Genetic experiments have suggested that a^, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-o-factor SpoIIAB and an anti-anti-cr factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of cr^-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-o-^ activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-cr^ activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-a capacity of SpoIIAA and hence the activity of a^.

F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [␥-32 P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.Early in the sporulation of Bacillus subtilis, the cell divides asymmetrically to form a small compartment, the prespore, and a large compartment, the mother cell. The two compartments embark on separate programs of gene expression, by means of which an endospore is formed. These programs are controlled in part by dedicated sigma factors, which become active in a compartment-specific way at defined times in sporulation (for a review, see reference 4). The first compartmentspecific sigma factor is F , the activity of which is carefully regulated. In the predivisional cell, the F protein is present in latent form, and after septation it becomes activated in the prespore but not in the mother cell; subsequent events in sporulation depend critically on this controlled activation (6). The regulation of F has been studied intensively in recent years, and we now know that regulation involves two proteins that are the products of genes in the operon (spoIIA) that also encodes F (SpoIIAC) itself. SpoIIAB is an inhibitor of F and is thus an anti-sigma factor, while SpoIIAA counteracts the inhibition by SpoIIAB and is thus an anti-anti-sigma factor (1-3, 7, 8, 10). Work in this laboratory has shown that SpoIIAB is also a phosphokinase, which catalyzes the transfer of phosphate from ATP to a serine residue of SpoIIAA (8), and we and our coworkers have recently outlined a model for the involvement of this phosphorylation in the regulation of F (2). We wanted to know which serine residue of SpoIIAA becomes phosphorylated in the phosphotransfer reaction catalyzed by SpoIIAB. Recent experiments have shown that mutagenesis of Ser-58 of SpoIIAA leads to a change in sporulation phenotype, whereas mutagenesis of two other residues (Ser-54 and Ser-84) has no phenotypic effect (2). These results are highly suggestive, but they do not constitute unequivocal proof that Ser-58 is phosphorylated by SpoIIAB or, if it is, that no other residue is phosphorylated. We now describe an experiment that identifies the site of phosphorylation by direct biochemical analysis.SpoIIAA and SpoIIAB were purified (2), and the proteins (20 g of each) were incubated for 30 min at 37ЊC in 100 l of phosphorylation buffer (8) containing 20 Ci of [␥-32 P]ATP with carrier ATP at a final concentration of 20 M. The mixture was subjected to high-pressure liquid chromatography on a size exclusion column, and the fractions containing phosphorylated SpoIIAA were pooled and dialyzed against three 1-liter changes of 100 mM Tris-HCl, pH 8.0. Endoproteinase Glu-C (2 g) was added to the Spo...

Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis. Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine. Study of the phosphorylation reaction showed that the K m for ATP and the K i for ADP were both about 1 M. The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase. In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex. The rate of this dissociation was not affected significantly by changes in the concentration of ATP. F is the first compartment-specific sigma factor that becomes active during sporulation of Bacillus subtilis (11,17). Since the demonstration some years ago by genetic means that F activity is regulated by spoIIAA, spoIIAB, and spoIIE (14, 19), this laboratory and others have been investigating the biochemical properties of SpoIIAA, SpoIIAB, and SpoIIE in order to uncover the molecular details of this regulation (1-3, 5-10, 12, 13, 15). Among the reactions involving these proteins is a phosphorylation of the Ser-58 residue of SpoIIAA catalyzed by SpoIIAB (15,16). That this phosphorylation is crucial for regulating F was inferred from experiments in which Ser-58 of SpoIIAA was changed to either alanine or aspartate, yielding the mutant proteins SpoIIAAS58A and SpoIIAAS58D (5); cells carrying these two mutations proved to have contrasting phenotypes with respect to F regulation, and the purified mutant SpoIIAA proteins behaved very differently from their wild-type counterpart in vitro (5, 10, 12). Recent work in this laboratory demonstrated that the overall rate of phosphorylation catalyzed by SpoIIAB is limited by a reaction involving the regeneration of the catalytically active species, but did not precisely define the rate-limiting step (13). We now report further studies of SpoIIAB that not only identify that step but also give evidence of conformational changes that occur in SpoIIAB.SpoIIAA strongly stimulates ATP binding to SpoIIAB. SpoI-IAB is a protein kinase, and as such must bind to ATP. In the first series of experiments, we used ultrafiltration to study this binding. SpoIIA proteins were prepared as previously described (12). SpoIIAB (3 M) was incubated in a Centricon 10 filter in buffer A (50 mM Tris-Cl [pH 7.5], 50 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol) with 6 M (a subsaturating amount) [␣- Ϫ1 in a total volume of 50 l, preincubated on ice in the dark for 10 min, and exposed to UV (254 nm) from a handheld UV Mineralight at a distance of about 2 cm. After 30 min of exposure, the samples were collected, boiled with sodium dodecyl sulfate and dithiothreitol, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% polyacrylamide). Figure 1 shows that ...

An intensive study is underway to evaluate different potential candidates for cell therapy of neurodegenerative disorders such as Parkinson's disease (PD). Availability and lower immunogenicity compared to other sources for stem cell therapy such as bone marrow have made human umbilical cord blood stem cells a considerable source for cell therapy. The present study aimed to investigate differentiation of recently introduced pluripotent cord blood stem cells, known as unrestricted somatic stem cells (USSCs), into cells with neural features in serum-withdrawal medium. Using reverse transcription polymerase chain reaction and immunocytochemistry assays, we have shown the expression of neuron-specific genes following a 2week treatment of USSCs in serum-withdrawal induction medium. In addition, we have found that USSCs and USSC-derived neuron-like cells express transcripts of genes associated with development and/or survival of dopaminergic mesencephalic neurons including En1, En2, Nurr1, Ptx3, Pax2, Wnt1 and Wnt3a. The expression of dopamine-associated genes suggests that these cells may be potential candidates to be used for cell therapy of PD.

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