ENLIGHT and LEIR biomedical facility.

Particle therapy (including protons and carbon ions) allows a highly conformal treatment of deep-seated tumours with good accuracy and minimal dose to surrounding tissues, compared to conventional radiotherapy using X-rays. Following impressive results from early phase trials, over the last decades particle therapy in Europe has made considerable progress in terms of new institutes dedicated to charged particle therapy in several countries. Particle therapy is a multidisciplinary subject that involves physicists, biologists, radio-oncologists, engineers and computer scientists. The European Network for Light Ion Hadron Therapy (ENLIGHT) was created in response to the growing needs of the European community to coordinate such efforts. A number of treatment centres are already operational and treating patients across Europe, including two dual ion (protons and carbon ions) centres in Heidelberg (the pioneer in Europe) and Pavia. However, much more research needs to be carried out and beamtime is limited. Hence there is a strong interest from the biomedical research community to have a facility with greater access to relevant beamtime. Such a facility would facilitate research in radiobiology and the development of more accurate techniques of dosimetry and imaging. The Low Energy Ion Ring (LEIR) accelerator at CERN presents such an opportunity, and relies partly on CERN's existing infrastructure. The ENLIGHT network, European Commission projects under the ENLIGHT umbrella and the future biomedical facility are discussed.

Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex.

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

The three-dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase-NADPH-L-2-amino-6-methylene-pimelate.

The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate D-dehydrogenase in a ternary complex with NADPH and L-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 A. L-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 microM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+ -dependent oxidation of the D-amino acid stereocenter of mesodiaminopimelate, and is the only enzyme known to catalyze the oxidative deamination of a D-amino acid. The enzyme is involved in the biosynthesis of meso-diaminopimelate and L-lysine from L-aspartate, a biosynthetic pathway of considerable interest because it is essential for growth of certain bacteria. The dehydrogenase is found in a limited number of species of bacteria, as opposed to the alternative succinylase and acetylase pathways that are widely distributed in bacteria and plants. The structure of the ternary complex reported here provides a structural rationale for the nature and potency of the inhibition exhibited by the unsaturated L-2-amino-6-methylene-pimelate against the dehydrogenase. In particular, we compare the present structure with other structures containing either bound substrate, meso-diaminopimelate, or a conformationally restricted isoxazoline inhibitor. We have identified a significant interaction between the alpha-L-amino group of the unsaturated inhibitor and the indole ring of Trp144 that may account for the tight binding of this inhibitor.

Structural symmetry: the three-dimensional structure of Haemophilus influenzae diaminopimelate epimerase.

The Haemophilus influenzae diaminopimelate epimerase was cloned, expressed, purified, and crystallized in the C2221 space group (a = 102.1 A, b = 115.4 A, c = 66.3 A, alpha = beta = gamma = 90 degrees). The three-dimensional structure was solved to 2.7 A using a single Pt derivative and the Se-Met-substituted enzyme to a conventional R factor of 19.0% (Rfree = 24.2%). The 274 amino acid enzyme consists of two structurally homologous domains, each containing eight beta-strands and two alpha-helices. Diaminopimelate epimerase is a representative of the PLP-independent amino acid racemases, for which no structure has yet been determined and substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. Cys73 of the amino terminal domain is found in disulfide linkage, at the domain interface, with Cys217 of the carboxy terminal domain, and we suggest that these two cysteine residues in the reduced, active enzyme function as the acid and base in the mechanism.

Substrate and inhibitor binding sites in Corynebacterium glutamicum diaminopimelate dehydrogenase.

