Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release

The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.     

The insulinotropic region of gastric inhibitory polypeptide; fragment analysis suggests the bioactive site lies between residues 19 and 30

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG) is a new viral thymidine kinase inhibitor that we tested for the ability to prevent recurrences of herpetic keratitis. Eighteen squirrel monkeys (Saimiri scuireus) were infected in both corneas with the Rodanus strain of herpes simplex virus type 1 (HSV-1). All corneas showed typical dendritic keratitis 3 days after infection, followed by spontaneous healing. On day 21, the monkeys were randomized into two coded groups and ocular examinations were begun. One group received intraperitoneal (i.p.) injections of HBPG, 150 mg/kg, in a corn oil suspension every 8 h, and the other group received i.p. injections of the corn oil vehicle only. On day 22, recurrences were induced by reducing the temperature of the room in the late afternoon so that a low of 18 degrees C was achieved during the night. After the morning treatment, room temperature was raised to the normal ambient temperature (24-27 degrees C), and treatment was discontinued. Treatment was reinstituted on day 27, the room temperature was lowered again on day 28, and treatment was again discontinued as before. Third and fourth cycles of treatment and cold stress were begun on days 34 and 69. Ocular examinations were continued until day 73, at which point the code was broken. We found that the HBPG treatment significantly reduced the number of corneas with recurrences during the treatment periods, compared with recurrences in untreated, cold-stressed animals (P = 0.01).     

Dynamic correction factors for K and G in high rate, SENB, impact tests

An analysis is given for the response of an impact loaded beam with a contact stiffness at the load point. The full frequency range is calculated and this is used to calculate the correction factors for the stress intensity factor and for the energy release rate as functions of loading time. These depend on the ratio of the contact to beam stiffness, , and both experimental and analytical schemes for finding are described. The procedures for finding are explored for one set of data on an epoxy resin in the speed range 0.2–28 ms^–1. Good agreement is found for these various methods. K _d shows a substantial increase at high speed while G _d varies only slightly. The analysis scheme seems to work well overall.     

Supramolecular control of reactivity in the solid state: from templates to ladderanes to metal-organic frameworks

We describe how reactivity can be controlled in the solid state using molecules and self-assembled metal-organic complexes as templates. Being able to control reactivity in the solid state bears relevance to synthetic chemistry and materials science. The former offers a promise to synthesize molecules that may be impossible to realize from the liquid phase while also taking advantage of the benefits of conducting highly stereocontrolled reactions in a solvent-free environment (i.e., green chemistry). The latter provides an opportunity to modify bulk physical properties of solids (e.g., optical properties) through changes to molecular structure that result from a solid-state reaction. Reactions in the solid state have been difficult to control owing to frustrating effects of molecular close packing. The high degree of order provided by the solid state also means that the templates can be developed to determine how principles of supramolecular chemistry can be generally employed to form covalent bonds. The paradigm of synthetic chemistry employed by Nature is based on integrating noncovalent and covalent bonds. The templates assemble olefins via either hydrogen bond or coordination-driven self-assembly for intermolecular [2 + 2] photodimerizations. The olefins are assembled within discrete, or finite, self-assembled complexes, which effectively decouples chemical reactivity from effects of crystal packing. The control of the solid-state assembly process affords the supramolecular construction of targets in the form of cyclophanes and ladderanes. The targets form stereospecifically, in quantitative yield, and in gram amounts. Both [3]- and [5]-ladderanes have been synthesized. The ladderanes are comparable to natural ladderane lipids, which are a new and exciting class of natural products recently discovered in anaerobic marine bacteria. The organic templates function as either hydrogen bond donors or hydrogen bond acceptors. The donors and acceptors generate cyclobutanes lined with pyridyl and carboxylic acid groups, respectively. The metal-organic templates are based on Zn(II) and Ag(I) ions. The reactivity involving Zn(II) ions is shown to affect optical properties in the form of solid-state fluorescence. The solids based on both the organic and metal-organic templates undergo rare single-crystal-to-single-crystal reactions. We also demonstrate how the cyclobutanes obtained from this method can be applied as novel polytopic ligands of metallosupramolecular assemblies (e.g., self-assembled capsules) and materials (e.g., metal-organic frameworks). Sonochemistry is also used to generate nanostructured single crystals of the multicomponent solids or cocrystals based on the organic templates. Collectively, our observations suggest that the organic solid state can be integrated into more mainstream settings of synthetic organic chemistry and be developed to construct functional crystalline solids.     

Primary surgical repair of traumatic lacerations of the lacrimal canaliculi

Normal human peritoneal cells collected during elective laparatomy from patients with gallbladder stones without clinically detectable inflammatory changes were characterized phenotypically with immunocytochemical method and flow cytometry, with special attention paid to the presence of memory cells. The responsiveness of normal PCs to mitogen and, specifically, the role of peritoneal macrophages in this process was studied. The peritoneal cells consisted of 45% of monocytes/ macrophages (CD68+), as many as CD2+ T lymphocytes, 8% CD57+ NK and K 2% CD22+ B, cells. The CD4/CD8 ratio was 0.4. The peritoneal cells did not express interleukin-2 (CD25+) and transferrin receptors (CD71+) on their surface. Approximately 49% of the peritoneal cells were class II MHC antigen positive cells. Two per cent of S100+ dendritic cells were found. Flow cytometric two-colour analysis revealed that the majority of peritoneal CD4+ (92.4%) and CD8+ (73.1%) lymphocytes, while only 50.2% of CD4+ and 30.1% CD8+ peripheral blood cells expressed simultaneously the CD45R0 (UCHL1) molecule, which is characteristic to the memory/effector T-cell subpopulation. Peritoneal T lymphocytes responded to the mitogens less than peripheral blood lymphocytes of the same individual. Supplementation of cell culture with anti-macrophage (anti-CD68) and anti-HLA-DR MoAb brought about a dose-dependent decrease of proliferative peritoneal cell response to ConA. The authors conclude that human peritoneal cell population comprises a high proportion of T lymphocytes and macrophages capable of presenting antigens to peritoneal lymphocytes. High prevalence of memory lymphocytes points to the preparedness of these cells to react with invading antigens most likely of gut bacterial origin.     

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.     

Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins

Kinetic studies have been conducted in pH 7.4 Hepes buffer at 25 degreesC on the removal of Fe(III) and Al(III) from the recombinant N-lobe half molecule of human serum transferrin (Tf/2N) and from the R124A, K206A, and K296A mutants of this protein. The rates of iron removal from Tf/2N by 3-hydroxypyridin-4-one (deferiprone) and nitrilotriacetic acid (NTA) are essentially identical with previous results on N-terminal monoferric transferrin (Tf-FeN). For both Tf/2N and Tf-FeN, iron removal by deferiprone follows simple saturation kinetics, while iron removal by NTA follows simple first-order kinetics. There is some discrepancy between the two proteins with respect to iron removal by PPi, but this may be due to differences in the chloride concentrations among different studies. The addition of Fe(NTA)2 to R124A at ambient bicarbonate concentrations forms the Fe-NTA-Tf ternary complex, but the usual Fe-CO3-Tf complex can be formed by adding ferrous ion in the presence of a larger excess of bicarbonate. This complex releases its iron very rapidly by a mechanism that is first-order with respect to the ligand. This suggests that the first-order component of metal release from transferrin involves the displacement of the synergistic carbonate anion. Since iron removal from K206A and K296A at pH 7.4 is extremely slow, studies have been conducted on the more labile Al3+ complexes of Tf/2N, K206A, and K296A. The removal of Al3+ from Tf/2N by PPi follows the same complex kinetic order with respect to the ligand concentration that is observed for iron removal, while the removal of Al3+ from both K206A and K296A reverts to a simple saturation process. The addition of perchlorate retards the removal of Al3+ from both K206A and K296A, suggesting that these lysine residues are not associated with the allosteric effects of inorganic anions on the rates of metal removal.     

Strain gauging for accurate determination of K and G in impact tests

Impact testing of materials is becoming increasingly important as a wide range of new materials are being developed for demanding high loading-rate working conditions. Charpy pendulum and many other impact-testing machines are being better instrumented to provide more information about the forces acting on a specimen up to and during fracture. Mostly, the force sensors are near the points of contact on the striker or the support and these can provide well for recording the overall forces acting on the specimen to be monitored. Of increasing interest is the distribution of stress and strain within the specimen during the initiation and propagation of fracture. This paper reports research using on-specimen strain-gauge sensors for impact testing of non-metallic specimens. Comparisons are made between force-time traces from sensors on the specimen and those located on the striker. Observations are made as to how the stresses relate to the fast crack in the core of the material specimen and those acting on the surface of the material about the crack, and also those acting on the plastic hinge formed on the compression side of the specimen. Optical and scanning electron microscopic studies are made of the crack surfaces and high-speed photography is used to observe the crack propagation in specimens with and without side-grooves to guide the crack and increase constraint.