Structure, function and control of complement C5 and its proteolytic fragments.

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.

The discovery of moriniafungin, a novel sordarin derivative produced by Morinia pestalozzioides.

A novel sordarin derivative, moriniafungin (1), containing a 2-hydroxysebacic acid residue linked to C-3' of the sordarose residue of sordarin through a 1,3-dioxolan-4-one ring was isolated from the fungus Morinia pestalozzioides. Isolation of moriniafungin employed a highly specific bioassay consisting of a panel of Saccharomyces cerevisiae strains containing chimeric eEF2 for Candida glabrata, Candida krusei, Candida lusitaniae, Crytpococcus neoformans, and Aspergillus fumigatus as well as wild type and human eEF2. Moriniafungin exhibited an MIC of 6 microg/mL versus Candida albicans and IC(50)'s ranging from 0.9 to 70 microg/mL against a panel of clinically relevant Candida strains. Moriniafungin was shown to inhibit in vitro translation in the chimeric S. cerevisae strains at levels consistent with the observed IC(50). Moriniafungin has the broadest antifungal spectrum and most potent activity of any natural sordarin analog identified to date.

The life and death of translation elongation factor 2.

eEF2 (eukaryotic elongation factor 2) occupies an essential role in protein synthesis where it catalyses the translocation of the two tRNAs and the mRNA after peptidyl transfer on the 80 S ribosome. Recent crystal structures of eEF2 and the cryo-electron microscopy reconstruction of its 80 S complex now provide a substantial structural framework for dissecting the functional properties of this factor. The factor can be modified by either phosphorylation or ADP-ribosylation, which results in cessation of translation. We review the structural and functional properties of eEF2 with particular emphasis on the unique diphthamide residue, which is ADP-ribosylated by diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa.

Identification of a role for actin in translational fidelity in yeast.

Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown. To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants. Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation. However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity. Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive. Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.

Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex.

In the elongation cycle of protein biosynthesis, the nucleotide exchange factor eEF1Balpha catalyzes the exchange of GDP bound to the G-protein, eEF1A, for GTP. To obtain more information about the recently solved eEF1A-eEF1Balpha structure, we determined the structures of the eEF1A-eEF1Balpha-GDP-Mg2+, eEF1A-eEF1Balpha-GDP and eEF1A-eEF1Balpha-GDPNP complexes at 3.0, 2.4 and 2.05 A resolution, respectively. Minor changes, specifically around the nucleotide binding site, in eEF1A and eEF1Balpha are consistent with in vivo data. The base, sugar and alpha-phosphate bind as in other known nucleotide G-protein complexes, whereas the beta- and gamma-phosphates are disordered. A mutation of Lys 205 in eEF1Balpha that inserts into the Mg2+ binding site of eEF1A is lethal. This together with the structures emphasizes the essential role of Mg2+ in nucleotide exchange in the eEF1A-eEF1Balpha complex.

Crystallization of the yeast elongation factor complex eEF1A-eEF1B alpha.

Crystals of the Saccharomyces cerevisiae elongation factor eEF1A (formerly EF-1 alpha) in complex with a catalytic C-terminal fragment of the nucleotide-exchange factor eEF1B alpha (formerly EF-1 beta) were grown by the sitting-drop vapour-diffusion technique, using polyethylene glycol 2000 monomethyl ether as precipitant. Crystals diffract to better than 1.7 A and belong to the space group P2(1)2(1)2(1). The unit-cell parameters of the crystals are sensitive to the choice of cryoprotectant. The structure of the 61 kDa complex was determined with the multiple anomalous dispersion technique using three selenomethionine residues in a 11 kDa eEF1B alpha fragment generated by limited proteolysis of full-length eEF1B alpha expressed in Escherichia coli.

Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1.

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.

Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha.

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.

[Prevalence of idiopathic scoliosis in the municipality of Hillerod]. / Praevalens af idiopatisk skoliose i Hillerød Kommune.

Among Scandinavian paediatric spinal surgeons there has been a debate whether the prevalence of idiopathic adolescent scoliosis (AIS) has declined. We examined all children in the town of Hillerød, Denmark attending third and fifth grade (age 10 and 12) with forward-bending-test using a scoliometer. All children with more than seven degrees of trunk inclination were referred to a PA radiogram of the spine. We found a 0.4 percent prevalence of AIS with Cobbangles greater than 19 degrees. This is similar to earlier findings, suggesting that the declining referral rate is due to late detection of idiopathic adolescent scoliosis.

High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP.

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1. 94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.

Low resolution X-ray structure of human methylamine-treated alpha 2-macroglobulin.

