Single lamella nanoparticles of polyethylene.

We present a complete analysis of the structure of polyethylene (PE) nanoparticles synthesized and stabilized in water under very mild conditions (15 degrees C, 40 atm) by a nickel-catalyzed polymerization in aqueous solution. Combining cryogenic transmission electron microscopy (cryo-TEM) with X-ray scattering, we demonstrate that this new synthetic route leads to a stable dispersion of individual PE nanoparticles with a narrow size distribution. Most of the semicrystalline particles have a hexagonal shape (lateral size 25 nm, thickness 9 nm) and exhibit the habit of a truncated lozenge. The combination of cryo-TEM and small-angle X-ray scattering demonstrates that the particles consist of a single crystalline lamella sandwiched between two thin amorphous polymer layers ("nanohamburgers"). Hence, these nanocrystals that comprise only ca. 14 chains present the smallest single crystals of PE ever reported. The very small thickness of the crystalline lamella (6.3 nm) is related to the extreme undercooling (more than 100 degrees C) that is due to the low temperature at which the polymerization takes place. This strong undercooling cannot be achieved by any other method so far. Dispersions of polyethylene nanocrystals may have a high potential for a further understanding of polymer crystallization as well as for materials science as, e.g., for the fabrication of extremely thin crystalline layers.

[CT-guided vertebroplasty and kyphoplasty: comparing technical success rate and complications in 101 cases]. / CT-gesteuerte Vertebro- und Kyphoplastie: Vergleichende Untersuchung zu technischem Erfolg und Komplikationen bei 101 Eingriffen.

PURPOSE: To compare the technical success and complication rates in CT-guided vertebroplasty and kyphoplasty. MATERIALS AND METHODS: From 2002 - 2005 69 patients (101 vertebrae) were treated with vertebroplasty (n = 82) or kyphoplasty (n = 19) using 4-slice MSCT with CT fluoroscopy as the sole guidance for the procedure. The underlying lesions were osteoporotic fractures in 78 vertebral bodies and 23 vertebral metastases. RESULTS: Technical success was achieved in all 101 procedures. Post-interventional CT demonstrated asymptomatic cement leakage in 51/101 vertebrae. Kyphoplasty resulted in leakage in 11/19 (57.9 %) and vertebroplasty in 40/82 (48.8 %) procedures. With p = 0.48 (Mann-Whitney Test) there was no significant difference between kyphoplasty and vertebroplasty with respect to the number of cement leakage occurrences. There was one minor complication of an L5 root irritation following radiofrequency ablation and vertebroplasty of a sarcoma metastasis which subsided without treatment after 8 weeks. There was one major complication of intraspinal cement leakage during tumor vertebroplasty causing T5 root compression and requiring laminectomy for cement removal. The overall rate of major complications requiring treatment was 0.99 %. CONCLUSION: Vertebroplasty and kyphoplasty can be safely performed using only MSCT fluoroscopy guidance. The rate of major complications is very low. There was a high rate of small asymptomatic cement leakages which may have remained undetected with conventional fluoroscopy (CF). There was no statistically significant advantage for kyphoplasty with respect to cement leakage and the technical success rate.

Transcatheter coil embolization of an aneurysm of the pancreatico-duodenal artery with occluded celiac trunk.

We report on a case of a wide-necked aneurysm of the pancreatico-duodenal artery with occlusion of the celiac trunk in an asymptomatic patient. The aneurysm was considered to be at high risk of rupture. Successful embolization after interdisciplinary consultation was followed with color-coded duplex ultrasound (CCDS) demonstrating significant flow reduction. Three weeks later CCDS and angiography demonstrated exclusion of the aneurysm and a patent arterial supply of the liver and spleen fed by superior mesenteric artery (SMA) collaterals. The patient has done well so far, without major adverse clinical events or evidence for tissue necrosis of the liver, pancreas or spleen. Discussion of the case and review of the literature indicate that transcatheter embolization is the therapy of choice even in complicated cases.

Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia.

The hereditary spastic paraplegias (HSPs; Strümpell-Lorrain syndrome, MIM number 18260) are a diverse class of disorders characterized by insidiously progressive lower-extremity spastic weakness (reviewed in refs. 1-3). Eight autosomal dominant HSP (ADHSP) loci have been identified, the most frequent of which is that linked to the SPG4 locus on chromosome 2p22 (found in approximately 42%), followed by that linked to the SPG3A locus on chromosome 14q11-q21 (in approximately 9%). Only SPG4 has been identified as a causative gene in ADHSP. Its protein (spastin) is predicted to participate in the assembly or function of nuclear protein complexes. Here we report the identification of mutations in a newly identified GTPase gene, SPG3A, in ADHSP affected individuals.

