Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.     

A new insulin-mimetic bis(allixinato)zinc(II) complex: structure–activity relationship of zinc(II) complexes

During the investigation of the development of insulin-mimetic zinc(II) complexes with a blood glucose-lowering effect in experimental diabetic animals, we found a potent bis(maltolato)zinc(II) complex, Zn(ma)₂, exhibiting significant insulin-mimetic effects in a type 2 diabetic animal model. By using this Zn(ma)₂ as the leading compound, we examined the in vitro and in vivo structure–activity relationships of Zn(ma)₂ and its related complexes. The in vitro insulin-mimetic activity of these complexes was determined by the inhibition of free fatty acid release and the enhancement of glucose uptake in isolated rat adipocytes treated with epinephrine. A new Zn(II) complex with allixin isolated from garlic, Zn(alx)₂, exhibited the highest insulin-mimetic activity among the complexes analyzed. The insulin-mimetic activity of the Zn(II) complexes examined strongly correlated (correlation coefficient=0.96) with the partition coefficient (logP) of the ligand, indicating that the activity of Zn(ma)₂-related complexes depends on the lipophilicity of the ligand. The blood glucose-lowering effects of Zn(alx)₂ and Zn(ma)₂ were then compared, and both complexes were found to normalize hyperglycemia in KK-A^y mice after a 14-day course of daily intraperitoneal injections. However, Zn(alx)₂ improved glucose tolerance in KK-A^y mice much more than did Zn(ma)₂, indicating that Zn(alx)₂ possesses greater in vivo anti-diabetic activity than Zn(ma)₂. In addition, Zn(alx)₂ improved leptin resistance and suppressed the progress of obesity in type 2 diabetic KK-A^y mice. On the basis of these observations, we conclude that the Zn(alx)₂ complex is a novel potent candidate for the treatment of type 2 diabetes mellitus.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0590-8     

Human bronchial smooth muscle cell proliferation via thromboxane A2 receptor

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.     

Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (Rfree=0.235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.     

NHE3 inhibition activates duodenal bicarbonate secretion in the rat

We examined the effect of inhibition of Na+/H+ exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using CO2-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 microM; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with Na+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 microM indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis, Na+-HCO3- cotransporter activation, or intracellular HCO3- formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function.