The three-dimensional structures of Corynebacterium glutamicum diaminopimelate dehydrogenase as a binary complex with the substrate meso-diaminopimelate (meso-DAP) and a ternary complex with NADP+ and an isoxazoline inhibitor [Abbot, S.D., Lane-Bell, P., Kanwar, P.S.S., and Vederas, J. C. (1994) J. Am. Chem. Soc. 116, 6513-6520] have been solved and refined against X-ray diffraction data to 2.2 A. Diaminopimelate dehydrogenase is a homodimer of approximately 35,000 molecular weight subunits and is the only dehydrogenase present in the bacterial diaminopimelate/lysine biosynthetic pathway. Inhibitors of the enzymes of L-lysine biosynthesis have been proposed as potential antibiotics or herbicides, since mammals lack this metabolic pathway. Diaminopimelate dehydrogenase catalyzes the unique, reversible, pyridine dinucleotide-dependent oxidative deamination of the D-amino acid stereocenter of meso-diaminopimelate to generate L-2-amino-6-oxopimelate. The enzyme is absolutely specific for the meso stereoisomer of DAP and must distinguish between two opposite chiral amino acid centers on the same symmetric substrate. The determination of the three-dimensional structure of the enzyme--meso-diaminopimelate complex allows a description of the molecular basis of this stereospecific discrimination. The substrate is bound in an elongated cavity, in which the distribution of residues that act as hydrogen bond donors or acceptors defines a single orientation in which the substrate may bind in order to position the D-amino acid center of meso-DAP near the oxidized nucleotide. The previously described isoxazoline inhibitor binds at the same site as DAP but has its L-amino acid center positioned where the D-amino acid center of meso-DAP would normally be located, thereby generating a nonproductive inhibitor complex. The relative positions of the N-terminal dinucleotide and C-terminal substrate-binding domains in the diaminopimelate dehydrogenase--NADP+, diaminopimelate dehydrogenase--DAP, and diaminopimelate dehydrogenase--NADP(+)--inhibitor complexes confirm our previous observations that the enzyme undergoes significant conformational changes upon binding of both dinucleotide and substrate.

Degradation of the morpholino ring in the crystal structure of cyanomorpholinodoxorubicin complexed with d(CGATCG).

The cyanomorpholino analogue of antitumor anthracycline doxorubicin possesses an intense potency and differs from the parent compound in cross-resistance and other biological properties. The induction by cyanomorpholinodoxorubicin of both DNA cross-links and strand scission suggests an altered mode of action relative to doxorubicin with a different DNA-interacting capacity. We have co-crystallized 3'-(3-cyano-4-morpholinyl)-3'-desaminodoxorubicin (CMD) with the DNA hexamer d(CGATCG) and have determined the crystal structure at 0.16-nm resolution. The complex crystallizes in the space group P4(1)2(1)2 and is similar to the previously reported anthracycline/DNA structures, with the drug intercalated at the CpG step, forming hydrogen bonds with the guanine residue. The structure reveals that the morpholino moiety has undergone a major rearrangement with loss of the cyano group and opening of the morpholino ring. The compound actually bound to DNA in the complex resembles N-(2-hydroxyethyl)doxorubicin, which was previously identified as a hydrolysis product of CMD. No DNA alkylation has been observed. However, the structure shows that the active site of the morpholino ring, after dissociation of the cyano group, lies in the minor groove in proximity of the A/T base pair. This may indicate that a C/G base pair next to the intercalation site, with NH2 group in the minor groove, is required for DNA alkylation.

2 angstrom X-ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro-binding mode.

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.

Medial fractures of the femoral neck in the elderly treated by SEM bipolar prosthesis.

The authors present 335 cases of medical fracture of the femoral neck in the elderly patient (mean age 80.5 years) treated by SEM type bipolar prosthesis. A total of 93 patients (28.3%) were followed-up for a total of 98 hips submitted to surgery (5 bilateral) after a mean period of 42 months, minimum 12 months, maximum 96. Clinical follow-up included these parameters: pain, movement, walking, according to Merle D'Aubigné. Radiographic follow-up consisted in standard views and maximum adduction and abduction. Pain was present in 49% of cases, although it did not significantly invalidate movement (quotients 6 and 5 in 96% of cases); in 60% of the cases there were problems with walking mostly due to the general conditions of the patient. Wear phenomena in the acetabulum were present in 32 hips (32.6%) with no correlation with clinical data. Dynamic x-rays showed that only 31% of the implants maintained intraprosthetic movement. What emerges from the study is the importance of adequate measurement of the prosthetic cupola to improve acetabular fit.

