Structural identifiability analysis of some highly structured families of statespace models using differential algebra

In this paper we identify biologically relevant families of models whose structural identifiability analysis could not be performed with available techniques directly. The models considered come from both the immunological and epidemiological literature.     

Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.     

Bacterial and viral DNA in periodontal disease: a study using multiplex PCR

Recent studies have suggested an association between periodontal disease and the presence of Herpesviruses, in particular: Epstein-Barr virus (EBV) and Human Cytomegalovirus (CMV) (Contreras et al., 1999--Contreras et al., 2000--Slots et al., 2000--Ting et al., 2000). In the work reported in this paper, we use a multiplex Polymerase Chain Reaction (PCR) to compare the presence of Herpesviruses and putative bacterial pathogens in patients with periodontal disease and in healthy individuals. Direct detection of microorganisms with PCR is shown to offer significant advantages in terms of time, effort and cost. The study detected no statistically significant differences between the prevalence of EBV and CMV in patients and controls. The failure to replicate previous findings may be due to differences in the age composition and the geographical and social origins of the study groups. The study detected a significant excess of HSV-1 in periodontal patients. This suggests that the role of Herpesviruses in the pathogenesis of periodontal disease deserves further investigation. The bacterial assay confirmed the results of previous studies showing a strong association between periodontitis and the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia.     

Gamma-COP appendage domain - structure and function

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α-, β'- and ε-COP subunits. Here, we present the structure of the appendage domain of γ-COP and show that it has a similar overall fold as the α-appendage of AP2. Again, like the α-appendage the γ-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ-COP, Sec21p, a second binding site is proposed to exist on the γ-COP appendage that interacts with the α,β',ε COPI subcomplex.     

Ion transport in the intestine of Gobius niger in both isotonic and hypotonic conditions

Ion transport in the intestine of Gobius niger, a euryhaline teleost, was studied in both isotonic and hypotonic conditions. Isolated tissues, mounted in Ussing chambers and bilaterally perfused with isotonic Ringer solution, developed a serosa negative transepithelial voltage and a short circuit current indicating a net negative current in absorptive direction. Bilateral removal of Cl- and Na+ from the bathing solutions as well as the luminal removal of K+in the presence of Ba2+(10(-3) M) almost abolished both Vt and Isc. Similar results were obtained by adding bumetanide (10(-5)M) to the luminal bath while other inhibitors of Cl- transport mechanisms were ineffective. These observations suggest that salt absorption begins with a coupled entry of Na+, Cl-, and K+ across the apical membrane; a Ba2+inhibitable K+ conductance, demonstrated also by micropuncture experiments, recycles the ion into the lumen. Salt entry into the cell is driven by the operation of the basolateral Na+/K(+)-ATPase since serosal ouabain (10(-4)M) completely abolished both Vt and Isc; this pump also completes the Na(+) absorption. The inhibitory effect of both serosal bumetanide (10(-4)M) and SITS (5 x 10(-4)M) suggests that Cl- would leave the cell via the KCl cotransport, the Cl/HCO3- antiport and/or conductive pathways. Bilateral exposure of tissues to hypotonic media produced a reduction of both the transepithelial voltage and the short circuit current probably due to the activation of homeostatic ionic fluxes involved in cell volume regulation. The results of experiments with both isolated enterocytes and intestine exposed to hypotonic solution suggested that the recovery of cell volume, after the initial cell swelling, involves a parallel opening of K+ and Cl- channels to facilitate net solute and water effluxes from the cell. J. Exp. Zool. 301A:49-62, 2004.     

Binding affinities for sulfonamide inhibitors with matrix metalloproteinase-2 using a linear response method

Due to their involvement in many pathological conditions, matrix metalloproteinases (MMPs), are very attractive therapeutic targets. Our study focuses on one of them, MMP-2, which is involved in tumor progression and metastasis. Recently, the solution structure of the catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020) was published by Feng et al. Using the Hanessian group published binding affinity data and the structure published by Feng as a basis, we have built a binding affinity model by targeting the S(2)' pocket of the enzyme with a set of nine alpha-N-sulfonylamino hydroxamic acid derivatives. Two binding geometries of each ligand have been generated corresponding to two binding modes denoted A and B, respectively, of which the first one is targeting the S(2)' pocket and the second one the S(1) pocket. For the binding affinity model developed for mode A the computed activities show a rmsd of 0.583 kcal/mol as compared with the experimental data, and a correlation coefficient r(2) of 0.779, while in the case of the binding mode B a rmsd of 0.834 kcal/mol and correlation coefficient r(2) of 0.500, respectively, were obtained. In conclusion, our data suggest a higher probability for the Phe(76) gated S(2)' open form pocket to accommodate the substituent alpha versus the wide solvent exposed S(1) subsite, probability which some research groups could have overlooked due to extensive use in their calculations of non revealing S(2)' pocket open state crystallographic structures instead of NMR ones.     

Redox state and carbonic anhydrase isozyme IX expression in human renal cell carcinoma: biochemical and morphological investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Carbonic anhydrases in chick extra-embryonic structures: a role for CA in bicarbonate reabsorption through the chorioallantoic membrane

The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.