Intermolecular Overhauser effects in fluoroalcohol solutions of cyclo-alanylglycine.

Interactions between the diketopiperazine cyclo-alanylglycine and four fluorinated alcohols in water-fluoroalcohol mixtures were examined by (1)H[(19)F] intermolecular nuclear Overhauser effects (NOE) experiments. The alcohols studied were trifluoroethanol, hexafluoroacetone trihydrate, 1,1,1,3,3,3-hexafluoroisopropanol and perfluoro-t-butanol. The experimental methods used permit detection of solvent-solute NOEs of 0.1% or less. Solute and solvent diffusion coefficients were determined and apparent molecular radii of the fluoroalcohols estimated. Using these data, it was shown that observed (1)H[(19)F] intermolecular NOEs are consistent with expectations based on theory. A method for extending conventional theory to take into account the shape of a solute and the exposure of its hydrogens to solvent is described. This approach gives reasonable agreement with experimental results, particularly if it is assumed that solute-solvent interactions take place in such a way that the fluorines of a fluoroalcohol are preferentially oriented toward the solute during solute-solvent encounters. The results support the suggestion that intermolecular (1)H[(19)F] NOEs may become a useful tool for studies of peptide and protein conformations in fluoroalcohol-water solvent mixtures.

Benign synthesis of 2-ethylhexanoic acid by cytochrome P450cam: enzymatic, crystallographic, and theoretical studies.

This study examines the ability of P450cam to catalyze the formation of 2-ethylhexanoic acid from 2-ethylhexanol relative to its activity on the natural substrate camphor. As is the case for camphor, the P450cam exhibits stereoselectivity for binding (R)- and (S)-2-ethylhexanol. Kinetic studies indicate (R)-2-ethylhexanoic acid is produced 3.5 times as fast as the (S)-enantiomer. In a racemic mixture of 2-ethylhexanol, P450cam produces 50% more (R)-2-ethylhexanoic acid than (S)-2-ethylhexanoic acid. The reason for stereoselective 2-ethylhexanoic acid production is seen in regioselectivity assays, where (R)-2-ethylhexanoic acid comprises 50% of total products while (S)-2-ethylhexanoic acid comprises only 13%. (R)- and (S)-2-ethylhexanol exhibit similar characteristics with respect to the amount of oxygen and reducing equivalents consumed, however, with (S)-2-ethylhexanol turnover producing more water than the (R)-enantiomer. Crystallographic studies of P450cam with (R)- or (S)-2-ethylhexanoic acid suggest that the (R)-enantiomer binds in a more ordered state. These results indicate that wild-type P450cam displays stereoselectivity toward 2-ethylhexanoic acid synthesis, providing a platform for rational active site design.

Rigid internal fixation of basilar fractures of the proximal phalanges by cancellous bone grafting only.

Ten patients with 13 basilar metaphyseal impaction fractures of the proximal phalanges of the fingers were treated with "rigid internal fixation" by bone grafting alone. When retrospectively reviewed at a mean follow-up of 32 months, bone healing had occurred without any relevant secondary displacement of the fracture fragments. The final ranges of motion were good and return to work was quicker than expected.

On the efficiency of hole and electron transfer from the hydration layer to DNA: An EPR study of crystalline DNA X-irradiated at 4 K.

The aim of this project was to gain an improved understanding of how the efficiency of hole and electron transfer from the solvation layer to DNA decreases as a function of distance from DNA. The packing of DNA in crystals of known structure makes it possible to calculate the degree of DNA hydration with a precision that is significantly greater than that achievable for amorphous samples. Previous work on oligodeoxynucleotide crystals has demonstrated that the efficiency of free radical trapping by DNA exposed to ionizing radiation at 4 K is relatively insensitive to base sequence, conformation, counterion, or base stacking continuity. Having eliminated these confounding variables, it is now possible to ascertain the degree of radical transfer that occurs from ionized water as a function of DNA hydration (Gamma, in mol water/mol nucleotide). EPR is used to measure the hydroxyl radical concentration in crystals irradiated at 4 K. From a lack of hydroxyl radicals trapped in the inner hydration mantle, we determine that hole transfer to DNA is complete for water molecules located within 8 A. This corresponds to Gamma = 9-11 and indicates that hole transfer is 100% (as efficient as direct ionization of DNA) for water molecules adjacent to DNA. Beyond approximately 8 A (Gamma > 10), hydroxyl radicals are observed; thus deprotonation of the water radical cation is seen to compete with hole transfer to DNA as soon as one water intervenes between the ionized water and DNA. The boundary for 0% hole transfer is projected to occur somewhere between 15 and 20 waters per nucleotide. Electron transfer, on the other hand, is 100% efficient across the entire range studied, 4.2

Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design.

Inosine monophosphate dehydrogenase (IMPDH) controls a key metabolic step in the regulation of cell growth and differentiation. This step is the NAD-dependent oxidation of inosine 5' monophosphate (IMP) to xanthosine 5' monophosphate, the rate-limiting step in the synthesis of the guanine nucleotides. Two isoforms of IMPDH have been identified, one of which (type II) is significantly up- regulated in neoplastic and differentiating cells. As such, it has been identified as a major target in antitumor and immunosuppressive drug design. We present here the 2.9-A structure of a ternary complex of the human type II isoform of IMPDH. The complex contains the substrate analogue 6-chloropurine riboside 5'-monophosphate (6-Cl-IMP) and the NAD analogue selenazole-4-carboxamide adenine dinucleotide, the selenium derivative of the active metabolite of the antitumor drug tiazofurin. The enzyme forms a homotetramer, with the dinucleotide binding at the monomer-monomer interface. The 6 chloro-substituted purine base is dehalogenated, forming a covalent adduct at C6 with Cys-331. The dinucleotide selenazole base is stacked against the 6-Cl-IMP purine ring in an orientation consistent with the B-side stereochemistry of hydride transfer seen with NAD. The adenosine end of the ligand interacts with residues not conserved between the type I and type II isoforms, suggesting strategies for the design of isoform-specific agents.

