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PubMed1999

Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR).

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [32P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNALysand the corresponding tRNALyscarrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNALeu(UUR)and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m2G content. Complementary sequencing of tRNALeu(UUR)has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.

Autoradiography↗

PubMed1993

Foraminal syringomyelia: suggestion for a grading system.

The standard treatment of foraminal syringomyelia includes foramen magnum decompression and duraplasty. Improvement or stabilization of the disease are achieved in most of cases. However, at least one third of patients are reported to receive little or no benefit. In this paper we retrospectively reviewed a series of 40 consecutive foramen magnum decompressions in order to identify the possible pre-operative outcome predictors. Based on clinical evolution, neurological impairment and radiological features, a scale of severity was fixed and retrospectively tested. A pre-operative score was obtained for each patient and was correlated with the surgical results. Then a four level grading system was derived. All grade I and grade II patients achieved good results (improvement or stabilization), whereas grade III patients showed intermediate behaviour and grade IV invariably worsened. On this basis, surgical results of foramen magnum decompression might be further improved provided that a careful pre-operative selection is made.

Adult↗

PubMed2003

Phylogeographic structure and cryptic speciation in the trans-Antarctic moss Pyrrhobryum mnioides.

Many bryophyte species have distributions that span multiple continents. The hypotheses historically advanced to explain such distributions rely on either long-distance spore dispersal or slow rates of morphological evolution following ancient continental vicariance events. We use phylogenetic analyses of DNA sequence variation at three chloroplast loci (atpB-rbcL spacer, rps4 gene, and trnL intron and 3' spacer) to examine these two hypotheses in the trans-Antarctic moss Pyrrhobryum mnioides. We find: (1) reciprocal monophyly of Australasian and South American populations, indicating a lack of intercontinental dispersal; (2) shared haplotypes between Australia and New Zealand, suggesting recent or ongoing migration across the Tasman Sea; and (3) reciprocal monophyly among Patagonian and neotropical populations, suggesting no recent migration along the Andes. These results corroborate experimental work suggesting that spore features may be critical determinants of species range. We use the mid-Miocene development of the Atacama Desert, 14 million years ago, to calibrate a molecular clock for the tree. The age of the trans-Antarctic disjunction is estimated to be 80 million years ago, consistent with Gondwanan vicariance, making it among the most ancient documented cases of cryptic speciation. These data are in accord with niche conservatism, but whether the morphological stasis is a product of stabilizing selection or phylogenetic constraint is unknown.

Antarctic Regions↗

PubMed1997

Template-based docking of a prolactin receptor proline-rich motif octapeptide to FKBP12: implications for cytokine receptor signaling.

A conserved proline-rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu-Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282-284). We have docked the prolactin receptor PRM (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833-844), energy minimization of the octapeptide conformers so obtained, template-based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506-peptide hybrid bound to FKBP12. Val5-Pro6 of the PRM was taken to be the biologically relevant Xaa-Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7-->O4 and N2H2-->O8, and two intermolecular ones, Ile56; N-H-->O = C:Pro6 and Tyr82:O-H-->O = C:Gly7. This conformer features a Type I beta-turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis-trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.

Amino Acid Sequence↗

PubMed1988

Characterization of porcine osteonectin extracted from foetal calvariae.

Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described 'culture shock', SPARC and BM-40 proteins, indicating that these are homologous proteins.

Amino Acid Sequence↗

PubMed1999

Sequence anomalies in the Cag7 gene of the Helicobacter pylori pathogenicity island.

The severity of Helicobacter pylori-related disease is correlated with a pathogenicity island (the Cag region of about 26 genes) whose presence is associated with the up-regulation of an IL-8 cytokine inflammatory response in gastric epithelial cells. Statistical analysis of the Cag gene sequences calculated from the complete genome of strain 26695 revealed several unusual features. The Cag7 sequence (1,927 aa) has two repeat regions. Repeat region I runs 317 aa in a form of AAA proximal to the protein N terminal; repeat region II extends 907 aa in the middle of the protein sequence consisting of 74 contiguous segments composed from selections among six consensus sequences and includes 58 regularly distributed cysteine residues with consecutive cysteines mostly 12, 18, or 24 aa apart. This "regular" cysteine arrangement may provide a scaffolding of linker elements stabilized by disulfide bridges. When Cag7 homologues from different strains are compared, differences were found almost exclusively in the repeat regions, resulting from deletion and/or insertion of repeating units. These observations suggest that the anomalous repetitive structure of the sequence plays an important role in the conformation of Cag7 gene product and potentially in the function of the pathogenicity island. Other facets of the Cag7 sequence show significant charge clusters, high multiplet count, and extremes of amino acid usage.

Amino Acid Sequence↗

PubMed2001

Stabilizing insulin-like growth factor-I in poly(D,L-lactide-co-glycolide) microspheres.

