (i.e., >0.5 µm) and meso-particles were investigated, and then coagulation-MF tests were conducted. As the result, irreversible membrane fouling was best controlled for both types of membranes, while meso-particle ZP approached zero at around pH 5.5 for both types of natural water. Since PN was greatest under these conditions, ZP is more important in determining the extent of irreversible membrane fouling than PN. However, the acidic condition to neutralize meso-particles is not suitable for actual operation, as considering residual aluminum concentration, pipe corrosion, and chlorination efficiency. It is therefore necessary to investigate coagulants or other methods for the appropriate modification of meso-particle characteristics.

Negative MRI findings in a case of degenerative myelopathy in a dog.

An 11-year-old male Rough collie was submitted with paraparesis, but did not respond to medical treatment. Clinical signs worsened and the dog displayed paralysis, inability to stand and loss of voluntary bladder control, whereupon magnetic resonance imaging (MRI) was performed. No significant abnormalities were identified from MRI, blood tests, cerebrospinal fluid tests or radiography. After MRI, the dog developed dyspnoea and died. Autopsy and subsequent histopathological examination led to a diagnosis of degenerative myelopathy.

Functional selectivity in cannabinoid signaling.

Cannabinoid (CB) agonists exhibit numerous potentially useful pharmacological properties, but unwanted side effects limit their use in clinical practice. Thus, novel strategies are needed to identify potential CB pharmaceuticals with fewer side effects. Activated CB receptors initiate multiple parallel intracellular signal transduction cascades. In the present paper we will review experimental data indicating that structurally different classes of CB agonists may exhibit selectivity toward individual subsets of intracellular signaling pathways. In support of this, recent findings indicate that chemically distinct classes of CB agonists frequently differ in their rank order of potency to produce analgesia versus other central nervous system effects in vivo. Structurally different agonists were also found to differ in their abilities to activate individual G protein types in vitro. Since it was suggested earlier that structurally distinct CB agonists may interact differently with the CB receptors, it has been hypothesized that different classes of cannabinoid agonists may stabilize unique active CB receptor conformations, leading to functional selectivity in CB receptor signaling. In order to obtain a direct proof for this hypothesis, we recently employed a highly sensitive biophysical method, plasmon-waveguide resonance (PWR) spectroscopy. PWR experiments have provided a direct proof that structurally different CB agonists produce qualitatively distinct changes in the shape and/or membrane orientation of the CB1 receptors, leading to functional selectivity in G protein activation. We expect that by identification of CB agonists that selectively activate preferred intracellular signaling pathways novel pharmacological lead structures can be identified for the design of improved CB analgesics with fewer side effects.

MRI findings of haematomyelia in a dog with spontaneous systemic haemorrhage.

A 7-year-old female cross-breed dog was brought to Nihon University Animal Medical Center for investigation of tetraplegia. Lameness in the pelvic limbs, that had developed 2 weeks previously, had progressed to tetraplegia. On magnetic resonance imaging of the spinal cord, isointensity was detected from C2 to C4 and T12 to T13, isointensity and hyperintensity were intermingled from L3 to L4, and hyperintensity was detected from L5 to L7 by T1-weighted imaging. On T2-weighted imaging, hyperintensity was detected in all regions described above. The dog recovered from anaesthesia, but died during the day from systemic bleeding as the result of a coagulopathy of unknown aetiology. Histopathological examination revealed haematomyelia in these regions of the spinal cord. This is the first report of magnetic resonance imaging findings of haematomyelia in canine spontaneous systemic haemorrhage. It appeared that the differences in the findings of T1-weighted imaging along the spinal regions reflected time-lags in the occurrence of bleeding.

Proline residue-modified polycationic analogs of gramicidin S with high antibacterial activity against both Gram-positive and Gram-negative bacteria and low hemolytic activity.

Novel polycationic analogs of the cyclic decapeptide antibiotic, gramicidin S, possessing NH(2), D/L-Phe-NH or L-Lys-NH groups at the 4alpha- or 4beta-positions of the L-Pro residues, were synthesized. While L-Pro(4alpha/beta-NH(2))-containing analogs exhibited much weaker antibacterial activity, the D/L-Phe and L-Lys-substituted analogs exhibited higher antibacterial activity against Gram-negative bacteria than the parent gramicidin S. All of these additional amino group-containing analogs showed substantially reduced toxicity against human blood cells.

Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil.

AIMS: To devise and evaluate a method for selective isolation of the less abundant actinomycetes, Nocardia spp. in soil. METHODS AND RESULTS: This newly developed method is based on differentiating Nocardia from other actinomycete taxa by centrifugation. A water suspension of air-dried soil is centrifuged through a gradient consisting of 10, 20, 30, 40 and 50% sucrose at 240 x g for 30 min. The 20% sucrose layer, which is enriched with Nocardia spp., is then diluted and plated on humic acid-vitamin agar supplemented with antibacterial agents. The proposed method consistently achieved selective isolation of Nocardia spp. in all 14 soil samples tested, which accounted for 5-89% of the total microbial population recovered. Tentative taxonomic characterization based on a restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal DNA suggested that many of the soil isolates could belong to N. asteroides, N. salmonicida or N. uniformis. CONCLUSIONS: Differential centrifugation can successfully and efficiently isolate soil Nocardia populations that are suppressed by conventional dilution plating approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to isolate less-explored actinomycete taxa is important for improving our knowledge about their taxonomy, ecology and industrial applications.

Myocardial hamartoma of the right atrium in a dog.

A myocardial hamartoma of the right atrium is described in an 8-year-old dog that died from pneumonia. At necropsy, a firm, mottled, dark-brown right atrial appendage, of normal shape but slightly enlarged, was found incidentally. On section, the right atrial appendage was composed of a grey-tan, solid mass. Histological features of the mass were as follows: the component cells were mature cardiac muscle cells; the mass contained all of the components of the normal heart wall (i.e., epicardium, myocardium and endocardium), but the arrangement of the component tissues was disorganized; growth of the mass was non-invasive, and continuity of the component cells with adjacent normal myocardial cells was evident, suggesting a congenital origin. This appears to be the first report of congenital myocardial hamartoma in any animal other than man.

Frequent expression of smooth muscle markers in malignant fibrous histiocytoma of bone.

BACKGROUND/AIMS: Malignant fibrous histiocytoma (MFH) of bone, a relatively rare primary malignant bone tumour, is a distinct clinicopathological entity as opposed to MFH derived from soft tissue. Although the true histogenesis of this condition is still controversial, a considerable number of cases of MFH in soft tissue show positive immunohistochemical reactivity for muscle markers such as desmin, common muscle actin (HHF35), and alpha smooth muscle actin (SMA), suggesting that MFH cells are myofibroblastic in nature. METHODS: This study investigated immunoreactivity for several different muscle markers in 19 cases of MFH of bone together with reverse transcription polymerase chain reaction (RT-PCR) analysis on frozen tissue samples that were available in four cases, and compared the data with those found in 11 cases of osteosarcoma and 11 cases of soft tissue MFH treated over the same period. RESULTS: Immunohistochemistry revealed that MFH of bone showed relatively frequent expression of smooth muscle markers, including calponin (nine cases), alpha-SMA (nine cases), and SM22alpha (18 cases), and this was confirmed by RT-PCR analysis. However, only one, two, and three cases of MFH of bone showed positive staining for desmin, MyoD1, and HHF35, respectively. Similarly, 11 osteosarcoma cases were relatively frequently positive for alpha-SMA (five cases), calponin (four cases), and SM22alpha (seven cases), and less frequently positive for desmin (one case), MyoD1 (none), and HHF35 (none). In contrast, very few MFH of soft tissue cases (n = 11) showed positive reactivity for all of these muscle markers. It has recently been reported that human bone marrow stromal cells also express various kinds of smooth muscle markers including alpha-SMA and calponin. CONCLUSIONS: These results suggested that MFH of bone may derive from mesenchymal stromal cells in bone marrow and has a more myofibroblastic differentiation than soft tissue MFH.

Cannabinoid CB(1) receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues.

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB(1) receptors in the trabecular meshwork and increase aqueous outflow.

Expression of alpha-transducin in Chinese hamster ovary cells stably transfected with the human delta-opioid receptor attenuates chronic opioid agonist-induced adenylyl cyclase superactivation.

