Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons.

PURPOSE: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells. MATERIALS AND METHODS: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy. RESULTS: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive. CONCLUSIONS: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.

Characterization of outward currents in interstitial cells from the guinea pig bladder.

PURPOSE: Outward currents were characterized from cells resembling interstitial cells of Cajal (ICCs) isolated from the detrusor of the guinea pig bladder. MATERIALS AND METHODS: ICC-like cells were studied using the whole cell patch clamp technique and K+ filled pipettes. Outward currents were evoked by stepping positively from a holding potential of -80 mV. RESULTS: ICC-like cells were distinguished from smooth muscle cells by the presence of lateral branches and an inability to contract spontaneously or when depolarized. Depolarization elicited large outward currents. Penitrem A, a blocker of large conductance, Ca activated K+ channels, significantly decreased the outward current. Its Ca dependence was demonstrated by significant inhibition with nifedipine and Ca-free solution. When large conductance, Ca activated K+ and Ca currents were blocked with penitrem A and nifedipine, a voltage dependent current was unmasked, which activated positive to -50 mV and displayed voltage dependent inactivation with half-maximal inactivation occurring at -71 mV. It was blocked in concentration dependent fashion by tetraethylammonium but unaffected by 4-aminopyridine, charybdotoxin or apamin, suggesting that small and intermediate conductance, calcium activated potassium channels, and Kv1.2 and Kv1.3 channels are unlikely to be involved. At maximal concentrations of tetraethylammonium a portion of the voltage dependent K+ current remained that was not affected by any of the blockers tested. CONCLUSIONS: ICC-like cells from the detrusor possess calcium activated and voltage dependent K+ currents.

Relativistic allowed and forbidden transition probabilities for fluorine-like Fe XVIII

Energy levels and the corresponding transition probabilities for allowed and forbidden transitions among the levels of the ground configuration and first 23 excited configurations of fluorine-like Fe XVIII have been calculated using the multiconfigurational Dirac-Fock GRASP code. A total of 379 lowest bound levels of Fe XVIII is presented, and the energy levels are identified in spectroscopic notations. Transition probabilities, oscillator strengths and line strengths for electric dipole (E1), electric quadrupole (E2) and magnetic dipole (M1) transitions among these 379 levels are also presented. The calculated energy levels and transition probabilities are compared with experimental data.

Electron impact ionization of hydrogen-like molybdenum ions

The electron impact ionization cross sections of hydrogen-like molybdenum ions were measured with an electron beam ion trap at the electron energies of 49.4, 64.4 and 79.6 keV The results are 2.82(22) x 10(-23), 3.13(29) x 10(-23) and 3.23(51) x 10(-23) cm(2), respectively. These results are compared with the experimental results measured previously. The agreement with the results obtained with, scaling formulae is also discussed.

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines. Author Keywords: Helminths; Trematodes; Parasites; Cathepsins; Proteases; Vaccines; Immunology; Biochemistry

Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells.

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of approximately 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 +/- 0.02-fold (P <0.005) relative to control with approximately 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like prolferation, differentiation and death in vitro

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula in mol% P2O5 (50)-CaO (50-X)-Na2O (X), where X was either 2, 4, 6, 8 or 10 were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion, proliferation and increased cell death in both cell types studied. There was no significant difference in %cell death between the PBGs which was significantly greater than the controls. However, compared to other PBGs, a greater number of cells was found on the 48 mol% CaO which may have been due to either increased adherence, proliferation or both. This composition was capable of supporting osteogenic proliferation and early differentiation and supports the notion that chemical modification of the glass could to lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

The construction of a whole-cell biosensor for phosphonoacetate, based on the LysR-like transcriptional regulator PhnR from Pseudomonas fluorescens 23F

The phnA gene that encodes the carbon-phosphorus bond cleavage enzyme phosphonoacetate hydrolase is widely distributed in the environment, suggesting that its phosphonate substrate may play a significant role in biogeochemical phosphorus cycling. Surprisingly, however, no biogenic origin for phosphonoacetate has yet been established. To facilitate the search for its natural source we have constructed a whole-cell phosphonoacetate biosensor. The gene encoding the LysR-type transcriptional activator PhnR, which controls expression of the phosphonoacetate degradative operon in Pseudomonas fluorescens 23F, was inserted in the broad-host-range promoter probe vector pPROBE-NT, together with the promoter region of the structural genes. Cells of Escherichia coli DH5a that contained the resultant construct, pPANT3, exhibited phosphonoacetate-dependent green fluorescent protein fluorescence in response to threshold concentrations of as little as 0.5 µM phosphonoacetate, some 100 times lower than the detection limit of currently available non-biological analytical methods; the pPANT3 biosensor construct in Pseudomonas putida KT2440 was less sensitive, although with shorter response times. From a range of other phosphonates and phosphonoacetate analogues tested, only phosphonoacetaldehyde and arsonoacetate induced green fluorescent protein fluorescence in the E. coli DH5a (pPANT3) biosensor, although at much-reduced sensitivities (50 µM phosphonoacetaldehyde and 500 µM arsonoacetate).

A 3-D well-differentiated model of pediatric bronchial epithelium demonstrates unstimulated morphological differences between asthmatic and nonasthmatic cells

There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

Differential Effect of IL-27 on Developing versus Committed Th17 Cells

IIL-27 counters the effect of TGF-beta+IL-6 on naive CD4(+) T cells, resulting in near complete inhibition of de novo Th17 development. In contrast, little is known about the effect of IL-27 on already differentiated Th17 cells. A better understanding of how IL-27 regulates these cells is needed to evaluate the therapeutic potential of IL-27 in Th17 cells-associated diseases. In this study, we show that IL-27 had surprisingly little effect on committed Th17 cells, despite its expression of a functional IL-27R. Contrary to de novo differentiation of Th17 cells, IL-27 did not suppress expression of retinoid-related orphan receptor (ROR)gammat or RORalpha in committed Th17 cells. Consistent with this finding, the frequency of committed Th17 cells and their cytokine secretion remained unaffected by IL-27. Both memory Th17 cells (CD4(+)CD25(-)CD62L(low)) that developed in vivo and encephalitogenic Th17 cells infiltrating the CNS of mice developing experimental autoimmune encephalomyelitis produced similar amounts of IL-17A when reactivated with IL-23 in the absence and presence of exogenous IL-27. Finally, IL-27 failed to suppress encephalitogenicity of Th17 cells in an adoptive transfer of experimental autoimmune encephalomyelitis. Analysis ex vivo of transferred Th17 cells in the spleen and CNS of recipient mice showed that cells retained similar phenotype irrespective of whether cells were treated or not with IL-27. Our data demonstrate that in contrast to inhibition of de novo differentiation of Th17 cells, IL-27 has little or no effect on committed Th17 cells. These findings indicate that therapeutic applications of IL-27 might have a limited efficacy in inflammatory conditions where aggressive Th17 responses have already developed.

Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.