This study aimed at developing a controlled drug delivery system for recombinant human insulin-like growth factor-I (IGF-I) for localized delivery in bone healing. IGF-I was microencapsulated into an end-group uncapped 14 kDa poly(D,L-lactide-co-glycolide) 50:50 (PLGA 50:50) by solvent extraction from a W(1)/O/W(2) dispersion. Prior to encapsulation, IGF-I was exposed to ultrasonication in a water/dichloromethane dispersion, and its stability tested in the presence and absence of various excipients in the W(1) phase. HPLC and RIA were used for the assessment of IGF-I stability. Microencapsulated IGF-I was tested again for its structural intactness and also for in vitro release from various formulations containing appropriate co-encapsulated excipients. A specific fat cell assay was used to determine the biological activity of released IGF-I. Moderate ultrasonic treatment of aqueous IGF-I/dichloromethane mixtures caused approx. 50% IGF-I degradation. However, IGF-I was fully protected when bovine serum albumin, succinylated gelatin or poly(ethyleneglycol) were added to the aqueous IGF-I. Co-encapsulation of these excipients protected efficiently the protein upon microencapsulation. IGF-I release from microsphere formulations was sustained for up to 13 days featuring a moderately pulsatile pattern, depending on the microsphere composition. Typically, the amounts of IGF-I released within the first 24 h (burst) and during the second release pulse were in the order of 20 and 40%, respectively, of the total dose. The biological activity of released IGF-I was confirmed at selected time-points by the fat cell assay. In conclusion, the developed microspheres proved to be suitable to release biologically intact IGF-I over up to 13 days, a time-period considered to be relevant to promote bone fracture healing.

Adipocytes↗

PubMed2003

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Algorithms↗

PubMed2006

Defining the role of vascular smooth muscle cell apoptosis in atherosclerosis.

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodelling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. Using transgenic mice with selective induction of VSMC apoptosis, a recent study has precisely determined the direct consequences of VSMC apoptosis in both normal vessels and atherosclerotic plaques. Surprisingly, normal arteries can withstand huge cell losses with little change in active or passive properties. Normal vessels demonstrate highly efficient clearance of apoptotic bodies, even in the absence of professional phagocytes. In contrast, VSMC apoptosis alone is sufficient to induce multiple features of vulnerability to rupture in plaques, identifying VSMC apoptosis as a critical process determining plaque stability.

Animals↗

PubMed2000

[Response of metastatic colorectal cancers to treatment with CPT11 (irinotecan): implications of the mismatched base repair system].

AIMS: The aim of our study was to assess the potential relationships between tumor responsiveness to CPT11, an analogue of camptothecin, which selectively inhibits DNA topoisomerase I, and the microsatellite instability, a feature of tumors with DNA mismatch repair defect. METHODS: We designed a retrospective clinical study including 35 patients with metastatic colorectal cancer treated with CPT11, for which we analyzed the expression of hMLH1 and hMSH2 in the tumor and determined microsatellite status of repeated mononucleotide tracts present in the coding region of RII-TGFB, BAX, hMSH3 and hMSH6 genes. RESULTS: A partial or minor response was observed in 9 patients, disease stabilization in 14 patients and progression in 12 patients. Staining of hMLH1 was undetectable in 2 of the 35 tumors, while only 1 tumor lacked hMSH2 expression. Four of the 31 tumors analyzed displayed intragenic microsatellite instability. We found a good correlation between inactivation of TGFB-RII, BAX or hMSH3 genes and tumor response to CPT-11 (P =0.002). CONCLUSION: Our preliminary data suggest that intragenic microsatellite instability may influence tumor response to CPT-11 in patients with colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Cobas Integra: clinical laboratory instrument with continuous and random-access capabilities.

The Cobas Integra from Roche Diagnostic Systems is a new clinical laboratory analyzer with continuous and random-access features for routine chemistries, specific proteins, electrolytes, hormones, therapeutic drugs, and drugs of abuse. The system maintains 68 test-specific reagent cassettes on board, along with multiple ion-selective electrodes (ISEs) for electrolyte determinations. This gives the Cobas Integra the capability of analyzing as many as 72 analytes without having to load additional reagents. We describe the basic analyzer configuration and the subsystems for absorbance, fluorescence polarization, and ISE measurements. Performance characteristics for precision, methods comparison, and on-board stability are given for assays representative of the various test groups. The 29 Cobas Integra tests evaluated in the present study show good agreement (r > or = 0.98 and slopes generally 0.90 to 1.12) with the respective methods available on either the Olympus AU5000, Hitachi 911 or 717, Behring BNA, or Abbott TDx systems. Total assay precision (CV) ranged from 0.8% to 8.5%, and calibration curves were stable for as long as 20 weeks. Test throughput, which is dependent on pipetting sequence, was determined to be up to 600 tests per hour without ISE and up to 750 tests per hour with ISE; the time to first result was 2.0-10.0 min.

Calibration↗

PubMed2001

The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.

BACKGROUND: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine. RESULTS: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. CONCLUSIONS: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.

Animals↗

PubMed1984

Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization.

