Open complex formation by DnaA initiation protein at the Escherichia coli chromosomal origin requires the 13-mers precisely spaced relative to the 9-mers

The 245 bp chromosomal origin, oriC, of Escherichia coli contains two iterated motifs. Three 13-mers tandemly repeated at one end of the origin and four 9-mers in a nearby segment of oriC are highly conserved in enteric bacteria, as is the distance separating these two sequence clusters. Mutant origins were constructed with altered spacing of the 9-mers relative to the 13-mers. Loss or addition of even a single base drastically reduced replication, both in vivo and in vitro. Spacing mutant origins bound effectively to DnaA protein but failed to support efficient open complex formation. These results suggest that interaction with the 9-mers positions at least one subunit of DnaA to recognize directly the nearest 13-mer for DNA melting.     

High-Resolution Structural and Functional Assessments of Cerebral Microvasculature Using 3D Gas ΔR2*-mMRA

The ability to evaluate the cerebral microvascular structure and function is crucial for investigating pathological processes in brain disorders. Previous angiographic methods based on blood oxygen level-dependent (BOLD) contrast offer appropriate visualization of the cerebral vasculature, but these methods remain to be optimized in order to extract more comprehensive information. This study aimed to integrate the advantages of BOLD MRI in both structural and functional vascular assessments. The BOLD contrast was manipulated by a carbogen challenge, and signal changes in gradient-echo images were computed to generate ΔR₂* maps. Simultaneously, a functional index representing the regional cerebral blood volume was derived by normalizing the ΔR₂* values of a given region to those of vein-filled voxels of the sinus. This method is named 3D gas ΔR₂*-mMRA (microscopic MRA). The advantages of using 3D gas ΔR₂*-mMRA to observe the microvasculature include the ability to distinguish air–tissue interfaces, a high vessel-to-tissue contrast, and not being affected by damage to the blood–brain barrier. A stroke model was used to demonstrate the ability of 3D gas ΔR₂*-mMRA to provide information about poststroke revascularization at 3 days after reperfusion. However, this technique has some limitations that cannot be overcome and hence should be considered when it is applied, such as magnifying vessel sizes and predominantly revealing venous vessels.     

Acylated flavanols and procyanidins from Salix sieboldiana

An homologous series of acylated flavan-3-ols and procyanidins have been isolated, together with the known procyanidins B-1, B-3 and trimer, from the bark of Salix sieboldiana. Chemical and spectroscopic evidence led to the assignments of their structures as the 3-O-(1,6-dihydroxy-2-cyclohexene-1-carboxylic acid ester) of (+)-catechin and the 1-hydroxy-6-oxo-2-cyclohexene carboxylic acid esters of (+)-catechin and procyanidins B-1, B-3 and trimer.     

Catabolism of the anion transport protein in human erythrocytes

We identified the catabolic products of protein 3 in human erythrocytes. Protein 3, the major protein of the erythrocyte membrane, functions in anion transport and reacts covalently with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid ([3H]DIDS), a very selective inhibitor of anion transport. In this study, [3H]DIDS was used to label protein 3 in the membranes of normal cells and those from a donor heterozygous for a variant of protein 3, defined by its elongated amino-terminal end. Both types of cells contained [3H]DIDS-labeled peptides other than protein 3. A protein fragment of 60K molecular weight was found in normal cells, whereas both 60K and 63K fragments were identified in cells from the heterozygote. These peptides are identical with those generated by treatment of intact erythrocytes with Pronase or chymotrypsin. A polyclonal rabbit antibody specific for the purified 60K fragment of protein 3 was used to detect this protein and its products in the erythrocyte membrane. Autoradiographs of membrane peptides that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and allowed to react with the monospecific antibody showed, in addition to protein 3, a 60K fragment and fragments in the 40K region and in the 20-30K region. Cells containing the protein 3 variant yielded two fragments showing a 3K difference in molecular weight in all three regions, demonstrating that degradation of protein 3 is identical in normal erythrocytes and those heterozygous for the variant. This observation also confirms the common derivation of the fragments from protein 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phospholipids on the specific binding of [3H]spiroperidol to the cholate extract of rat brain synaptic membranes

Effects of phospholipids including PC, PE, PI, and PS on the specific [3H]SPD binding to the solubilized dopamine receptors were examined in the cholate extracts of the cortical and striatal synaptic membranes (P2M) of the rat brain. PC and PS, but not PE or PI, at 0.4 mM greatly enhanced the specific [3H]SPD binding to the cholate extracts of both cortical and striatal P2M fractions. PC and PS did not enhance the specific [3H]DA binding to the same cholate extracts. The enhancing effects of PC and PS were temperature-dependent and in a dose-response manner peaking at 0.4 mM and 0.2 mM respectively. Such temperature dependence indicated that the PC effects were not due to trapping of [3H]SPD by PC but represented a possible DAR-PC complex formation that allowed higher binding for the ligand. Failure of natural cerebellar P2M in enhancing the [3H]SPD binding to the cholate extract supports the notion that fluidity of the phospholipids is required for the binding or the formation of the DAR-PC (or PS) complex. Scatchard analysis of the [3H]SPD binding to the cholate extract in the absence or presence of PC or PS indicated that the PC or PS enhancement of the ligand binding may be mainly due to an increase in the number of binding sites since both PC and PS significantly increased the Bmax but not the Kd of the binding.     

Constitutively phosphorylated residues in the NS protein of vesicular stomatitis virus

The NS protein of vesicular stomatitis virus is an auxiliary protein in the virus core (nucleocapsid) that plays a role in virus-specific RNA synthesis. NS exhibits a variety of phosphorylated forms, and the degree of phosphorylation correlates with the rate of RNA synthesis. However, chymotryptic peptide mapping has indicated that all forms of NS share a common cluster of phosphorylated residues. To locate these residues in the primary structure of the molecule, we performed a series of residue-specific chemical and enzymatic cleavages and separated radiophosphate-labeled peptides by gel electrophoresis. The data indicate that the constitutively phosphorylated sites in NS molecules reside in the amino-terminal region of the molecule, between residues 35 and 78. The previously reported resistance of the phosphoamino acids in this region to dephosphorylation by exogenous phosphatase suggests that this domain is embedded within the tertiary structure of the molecule or involved in quaternary interactions. In contrast, the amino acid residues that are phosphorylated secondarily, making NS more active in RNA synthesis, reside in more exposed regions of the molecule.     

Preparation and characterization of recombinant DNA-derived human interferon-gamma

An attempt was made to prepare a highly purified, active recombinant DNA-derived human interferon-gamma. When the protein was denatured in urea and refolded, gel filtration and sedimentation velocity experiments indicated the presence of two forms, which are different in size and are not in a rapid reversible equilibrium. The two forms could be chromatographically separated. Far-UV circular dichroic spectra indicated the presence of secondary structures for both forms. Near-UV circular dichroic spectra revealed that the smaller form is folded into a rigid tertiary structure. The antiviral activity of the two forms of interferon-gamma showed a significant difference, i.e. the smaller form was 4-8-fold more active than the larger form. A variety of experiments show that the smaller form is more active, homogeneous, soluble, and stable than the larger form.