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PriB is one of the Escherichia coli X-type primosome proteins that are required for assembly of the primosome, a mobile multi-enzyme complex responsible for the initiation of DNA replication. Here we report the crystal structure of the E. coli PriB at 2.1 Å resolution by multi-wavelength anomalous diffraction using a mercury derivative. The polypeptide chain of PriB is structurally similar to that of single-stranded DNA-binding protein (SSB). However, the biological unit of PriB is a dimer, not a homotetramer like SSB. Electrophoretic mobility shift assays demonstrated that PriB binds single-stranded DNA and single-stranded RNA with comparable affinity. We also show that PriB binds singlestranded DNA with certain base preferences. Based on the PriB structural information and biochemical studies, we propose that the potential tetramer formation surface and several other regions of PriB may participate in protein-protein interaction during DNA replication. These findings may illuminate the role of PriB in X-type primosome assembly.

Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alpha(IIb)beta3 of platelets. Rho produced in Escherichia coli inhibited platelet aggregation with a K(I) value of 263 nM. Although functional, Rho produced in E. coli is misfolded based on our 2D and 3D NMR studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris. The recombinant Rho expressed in P. pastoris inhibited platelet aggregation with a resulting K(I) value of 70 nM. This is the same potency as that of native Rho. CD analysis showed that the secondary structures of Rho are pH-independent and contain 3.5-7.9% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-edited NMR spectra. These results provide the first direct evidence that highly disulfide-bonded disintegrin can be expressed in P. pastoris with the correct fold. This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants.

Our experience with 73 transverse rectus abdominis myocutaneous (TRAM) flap transfers was reviewed to see the variance in the incidence of complications among three groups of patients undergoing different types of surgical techniques. The TRAM flap was transferred as a free flap in 26 patients, a unipedicled flap in 25 patients, and a microvascularly augmented pedicled flap in 22 patients. Our data demonstrated that the incidence of partial flap loss and fat necrosis in the microvascularly augmented group was significantly lower than that in the unipedicled flap group (P < 0.01), and also lower than that in the free flap group with a statistically marginal significance (P = 0.055). Supplemental surgery is less often required in the microvascularly augmented group than in the conventional TRAM group (P = 0.002). Substantial increase in venous O(2) concentration (P = 0.03), O(2) saturation level (P = 0.007), and pH value (P = 0.002) was noticed following supercharge, and this very fact testifies to the perfusion-promoting effect of the microvascular augmentation maneuver.

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