To investigate the role of G-protein beta gamma subunits in delta-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO cells) with the G(alpha)-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase C beta (PLC beta) activation was measured in each of these cell lines. Because PLC beta(3) activation in CHO cells has been shown to be mediated by free G(beta gamma) subunits derived from G(alpha i/o), the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-mediated maximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 +/- 12% compared with control cells. The acute inhibition of cAMP formation was unchanged between control and transducin-expressing cells. We show that cells stably expressing the human delta-opioid receptor exhibited a pertussis toxin-sensitive cAMP overshoot in response to chronic application of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 229 +/- 37% compared with buffer-treated cells. Expression of transducin in hDOR/CHO cells diminished this response: hDOR/T1/CHO cells showed no significant change in maximal forskolin-stimulated cAMP formation after pretreatment and washout. These data indicate that the expression of alpha-transducin scavenges free G(beta gamma) subunits and, furthermore, that free G(beta gamma) subunits play a role in opioid-mediated PLC beta activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.

Syk expression in endothelial cells and their morphologic defects in embryonic Syk-deficient mice.

Mice deficient in the Syk tyrosine kinase showed severe petechiae in utero and died shortly after birth. The mechanism of this bleeding, however, remains unknown. Here it is shown that this bleeding is caused by morphologic defects of Syk-deficient endothelial cells during embryogenesis. Immunoblot and reverse transcriptase-polymerase chain reaction Northern blot analysis indicated that Syk is expressed in several endothelial cell lines. Immunocytochemical analysis also confirmed that Syk is expressed in the normal embryonic endothelial cells and is absent in Syk-deficient mice. Furthermore, electron microscopic analysis of Syk-deficient mice revealed an abnormal morphogenesis and a decreased number of endothelial cells. The results indicate a critical role for Syk in endothelial cell function and in maintaining vascular integrity in vivo.

Syk expression and novel function in a wide variety of tissues.

Syk protein-tyrosine kinase has been implicated in a variety of hematopoietic cell responses, in particular immunoreceptor signaling events that mediate diverse cellular responses including proliferation, differentiation, and phagocytosis. On the other hand, Syk exhibits a more widespread expression pattern in nonhematopoietic cells like fibroblasts, epithelial cells, breast tissue, hepatocytes, neuronal cells, and vascular endothelial cells and has been shown to be functionally important on these cell types. Thus, Syk appears to play a general physiological function in a wide variety of cells. In this article, we briefly review the current literature regarding the expression and novel function of Syk in various cells and tissues.

Adhesion via CD43 induces Syk activation and cell proliferation in TF-1 cells.

The effect of adhesion via CD43 (leukosialin, sialophorin) on cell proliferation and phosphorylation signaling were examined in a growth factor-dependent hematopoietic progenitor cell line, TF-1. TF-1 cells promptly resulted in death after withdrawal of growth factors. However, the viable cell number increased when TF-1 cells were cultured on anti-CD43 monoclonal antibody-coated plates. In this case, sustained activation of protein tyrosine kinase Syk and extracellular signal-regulated kinase (Erk) 1/2 were detected. Overexpression of exogenous Syk on TF-1 cells by the adenovirus vector system induced enhancement of the cell proliferation accompanied with enhancement of the Erk activation by a dominant-positive effect. The signal transducer and activator of transcription (STAT) 5 seemed not to be associated with the CD43-mediated cell proliferation. These results indicated that adhesion via CD43 induces the proliferation of TF-1 cells in the absence of growth factors in part by Syk-dependent Erk 1/2 signaling.

A critical role for Syk in endothelial cell proliferation and migration.

Syk is a protein-tyrosine kinase that is widely expressed in haematopoietic cells and involved in coupling activated immunoreceptors to downstream signaling. On the other hand, Syk-deficient mice showed severe petechiae in utero and died shortly after birth. Recently we have shown the expression of Syk in endothelial cells and morphological defects of these cells in embryonic Syk-deficient mice. Here we report that both proliferation and migration of human umbilical vein endothelial cells were severely impaired by adenovirus-mediated expression of Syk dominant negative mutants. Furthermore, a close relationship between Syk kinase activity and extracellular signal-regulated kinase activation was suggested. Our results indicate that Syk plays a critical role in endothelial cell functions, including morphogenesis, cell growth, migration, and survival, and contributes to maintaining vascular integrity in vivo.

The structure of physalin T from Physalis alkekengi var. franchetti.

A new steroidal constituent named physalin T (3) was isolated from the aqueous extract of Physalis alkekengi var. francheti. Based on 1H and 13C NMR spectral studies the structure was assigned as 2,3-dihydrophysalin D, i.e., 5alpha,6beta-dihydroxy-2,3,5,6-tetrahydrophysalin B, which is the first example of a natural physalin possessing a saturated ring A moiety. The structure was confirmed by the chemical transformation from the known physalin D (2) to physalin T.