Synthesis and characterization of homochiral polymeric S-malato molybdate(VI): toward the potentially stereospecific formation and absolute configuration of iron-molybdenum cofactor in nitrogenase

Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0–7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na₃[MoO₂H(S-mal)₂] and K₃[MoO₂H(S-mal)₂]·H₂O (H₃mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO₂···H··· O₂C-hydrogen bond [2.432(5) Å], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and -carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Λ and the homochirality within the chain is established as a homochiral form ···Λ_S–Λ_S–Λ_S–Λ_S···. It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV⁻ FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as Δ_R.     

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an (125)I(-) efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a (86)Rb(+) efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca(2+) concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger (125)I(-) efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I(sc) increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl(-) gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I(sc) increase from apically permeabilized PDEC subject to a luminal-to-serosal K(+) gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca(2+)-activated apical Cl(-) channels and basolateral K(+) channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.     

Role of ICAM-1 in chronic hepatic allograft rejection in the rat

The pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecule-1 (ICAM-1)-mediated cell recruitment in chronic hepatic rejection. Liver transplantation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg. kg(-1). day(-1) x 6 days ip) or a control preparation (either ICAM-1 missense oligonucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemistry, and survival rates were also examined. Leukocyte adhesion in terminal hepatic venules was significantly increased in the rejection group compared with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the antisense-treated groups compared with control-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 +/- 1.2 days) compared with the controls (25.2 +/- 2.7 days). These results showed that early leukocyte recruitment in the hepatic microvasculature of rejecting allografts is ICAM-1 dependent and suggest that impacting on early cell recruitment can significantly ameliorate chronic rejection.     

An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry

T-cell lymphoproliferative disorders are among the most challenging diagnoses in hematopathology. Unlike the more common B-cell disorders, in which clonality is often readily discernible by surface immunoglobulin light chain restriction, there is no specific immunophenotypic signature that is diagnostic of a clonal T-cell population. Immunophenotypic criteria that are helpful in the diagnosis of T-cell neoplasms include T-cell subset antigen restriction, anomalous T-cell subset antigen expression, deletion or diminution of one of the pan T-cell antigens, a precursor T-cell phenotype, and expression of additional markers (e.g., CD30, CD20, major myeloid antigens, and TCRgammadelta). Analysis of the inherent forward and orthogonal light scatter properties of the cell can also provide important diagnostic clues. None of these features is 100% specific, however, for aberrant expression of pan-T antigens may be seen in viral infections, B-cell malignancies, or in reactive changes following administration of certain medications. An increased CD4:CD8 ratio is often observed in Hodgkin's lymphoma. Based on the analysis of 87 neoplastic and 80 control cases, we conclude that flow cytometric features that are most suspicious for malignancy include the loss or markedly dim expression of CD45; complete loss of one or more pan-T antigens; diminished expression of more than two pan-T antigens in conjunction with altered light scatter properties; and CD4/CD8 dual-positive or dual-negative expression (except thymic lesions).     

Gastric H-K-ATPase and acid-resistant surface proteins

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta-subunit (HKbeta), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta-subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta. However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta, 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.     

RSC2, encoding a component of the RSC nucleosome remodeling complex,is essential for 2 microm plasmid maintenance in Saccharomyces cerevisiae

The stable maintenance of the 2 microm circle plasmid depends on its ability to overcome intrinsic maternal inheritance bias, which in yeast normally results in the failure to transmit DNA molecules efficiently to daughter cells. In addition to the plasmid proteins Rep1 and Rep2 acting on the plasmid DNA locus STB, it is likely that other chromosomally encoded yeast proteins are required. We have isolated mutants of yeast unable to maintain 2 microm and found that RSC2 is essential for 2 microm to overcome maternal inheritance bias. Rsc2 is part of a multisubunit RSC chromatin remodeling complex, and we show that in the absence of Rsc2 the chromatin structure of the STB region is significantly altered and the Rep1 protein loses its normal localization to subnuclear foci. Rsc1, a closely related homolog of Rsc2 present in an alternative form of the RSC complex, is not required for 2 microm maintenance and does not replace the requirement for Rsc2 when overexpressed. This represents the first specific role for Rsc2 that has been related to a change in chromatin structure, as well as the first direct evidence linking chromatin structure to 2 microm segregation.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.