Phosphorylation of the β1 Integrin Cytoplasmic Domain: Toward an Understanding of Function and Mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the β1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of β1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in β1 phosphorylation and selective loss of β1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with β1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with β1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with β1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with β1. A possible role for regulated β1 phosphorylation in cell migration is discussed.     

The AidB component of the Escherichia coli adaptive response to alkylating agents is a flavin-containing, DNA-binding protein

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. In order to begin to identify the role of AidB in the cell, the protein was purified to homogeneity, shown to possess stoichiometric amounts of flavin adenine dinucleotide (FAD), and confirmed to have low levels of isovaleryl-coenzyme A (CoA) dehydrogenase activity. A homology model of an AidB homodimer was constructed based on the structure of a four-domain acyl-CoA oxidase. The predicted structure revealed a positively charged groove connecting the two active sites and a second canyon of positive charges in the C-terminal domain, both of which could potentially bind DNA. Three approaches were used to confirm that AidB binds to double-stranded DNA. On the basis of its ability to bind DNA and its possession of a redox-active flavin, AidB is predicted to catalyze the direct repair of alkylated DNA.     

Prior exposure to methylenedioxyamphetamine (MDA) induces serotonergic loss and changes in spontaneous exploratory and amphetamine-induced behaviors in rats

The substituted amphetamine 3,4 methylenedioxyamphetamine (MDA) is a popular recreational drug of abuse. Administration of MDA to experimental animals has been shown to induce damage to serotonergic axons and nerve terminals. However, there is a lack of information on whether these treatments can produce any long-term changes in behavioural performance particularly under stressful conditions. In the present study, MDA (7.5 mg/kg; i.p.) was administered twice daily to adult male Sprague Dawley rats for four days. Four weeks following the last dose, spontaneous behaviors of these animals were tracked and scored in a novel "open field" environment using an automated video registration and computer interpretation system. Changes in behavior were observed in MDA treated animals including reductions in exploratory oriented behaviors (locomotion and rearing) and increases in grooming behavior when compared to vehicle treated controls. MDA-treated animals also displayed an enhanced locomotor and stereotyped response to d-amphetamine (12 mg/kg; i.p.). Significant reductions in 5-HT concentrations (20-30%) were observed in the frontal cortex, amygdala, striatum, and hypothalamus as a result of MDA treatment. In addition, [3H] paroxetine binding was reduced by (40%) in the cortex of MDA treated rats indicating that the decrease in 5-HT concentrations were accompanied by a reduction in intact presynaptic 5-HT nerve terminals. Changes in behavioural performance in a novel "open field" environment and following d-amphetamine challenge might be considered as a behavioural model of serotonergic deficit induced by MDA. The findings of this study also suggest that MDA use may increase both the abuse potential, and the propensity to develop psychosis as a result of abusing other psychostimulants such as d-amphetamine.     

13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins.

Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.). The data are compared with published (31)P-NMR data of enzymes closely related to TrxR. (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5). The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from those of the other enzymes. From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data. In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein. As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized. The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin. In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e. in the neutral state. Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins. The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins. This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation. The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.     

Roles for Rho/ROCK and vinculin in parietal endoderm migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.     

Metal ion dependence of recombinant Escherichia coli allantoinase

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess approximately 1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (k(cat) = 34.7 min(-1)) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (k(cat) values of 5,000 and 28,200 min(-1) and 200 min(-1), respectively). These rates and corresponding K(m) values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K(i) differences for the zinc and cobalt species (237 and 795 micro M, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.