Methotrexate-resistant Leishmania tropica contain two separate regions of DNA amplification, one encoding the bifunctional thymidylate synthetase-dihydrofolate reductase (TS-DHFR) characteristic of protozoans and the other of yet unknown function. The amplified DNAs are initially found as extrachromosomal closed circular forms, which are unstable in the absence of selection. After prolonged culture in methotrexate the amplified DNAs are found as repetitive arrays associated with the chromosomal DNA fraction after CsCl-ethidium bromide density gradient centrifugation, and are stable once selection is removed. The molecular description of gene amplification in Leishmania thus closely parallels the cytological features of gene amplification in cultured mammalian cells.

Animals↗

PubMed1992

New IS10 transposition vectors based on a gram-positive replication origin.

We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria. These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.

Bacillus subtilis↗

PubMed1999

Effect of 0.2 T static magnetic field on human neurons: remodeling and inhibition of signal transduction without genome instability.

We describe the effect of the static magnetic field generated by a 0.2 T magnetic resonance tomograph on a normal human neuronal cell culture (FNC-B4). After 15 min exposure cells showed dramatic changes of morphology: they formed vortexes of cells and exposed branched neurites featuring synaptic buttons. At the same time, thymidine incorporation and inositol lipid signaling were significantly reduced. Control (sham exposed) or non-neuronal cells (mouse leukemia, and human breast carcinoma cells) did not show any alteration following exposure. Endothelin-1 release from FNC-B4 cells was also dramatically reduced after 5 min exposure. However, PCR analysis of 12 DNA microsatellites selected as gauges of genome instability, did not reveal any alteration following exposure, thus ruling out a direct effect of the magnetic field on DNA stability. These data can be interpreted as a specific effect of the static magnetic field on human neuronal cells and are consistent with the induction of remodeling and differentiation; they demonstrate that fields below 0.5 T have significant biological effects on human neurons.

Cell Line↗

PubMed1999

Selective assembly of cyclodextrins on poly(ethylene oxide)-poly(propylene oxide) block copolymers.

This paper presents a computational study on the formation of a molecular necklace formed by specific threading of cyclodextrins (CDs) on block copolymers. Structural as well as energetic principles for the selective complexation of alpha- and beta-cyclodextrin with poly(ethylene oxide)-poly(propylene oxide) block copolymers (PEO-PPO) are elucidated considering a diblock copolymer of equimolecular composition (PEO)4-(PPO)4 as guest. A non-statistical distribution of CDs, i.e. alpha-CDs primarily located on the PEO chain and beta-CDs on PPO blocks of the polymer, is based on a variety of structural features and energetic preferences considering both potential as well as solvation energies. This selectivity becomes already obvious considering 1:1 complexes between PEO and PPO monomers and the two CDs, but is increasingly evident when calculating higher order ensembles. Besides the host-guest interaction, docking between CDs themselves is an important, also non-statistical, prerequisite for the self-assembly of highly ordered tubes. The formation of intermolecular hydrogen bonds between adjacent CDs in a tubular aggregate gives an important contribution to the overall stability of the molecular necklace. The net effect, based on the preferential interaction between host and guest as well as between the host molecules themselves, results in the formation of a stable, highly ordered macromolecular, multicomponent aggregate.

Cyclodextrins↗

PubMed2004

[Tarsometatarsal injuries--an often neglected entity].

Tarsometatarsal dislocations or fracture dislocations represent infrequent, but severe injuries which endanger the structural and mechanical integrity of the midfoot if the diagnosis is missed initially. Delayed diagnosis may result in painful and disabling arthritis and the need for salvage reconstructive surgery. As such, the rationale of treatment should follow the principles of reconstruction of weight-bearing joint injuries. The degree of instability and dislocation will guide the decision for surgical intervention and anatomic reconstruction. As the clinical features of Lisfranc injuries are rarely conclusive an adequate radiographic examination of the foot employing three standard projections (dorso-plantar, lateral and 45 degrees oblique) usually supplemented by CT scans and/or MRI is decisive for a correct analysis of the injury components and an optimum selection of treatment options. Anatomic reduction and alignment are prerequisites for a good functional outcome. The reduction of the second metatarsal ray is the keystone and the first step of surgical reconstruction followed by the other structures involved. Since adequate stability is needed until definite healing has taken place the temporary transfixation of the corresponding tarsometatarsal joints employing small fragment positioning screws has substantial advantages compared with the traditional temporary K-wire arthrodesis.

Arthrodesis↗

PubMed1997

X-ray structure of Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP-NH2), a highly potent mu-receptor selective opioid agonist. Comparison with proposed model structures.

Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the mu-receptor. Its conformational features have been studied using a combination, a solid-state (X-ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X-ray study showed two distinct conformers for D-TIPP, with the main differences lying in the orientation of the Tyr side-chain and the presence of both D-Tic(+) and D-Tic(-) conformations for the D-Tic residue. The peptide backbone is folded and stabilized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D-Tic residue are found in the lowest-energy structures. Comparison of the X-ray and modeling results shows many similarities especially around the D-Tic residue.

Crystallography, X-Ray↗