Substituent effects on the enantioselective retention of anti-HIV 5-aryl-delta 2-1,2,4-oxadiazolines on R,R-DACH-DNB chiral stationary phase.

A series of racemic 3-phenyl-4-(1-adamantyl)-5-X-phenyl- delta 2-1,2,4-oxadiazo lines (PAdOx) were directly resolved by HPLC using a Pirkle-type stationary phase containing N,N'-(3,5-dinitrobenzoyl)-1(R),2(R)-diaminocyclohexane as chiral selector. The more retained enantiomers have S configuration, as demonstrated by X-ray crystallography and circular dichroism measurements. The influence of aromatic ring substituents on enantioselective retention was quantitatively assessed by traditional linear free energy-related (LFER) equations and comparative molecular field analysis (CoMFA). In good agreement with previous findings, the results from this study indicate that the increase in retention (k') is favoured mainly by the phi-basicity and the hydrophilicity of solute, whereas enantioselectivity (alpha) can be satisfactorily modeled by electronic and bulk parameters or CoMFA descriptors. The LFER equations and CoMFA models gave helpful insights into chiral recognition mechanisms.

Interactions between morpholinyl anthracyclines and DNA. The crystal structure of a morpholino doxorubicin bound to d(CGTACG).

Anthracycline antibiotics daunomycin and adriamycin are among the most widely used in cancer chemotherapy and DNA is believed to be the primary target of their biological action. The crystal structure of a morpholino derivative of adriamycin bound to the DNA hexamer d(CGTACG) has been determined at 1.5 A resolution. The complex crystallizes in space group P1 with unit cell dimensions a = 18.01 A, b = 18.83 A, c = 27.65 A, alpha = 92.6 degrees, beta = 100.5 degrees, gamma = 94.9 degrees and there are two drug molecules bound per duplex. Morpholino derivatives differ greatly from their parent compounds in their biological and pharmacological properties. Structural comparison of this complex with the series of previously reported anthracycline-DNA complexes offers an opportunity for studying relationships between structure and function. The anthracycline chromophore intercalates at the CpG step and DNA distortions from a B-type conformation are similar to those observed in the other DNA-anthracycline complexes. Interactions between drug and DNA show no differences at the intercalation site, while in the minor groove they are significantly affected by the presence of the bulky morpholinyl moiety on the anthracycline amino sugar. The binding site involves four base-pairs and the absence of a positive charge on the amino sugar appears to influence the hydration pattern on both grooves. The two halves of the duplex are symmetrically related by a non-crystallographic 2-fold axis but they are not equivalent. In one half, one magnesium cluster bridges both drug and DNA, further stabilizing the complex.

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole.

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.

[Post-stenotic aneurysm following incarceration of the popliteal artery. Description of a case]. / L'aneurisma post-stenotico da incarceramento dell'arteria poplitea. Descrizione di un caso.

The paper reports a case of post-stenotic aneurysm following an Inshua type II incarceration of the popliteal artery which was treated surgically, and associated with concomitant popliteal venous thrombosis, with complete remission of all clinical symptoms. The paper underlines the rarity of this pathology and the even rarer association with homolateral thrombosis of the popliteal vein. Anomalies which may occur following the incarceration of the popliteal artery are discussed, as are the causes for dilatory degeneration.

[Apophysis detachment in adolescents]. / I distacchi apofisari dell'adolescente.

The authors present a review of 37 patients (mean age 15 years), treated in our Institute from 1974 to 1984 for an apophysis detachment. The lesions were mainly due to indirect trauma. The results verify that a complete functional activity may be achieved also by an incomplete reduction of bone fragments and that conservative treatment is sufficient for almost all patients.