The death domain superfamily: a tale of two interfaces?

The death domain superfamily, composed of the death domain (DD), death effector domain (DED) and caspase recruitment domain (CARD) families of proteins, plays a pivotal role in signaling events that regulate apoptosis. This review compares and contrasts the ten superfamily members with known structures. In particular, the two heterodimerization modes described to date, the CARD-CARD interaction between human Apaf-1 and procaspase 9, and the DD-DD interaction between Drosophila Pelle and Tube, are examined. The dimerization modes are strikingly different and, importantly, are not mutually exclusive. In fact, a trimer can be formed using both interactions.

A docking model of key components of the DISC complex: death domain superfamily interactions redefined.

Apoptosis is mediated by a highly regulated signal transduction cascade that eventually leads to precisely directed cell death. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. We have docked the experimentally determined structures of the Fas and FADD death domains into a model of a partial DISC signaling complex. The arrangement of Fas and FADD was determined using the interaction modes of the two heterodimer crystal structures determined to date, Pelle/Tube and Apaf-1/procaspase-9. The proposed model reveals that both interactions can be accommodated in a single multimeric complex. Importantly, the model is consistent with reported site-directed mutagenesis data indicating residues throughout the domain are critical for function. These results imply that members of the death domain superfamily have the potential for multivalent interactions, offering novel possibilities for regulation of apoptotic signaling.

Refined structures of oxidized flavodoxin from Anacystis nidulans.

Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the first member of the flavodoxin family to be characterized, and is the structural prototype for the "long-chain" flavodoxins that have molecular masses of approximately 20 kDa. Crystal structure analyses and refinements of three orthorhombic forms of oxidized A. nidulans flavodoxin are reported, and salient features of the fold and the FMN binding site are compared with other flavodoxins. The structure of form I (wild-type: P212121, a=57.08 A, b=69.24 A, c=45.55 A), determined initially by multiple isomorphous replacement, has been refined to R=0.183 and R(free)=0.211 for data from 10.0 to 1.7 A resolution. Structures of form II (wild-type: P212121, a=60.05 A, b=65.85 A, c=51.36 A) and form III (Asn58Gly: P212121, a=51.30 A, b=59.15 A, c=94.44 A) have been determined by molecular replacement and refined versus data to 2.0 A and 1.85 A, respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermolecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper.

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials.

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.

A prototypical cytidylyltransferase: CTP:glycerol-3-phosphate cytidylyltransferase from bacillus subtilis.

BACKGROUND: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, which utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus inorganic pyrophosphate. Several cytidylyltransferases are related and constitute a conserved family of enzymes. The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic domains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltransferase (GCT), contains only the catalytic domain. Thus, GCT provides an excellent model for the study of catalysis by the eukaryotic cytidylyltransferases. RESULTS: The crystal structure of GCT from Bacillus subtilis has been determined by multiwavelength anomalous diffraction using a mercury derivative and refined to 2.0 A resolution (R(factor) 0.196; R(free) 0.255). GCT is a homodimer; each monomer comprises an alpha/beta fold with a central 3-2-1-4-5 parallel beta sheet. Additional helices and loops extending from the alpha/beta core form a bowl that binds substrates. CTP, bound at each active site of the homodimer, interacts with the conserved (14)HXGH and (113)RTXGISTT motifs. The dimer interface incorporates part of a third motif, (63)RYVDEVI, and includes hydrophobic residues adjoining the HXGH sequence. CONCLUSIONS: Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity. The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.

Agarose-embedded tissue arrays for histologic and genetic analysis.

To facilitate the histologic analysis of large numbers of 7-day-old zebrafish (Danio rerio), a method has been developed to process them in agarose-embedded arrays. Using thin tissue sections, the morphology of cells and tissues can be examined microscopically to investigate a variety of biologic processes. Because of their small size, precise arrangement of the larvae is necessary to section them simultaneously. A technique was designed to embed groups of zebrafish larvae in a single plane in agarose before sectioning. Stained tissue sections of thousands of larvae can be examined efficiently using this embedding method. In addition to histologic analysis, PCR-based genotypic analysis of DNA from individual larval sections is also possible. This technique can be modified to accommodate any study that requires the histologic examination of many pieces of tissue.

Papillary cystic and solid tumor of the pancreas.

