DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis

Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1–300 and another between residues 365–428, and a dsDNA-binding site within residues 365–428. Tetramerization of DnaB is mediated within residues 1–300, and DNA-dependent oligomerization within residues 365–428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.     

Conflicting evidence regarding the transport of alpha-glutamyl-dipeptides by human erythrocytes.

A novel ninhydrin-positive compound, N-methyl-D-aspartic acid, was identified in the muscle extracts of the blood shell, Scapharca broughtonii. This compound is already known to have potent neuroexcitatory activity, inducing hypermotility and strong releasing action of serum luteinizing hormone in mammals. This may be, however, the first finding of N-methyl-D-aspartic acid in natural products.     

The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Plasma concentrations of temazepam,a 3-hydroxy benzodiazepine,determined by electron-capture gas—liquid chromatography

A simple and sensitive high-performance liquid chromatographic assay of methotrexate (MTX) and its two active metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-di-amino-N¹⁰-methylpteroic acid (APA) in plasma, saliva and urine was developed. The method involved deproteinization with acetonitrile followed by addition of isoamyl alcohol and ethyl acetate. After extraction the sample was chromatographed on a cation-exchange column and monitored at 313 nm. The retention times were 5, 7 and 9 min and detection limits 20, 10 and 5 ng/ml for 7-OH-MTX, MTX and APA, respectively. For concentrations greater than 100 ng/ml one-step deproteinization of 0.1 ml sample with 0.25 ml acetonitrile was satisfactory for sample preparation. The method has been evaluated in samples from patients and rabbits receiving MTX.     

Metabolism of linoleic acid by prostaglandin endoperoxide synthase from adult and fetal blood vessels

Linoleic acid (18:2) is converted by prostaglandin endoperoxide synthase in particulate fractions and homogenates of fetal calf aorta to its 9- and 13-hydroperoxy metabolites. These intermediates are then either dehydrated to the corresponding oxo compounds or reduced to monohydroxy products. Alternatively, the hydroperoxyoctadecadienoic acids can be converted to epoxyhydroxyoctadecenoic acids, which are hydrolyzed to trihydroxy metabolites by epoxide hydrolases present in both particulate and cytosolic fractions from aorta. Linoleic acid (Km, 442 microM) is a much poorer substrate for prostaglandin endoperoxide synthase than is arachidonic acid (20:4) (Km, 48 microM). However, the oxygenation of 18:2 by particulate fractions from aorta is linear with time for at least 5 min, whereas the oxygenation of 20:4 is linear for only 15 s. Arachidonic acid strongly inhibits the conversion of 18:2 to monohydroxy (ID50, 10 microM) and trihydroxy (ID50, 140 microM) products. Linoleic acid has a similar, but much weaker effect on the formation of 6-oxoprostaglandin F1 alpha from 20:4. Substantial amounts of both the monohydroxy (9-hydroxy-10, 12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid) and trihydroxy (9,10,11-trihydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid) metabolites of 18:2 were shown by gas chromatography-mass spectrometry to be formed from endogenous substrate during incubation of slices of fetal calf aorta in physiological medium. This raises the possibility that some of these products or their hydroperoxy precursors may have some biological significance.