The structure of methylamine-treated human alpha 2-macroglobulin (alpha 2M-Ma), a 720-kDa tetrameric inactivated proteinase inhibitor from plasma, has been determined to a resolution of 10 A. Data were collected with synchrotron radiation at 120 K, and phases were calculated by multiple isomorphous replacement and solvent flattening. A novel feature of the structure of alpha 2-M is present in its proteinase-binding cavity, dividing it into two compartments. The potential sites for proteinase entrapment in these compartments are sterically restricted. The positions of the thiol groups appearing from the functional important thiol esters upon their cleavage have been determined. They are found at the walls of the compartments at the center of the structure. The overall structure of alpha 2M-MA is much more sphere-like than previously inferred from electron microscopy studies. However, several aspects of the structure are well described by recent three-dimensional reconstructions. Possible models for the monomer, the disulfide bridged dimer, and native alpha 2M are discussed.

Crystallisation and preliminary X-ray analysis of the receptor-binding domain of human and bovine alpha 2-macroglobulin.

The receptor-binding domains (RBDs) of human and bovine alpha 2-macroglobulin (alpha 2M) have been isolated after limited proteolysis of methylamine-treated alpha 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine alpha 2M give diffraction patterns suitable for X-ray analysis, and a complete dataset with a maximum resolution of 2.3 A has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3(1)21 or P3(2)21 with cell parameters a = b = 106.8 A, c = 72.2 A.

Functional and social long-term results after free tissue transfer to the lower extremity.

We report the long-term social and functional results in 53 patients receiving free tissue transfer after trauma to the lower extremity. The results are compared with those of a matched group of patients receiving primary amputation. The microsurgically treated patients had significantly more complaints over pain during walk (p = 0.02) and edema (p < 0.00005). Regarding social results, no significant differences between the two groups were found. Time until surgery, infection, or bone defect before free flap surgery did not alter the overall results significantly. It is concluded that the long-term functional and social results after free tissue transfer are almost the same as those achieved after simple amputation. Because the median time until free flap surgery in this series was 158 days, early limb-saving procedures could possibly improve the long-term results.

Selection of fusion levels in idiopathic adolescent scoliosis treated by Harrington-DDT instrumentation: a short-term radiologic study.

Clinical records and radiographs of 106 patients treated by Harrington-dorsal transverse traction (DDT) instrumentation for idiopathic adolescent thoracolumbar scoliosis were reviewed. Our strategy was to fuse from one vertebra above the measured curve to two vertebrae below the curve, but to avoid fusions below the third lumbar vertebra. With this strategy, the lower level of fusion rarely coincided with the stable vertebra. In King type 2 and type 3 scolioses, the best results were obtained when the lower fusion level coincided with the stable vertebra. In King type 4 and in most King type 5 scolioses, the lower level of fusion was two or three vertebrae short of the stable vertebra; nevertheless, we obtained good corrections. We conclude that in King type 4 and type 5 scolioses extensive lumbar fusion can be avoided.

Crystallization of human methylamine-treated complement C3 and C3b.

Human methylamine-treated complement C3 (C3-MA) and C3b (C3b-MA) have been crystallized using ammonium sulfate as precipitant. The crystals of the two compounds are morphologically indistinguishable though they belong to different space groups. We show that only minor alterations in packing are responsible for the change in space group. Crystals of C3-MA are tetragonal [P4(1(3))22, a = b = 135, c = 610 A] with two molecules per asymmetric unit. Crystals of C3b-MA are also tetragonal [P4(1(3))2(1)2, a = b = 191, c = 610 A] with four molecules per asymmetric unit. The maximum diffraction observed is 7.7 A at cryogenic temperature using synchrotron radiation.

Low-resolution X-ray diffraction data obtained from hexagonal crystals of methylamine-treated alpha2-macroglobulin.

Two hexagonal crystal forms of tetrameric human methylamine-treated alpha(2)-macroglobulin have been grown by vapour diffusion. One of the crystal forms diffracts X-rays beyond 9 A resolution. The space group of this form is P6(2)22 or P6(4)22 with a = b = 327, c = 219 A and with one dimer of alpha(2)-macroglobulin in the asymmetric unit. Several data sets have been collected by the use of synchrotron radiation at cryogenic temperature. A native data set extending to 10 A resolution has been obtained. The merging R factor of these data is 10.3%.

[Idiopathic scolioses]. / Idiopatiske skolioser.

The article summarizes the current theories on the aetiology, natural history, diagnosis and treatment of adolescent idiopathic scoliosis. Much remains unknown concerning the genesis of scoliosis. The current treatment with bracing or spinal fusion is effective.

[Surgical management of idiopathic scoliosis using Harrington DTT instruments]]. / Idiopatiske skolioser opereret a.m. Harrington-DTT.

We reviewed the clinical charts and roentgenogram of 111 patients operated with Harrington-DTT instrumentation for idiopathic adolescent scoliosis at Copenhagen University Hospital from 1983 to 1989. Male/female ratio was 1:9. Median age was 14.5 (11-21) years at the time of surgery. Median follow-up time was 4.0 (1-7) years. Of the 111 patients, complications were registered in fifteen. Seven were reoperated, four due to gliding of the upper hook, three due to fatigue fracture of the Harrington rod before union. We found no deep infections or persisting neurological damage.