[Management of lateral type B malleolar fracture with penetrating wires, cerclage and hemicerclage]. / Versorgung der lateralen Malleolarfraktur Typ B mit Spickdrähten, Cerclagen und Hemicerclagen.

In this retrospective study 52 cases of malleolar fractures type B, that were treated with Kirschner wires, cerclages and hemicerclages, between 1981 and 1991 at the Männedorf Hospital, were compiled and completed with the corresponding synopsis. The results show that the osteosynthesis technique, in certain cases, is absolutely justifiable and represents a reliable alternative to tensile screw osteosynthesis. This procedure of osteosynthesis is a good method in comminuted fractures as well as osteoporotic bone, in which the screws grip badly. A minimum of osteosynthesis material is implanted in precarious soft tissue conditions. As in the tensile screw osteosynthesis, the removal of the material can be done ambulatory under local anaesthetic with stab incision. The functional after-treatment is not recommended. We suggested a postoperative plaster of Paris treatment for 6 weeks (2 weeks without and 4 weeks with strain). As the postoperative course shows comparable results, osteosynthesis of the lateral malleolar fracture type B with Kirschner wires, cerclages and hemicerclages represents a good variation to plate osteosynthesis and tensile screw osteosynthesis.

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.

Rapid isolation of thymosin beta 4 from thymosin fraction 5 by preparative high-performance liquid chromatography.

We have developed a rapid, efficient, and reproducible two-step method for the purification of thymosin beta 4 (T beta 4) from thymosin fraction 5 (TF5). This purification is based on the use of high-performance preparative/semi-preparative and analytical reversed-phase (Delta-Pak C18) chromatographic columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid compositional analysis have shown that natural, purified T beta 4 is identical to synthetic T beta 4. This procedure can be used to isolate other biologically active peptides from TF5 in sufficient quantity for characterization.

Scale-up methodology for the preparative purification of peptide M.

An octadecapeptide, peptide M, the epitope of a retinal protein that induces experimental autoimmune uveitis, was synthesized and purified by preparative reversed-phase chromatography. The flow-rate and gradient conditions for maximum separation of impurities were determined on a 30 x 0.39 cm I.D. column of Delta Pak (15-microns spherical C18-bonded silica with 300-A pores). The maximum amount of peptide that was resolved under these conditions was then determined experimentally. Using a scale factor dependent on the square of the column diameters, the flow-rate and amount loaded were increased 164 times on a 30 x 5 cm I.D. column of the same packing. The same resolution was achieved. Batches of 200-342 mg were chromatographed with reproducible results, providing a total yield of 394 mg of pure peptide.

Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies.

Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes. Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA). None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality. The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity. Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins. Many of the antibodies bound with higher avidity to toxin A than to toxin B. In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B. When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities. Further structural comparisons were made by investigation of mol. wts, subunit structures and amino acid compositions. The native mol. wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively. Denatured and reduced toxins each had a single subunit of 315,000. Both toxins had about 50% hydrophobic amino acids.

Hereditary warfarin resistance. Investigation of a rare phenomenon.

A 57-year-old black woman required a daily dosage of 50 mg of warfarin sodium to maintain her prothrombin time in a therapeutic range. The central volume of distribution and clearance of warfarin were normal for this patient. These findings, combined with the patient's requirement for plasma warfarin levels four times greater than those usually required to achieve adequate anticoagulation, indicated that the relative resistance was due to altered pharmacodynamics of warfarin. The only child of the propositus, a daughter, showed a similar relative resistance, confirming that this family is the third to be reported with hereditary resistance to warfarin.

Cerebro-craniofacial and craniofacial malformations: an embryological analysis.

A macro- and/or microscopical study on the normal and abnormal development of the forebrain with the eyes, nose, and cranium, was performed in 139 mouse embryos, 120 normal and 19 abnormal human embryos and fetuses, and in about 2,300 human skulls. The results suggest that from the embryological point of view, a distinction should be made between facial defects involving the brain and/or the neural elements of the eyes, i.e., the cerebro-craniofacial dysplasias, and malformations of the face and cranium only, called the craniofacial dysplasias. Both groups can be subdivided into early or primary defects (in embryos less than or equal to 17 mm C-RL) and late or secondary defects (in embryos greater than or equal to 17 mm C-RL). Almost all of the primary defects can be considered to originate from disorders occurring during the transformation of the brain and face. The secondary defects concern defective differentiation of neurectoderm and of the mesenchyme into bone centers, cartilage, and muscles. All of the defects in question can be explained by insufficient cell proliferation, degeneration, and/or differentiation. New terminology is proposed.

Femtomolar ion-pair high-performance liquid chromatographic method for determining Dns-polyamine derivatives of red blood cell extracts utilizing an automated polyamine analyzer.

An ultra-sensitive automated method for the determination of polyamines in red blood cell extracts by ion-pair reversed-phase high-performance liquid chromatography is described. The 5-dimethylaminonaphthalene-1-sulfonyl derivatives of putrescine, 1,6-diaminohexane, spermidine, and spermine are separated on a muBondapak C18 column using 1-heptanesulfonic acid and acetonitrile as the mobile phase. All compounds are eluted within 28 min subsequent to the initial injection. The method has a lower detection limit of 25 fmoles on column. Because of the simplicity and ease of operation, the method is applicable for use in either the research or clinical laboratory.