The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3.

A novel peptide (designed to bind to alphaIIbbeta3) caused platelet aggregation and aggregation-independent secretion of the contents of alpha-granules in an alphaIIbbeta3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, alphaIIbbeta3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized alphaIIbbeta3.

Oxygen delivery, oxygen consumption, and gastric intramucosal pH are not improved by a computer-controlled, closed-loop, vecuronium infusion in severe sepsis and septic shock.

Objective: To investigate the influence of the neuromuscular blocking agent vecuronium on oxygen delivery (DO2), oxygen consumption (VO2), oxygen extraction ratio, and gastric intramucosal pH in heavily sedated patients with severe sepsis or septic shock.

Design: Prospective, randomized, placebo-controlled, cross-over trial.

Setting: University hospital intensive care unit.

Steroid recognition by chloramphenicol acetyltransferase: engineering and structural analysis of a high affinity fusidic acid binding site.

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CATI). In the absence of crystallographic data for CATI, the structural determinants of steroid binding were identified by (1) construction in vitro of genes encoding chimaeric enzymes containing segments of CATI and the related type III variant (CATIII) and (2) site-directed mutagenesis of the gene encoding CATIII, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CATIII (Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CATI yields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol (Ki = 5.

Model for the complex between protein G and an antibody Fc fragment in solution.

Background: Streptococcal protein G and staphylococcal protein A are bacterial antibody-binding proteins, widely used as immunological tools, whose antibody-binding domains are structurally quite different. The binding of protein G to Fc fragments is competitive with respect to protein A, suggesting that the binding sites for protein A and protein G on Fc overlap, notwithstanding the fact that they lack sequence or structural similarity.

Results: To resolve this issue, the residues involved in the interaction between an IgG-binding domain of protein G (domain II) and the Fc fragment of mouse IgG2a have been identified by use of 13C and 15N NMR.

The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab.

Protein G is a cell surface-associated protein from Streptococcus that binds to IgG with high affinity. We have determined the X-ray crystal structures of the third IgG-binding domain (domain III) alone to a resolution of 1.1 A (final R-factor of 19.

Complementary peptides that interfere with platelet aggregation and adherence.

This article describes the application of the molecular recognition hypothesis to the critically important process of fibrinogen binding to platelets, a process that is the subject of extensive and intensive basic and clinical research. The objectives of the studies summarized below were to design, synthesize, and characterize peptides that can inhibit the binding of fibrinogen and related ligands to human platelets and thereby prevent platelet aggregation, adhesion, and clot retraction. The purpose of doing this work was twofold: first, to determine whether the molecular recognition hypothesis could serve as a useful rationale for the design of peptides that can specifically inhibit the binding of fibrinogen and related ligands to platelets; and second, to use these peptides to try to learn where fibrinogen binds to the platelet fibrinogen receptor.

Characterization of adhesion of non-exogenously stimulated and resting platelets in normal plasma to fibrinogen and its fragments.

Platelet adhesion to various forms of fibrinogen was studied using platelets in plasma and washed platelets. The study was designed to determine if platelets prepared with minimal handling in plasma at physiological pH containing normal levels of Ca2+ have different requirements for adhesion to immobilized fibrinogen than do washed platelets tested in the absence of plasma. Exposure of platelets to citrate and low pH did not seem to affect the requirements of washed platelets for adhesion to fibrinogen.

Analysis of hydrogen bonding in enzyme-substrate complexes of chloramphenicol acetyltransferase by infrared spectroscopy and site-directed mutagenesis.

Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) or 1-acetylchloramphenicol (1AcCm). The products of the forward reactions, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol (1,3Ac2-Cm), are the substrates for the reverse reaction. The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm and 1,3Ac2Cm to CAT has been investigated using infrared spectroscopy.

Antibodies to GPIIb alpha (300-312) inhibit Fg binding, clot retraction, and platelet adhesion to multiple ligands.

We previously reported that a peptide with the sequence Gly-Ala-Pro-Leu (GAPL) found as residues 309-312 in glycoprotein IIb alpha (GPIIb) comprises at least part of a Fg binding site on GPIIb (1). Subsequent studies demonstrated that a peptide corresponding to residues 300-312 of GPIIb alpha can bind to Fg and Vn, and is a potent inhibitor of platelet aggregation and the adhesion of activated platelets to at least four adhesive ligands: Fg, Fn, Vn, and vWf (2). Here, the production and initial characterization of polyclonal antibodies against this peptide are described.

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300-312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the gamma chain of Fg.

Developing a standard of practice while undergoing a facility merger.

While the three facilities that eventually became Centennial Medical Center in Nashville were in the process of merging, it became necessary to merge all of the nursing policies and procedures to establish one standard of practice. Many areas of nursing are not accountable to the vice president of nursing, yet she is responsible for nursing standards of care and practice. To bring it all together, the author formed a council of all the chairs of the manual revision process; this council condensed 35 manuals to 19 manuals, developed historical files on all active and inactive policies, developed a flow chart for new policy manual assignments, and developed one standard of practice for the medical center.

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints.

Crystal structure of a streptococcal protein G domain bound to an Fab fragment.

Protein G is a cell-surface protein from Streptococcus which binds to IgG molecules from a wide range of species with an affinity comparable to that of antigen. The high affinity of protein G for the Fab portion of IgG poses a particular challenge in molecular recognition, given the variability of heavy chain subclass, light chain type and complementarity-determining regions. Here we report the crystal structure of a complex between a protein G domain and an immunoglobulin Fab fragment.

Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution.

Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited 1H NMR and site-directed mutagenesis.

The binary complex of diacetylchloramphenicol and chloramphenicol acetyltransferase (CAT) has been studied by a combination of isotope-edited 1H NMR spectroscopy and site-directed mutagenesis. One-dimensional HMQC spectra of the complex between 1,3-[2-13C]diacetylchloramphenicol and the type III natural variant of CAT revealed the two methyl 1H signals arising from each 13C-labeled carbon atom in the acetyl groups of the bound ligand. Slow hydrolysis of the 3-acetyl group by the enzyme precluded further analysis of this binary complex.

Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G.

Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by 1H NMR. Two-dimensional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the 1H NMR spectrum of the peptide.

Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method.

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75,000, [ring 2-13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring.

The serine acetyltransferase from Escherichia coli. Over-expression, purification and preliminary crystallographic analysis.

An expression vector has been constructed which increases the expression of serine acetyltransferase (SAT) from E. coli to 17% of the soluble cell protein. A novel purification procedure, using dye-affinity chromatography, allows purification of SAT to homogeneity.

L-carnitine acyltransferase in intact peroxisomes is inhibited by malonyl-CoA.

Inhibition of the overt mitochondrial carnitine palmitoyltransferase by malonyl-CoA is important in the regulation of fatty acid oxidation. In the past, the contribution of peroxisomal carnitine acyltransferase activity to the generation of medium- and long-chain acylcarnitines in the cytoplasm has been ignored. On the basis of marker enzyme levels, we now estimate that peroxisomal palmitoyltransferase activity constitutes about 20% of the peroxisomal plus overt-mitochondrial pool in fed rat liver.

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle.

A 20-year-old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10-3H]-palmitate or of [1-14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patient's muscle was inhibited by malonyl-CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA.