Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells

Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with beta-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites.     

Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis

The mannose-binding lectin (MBL) (also known as the mannose-binding protein) is a serum protein that plays a role as an "ante-antibody" in innate immunity. In man, MBL is encoded by a single gene, whereas in mice there are two homologous proteins, MBL-A and MBL-C. In order to evaluate the relative roles of these two forms of MBL, we created MBL-A null mice that were MBL-C sufficient. We found MBL-A null mice had enhanced survival in a septic peritonitis model compared to wild-type mice and complement 3 null mice at 24 h, 48 h and 10 d (P < 0.05). Reconstitution of these mice with human MBL reversed the phenotype. Surviving mice had significantly decreased TNF-alpha and IL-6 levels in the blood and peritoneal cavity (P < 0.01). In vitro studies indicate that bacteria opsonized with MBL-A-deficient serum induced significantly less cytokine by peritoneal macrophages compared to those with wild-type serum. Our results indicate that MBL-A is a modulator of inflammation in vivo and in vitro in the mouse and that the role of MBL may extend beyond its role as an opsonin.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.     

Proteinase K Processing of Rabbit Muscle Creatine Kinase

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A₃₂₈ and A₃₂₄, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A₃₂₈–K₃₈₀). However, the C-terminal end of the K1 fragment is not A₃₂₇ as expected, but D₃₂₅. Thus, the amino acids residues T₃₂₆ and A₃₂₇ have been eliminated by the protease.     

Natural occurrence of Fusarium toxins in barley harvested during five years in an area of southwest Germany

A total of 44, 40, 47, 51, and 58 barley samples for feed use were collected randomly after the 1987, 1989, 1990, 1991, and 1992 crops, respectively, from farms located in an area of southwest Germany. The sum of precipitation from May to September was high in 1987 and markedly lower in 1989–1992. Deoxynivalenol, 3-. and 15-acetyldeoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, HT-2 toxin and diacetxyscirpenol were determined by gas chromatography with mass selective detection (GC-MS), zearalenone,α- and β-zearalenol by GC-MS or by HPLC. Deoxynivalenol was the major toxin with incidences at 71–98% and mean contents at 42–400 μg/kg. In contrast, incidences of zearalenone, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin were at 7–68, 7–48, 11–41, 0–9, and 2–29%, respectively; with mean contents at 3–146 μg/kg. α- and β-zearalenol and diacetoxyscirpenol were not detected in any sample. 15-acetyldeoxynivalenol and fusarenon-X were assayed in samples from 1987, 1991 and 1992. 15-acetyldeoxynivalenol was detected in 30, 0 and 2% of samples, respectively, with an average content of positive samples at 8 and 4 μg/kg, fusarenon-X was not detected. Over the years, incidences and levels of toxins remained constant, decreased or increased. The correlation between the occurrence of toxins and level of precipitation is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic therapy, person-regarding reasons and the determination of identity -- a reply to Robert Elliot

I have outlined two ways of defending the claim that there are so-called person-regarding reasons for practising gene therapy on human conceptuses. One is metaphysical and concerns our nature and identity. The upshot of it is that, in cases of most interest, this therapy does not affect our identity, by bringing into existence anyone of our kind who would not otherwise have existed. The other defence is value theoretical and claims that even if genetic therapy were to affect the identity of beings our kind, there could still be person-regarding reasons for performing it, since we can be benefited and harmed by being caused to exist or not to exist. Robert Elliot has attacked both of these lines, and my present objective is to show how his criticisms can be deflected.