The papillary cystic and solid tumor of the pancrease (PCSTP) is a primary pancreatic neoplasm of unknown etiology occurring most commonly in young women and regularly containing hemorrhagic areas. Clinical symptoms are non-specific. Although these tumors reach an average diameter of 10 cm, they are often discovered by accident. Because patients with surgically resected PCSTP have very good prognoses, it is important to distinguish these tumors from other growths in the pancreas, for example mucinous cystadenoma. Whereas, due to the variable proportion of fluid components, these tumors offer non-characteristic structure at ultrasonography, computed tomography (CT) possesses high specificity for PCSTP, particularly when calcifications are present. Angiography distinguishes these tumors from hypervascular neoplasms, such as the endocrinologically inactive islet cell tumor. Magnetic resonance tomography (MRT) is especially suited for imaging the hemorrhagic areas in solid tumor formations, as well as hemorrhagic debris in the fluid portions and layer phenomena are frequently observed.

Inferred protein content and distribution from density measurements of calcitic and aragonitic otoconia.

Otoconia from the peripheral portion of the vestibular system contain specific proteins and are mineralized by several polymorphs of calcium carbonate. To infer the internal distribution of their mineral and protein components, we have measured the densities, by equilibrium centrifugation, and the lengths of aragonitic otoconia from the African clawed frog (Xenopus laevis) and calcitic otoconia from the Norway rat (Rattus norvegicus). The densities are statistically dependent on the length of the otoconia. Further, this dependence is not the same for aragonitic and calcitic otoconia. Aragonitic otoconia are statistically more dense when smaller and less dense when larger. For calcitic otoconia the opposite is true; the smaller otoconia are statistically less dense than larger otoconia. Because the organic and inorganic phases have different densities, this indicates that the otoconial proteins and the mineral phases have different distributions within these two types of otoconia.

Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli.

Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts. To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required. A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis. For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR. By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E. coli. The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.

Successful restoration of fertility twenty-nine years after bilateral vasal injury in infancy.

Successful correction twenty-nine years after bilateral injuries to the vasa deferentia sustained at the time of infant hernia repairs, resulting in a fertile semen analysis and pregnancy is reported. This appears to be the first such case reported in the English literature.

Accuracy of preoperative studies in staging nonseminomatous germ cell testicular tumors.

The preoperative staging procedures used in 45 patients with surgical Stage I and Stage II nonseminomatous germ cell tumors of the testis were analyzed retrospectively. Our results indicate that gallium-67-citrate scan and supraclavicular lymph node biopsy add little information in the routine preoperative evaluation of these patients. Bipedal lymphangiography, in our experience, was no more accurate than excretory urograms in selecting patients with retroperitoneal disease and in addition provides no information regarding the status of the upper urinary tract. Thus, we suggest that physical examination, excretory urography, chest x-ray film, whole lung tomography, serum tumor markers, and liver scan provide sufficient information to proceed with surgical treatment when appropriate.

Anorchism versus cryptorchidism: the importance of a diligent search for intra-abdominal testes.

We reviewed 13 cases of testicular tumors arising in cryptorchid, intra-abdominal testes. All types of germinal neoplasms were found, seminoma being the most common. Of the 13 patients 5 had undergone limited exploration previously, with no testis being identified and, thus, were thought to have had unilateral anorchism. The importance of a thorough exploration of the peritoneal cavity is emphasized.

Oligospermia secondary to müllerian duct cyst. Simple surgical cure.

Fertility evaluation of a twenty-three-year-old male with severe oligospermia revealed a müllerian duct cyst that appeared to compress both ejaculatory ducts. Simple endoscopic unroofing of the cyst was followed by disappearance of the mass felt rectally, improvement of the radiographic appearance of the seminal tracts, and a return of the seminal analyses to well within fertile range. Müllerian duct cysts may be a rare cause of surgically correctable male infertility. Endoscopic unroofing of small müllerian duct cysts appears to be the treatment of choice in young males in whom there is concern for preserving fertility and potency.

Carcinoma in the defunctionalized bladder: report of a case and review of the literature.

There are few reports in the literature of carcinoma developing in the post-diversionary bladder. The sixth such case is reported and the pertinent literature is reviewed. All cases of carcinoma in defunctionalized bladders have been associated with chronic inflammation of the bladder and all tumors became apparent within 4 years of diversion. Simple cystectomy at the time of diversion should be considered for patients with a long history of bladder inflammation.

Segmental replacement of the ureter using Sparks' mandrils. An experimental study.

Segmental ureteral replacement with free autogenous fibroelastic Dacron tube grafts in three dogs was unsuccessful during preliminary trials. However, a proliferation of urothelium, smooth muscle, and blood vessels throughout the length of the grafts was demonstrated, providing an encouraging potential for further investigations. Technical refinements are suggested that may provide for improved success with experimental ureteral replacement.