Non-enzymatic glycation of type I collagen diminishes collagen-proteoglycan binding and weakens cell adhesion

Non-enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans(1) (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell-collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG-collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content-effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B-cells expressing the cell surface heparan sulfate PG syndecan-1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein-collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG-collagen interactions, thereby disrupting matrix integrity and cell-collagen interactions, adhesion, and migration.     

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability,Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Abstract

Oligodeoxyribonucleotides containing dA·dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA·oligomer. Thermal denaturation studies show that these parallel dAdU sequences are significantly less stable than their dA·analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5- methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA·and APS dA·dU sequences. However, binding to the PS dA·hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmoF reduced free energy of binding for the PS dA·sequence. Replacement of the bulky methyl group with a hydrogen (ie. T -> U) results in significantly stronger Hoechst 33258 binding to the parallel dA·dU sequences with a penalty of only 4.1 kJmol^?1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA·dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.     

Characterization of a parallel-stranded DNA hairpin

Recently we have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present study, we have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T)8C4(A)8-3' and the related antiparallel (APS) hairpin 5'-d(T)8C4(A)8-3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. However, the small chemical shift dispersion observed in proton NMR spectra of the PS hairpin suggests that the stem of this hairpin is more regular than that of the APS hairpin. On the basis of NOESY measurements, we find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of a multidrug resistance-adenosine deaminase fusion gene

A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA. The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells. Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed. A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity. The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells.     

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

CT-Guided Biopsy in Suspected Spondylodiscitis – The Association of Paravertebral Inflammation with Microbial Pathogen Detection

Objectives

To search for imaging characteristics distinguishing patients with successful from those with futile microbiological pathogen detection by CT-guided biopsy in suspected spondylodiscitis.

Methods

34 consecutive patients with suspected spondylodiscitis underwent CT-guided biopsy for pathogen detection. MR-images were assessed for inflammatory infiltration of disks, adjacent vertebrae, epidural and paravertebral space. CT-images were reviewed for arrosion of adjacent end plates and reduced disk height. Biopsy samples were sent for microbiological examination in 34/34 patients, and for additional histological analysis in 28/34 patients.

Results

Paravertebral infiltration was present in all 10/10 patients with positive microbiology and occurred in only 12/24 patients with negative microbiology, resulting in a sensitivity of 100% and a specificity of 50% for pathogen detection. Despite its limited sensitivities, epidural infiltration and paravertebral abscesses showed considerably higher specificities of 83.3% and 90.9%, respectively. Paravertebral infiltration was more extensive in patients with positive as compared to negative microbiology (p = 0.002). Even though sensitivities for pathogen detection were also high in case of vertebral and disk infiltration, or end plate arrosion, specificities remained below 10%.

Conclusions

Inflammatory infiltration of the paravertebral space indicated successful pathogen detection by CT-guided biopsy. Specificity was increased by the additional occurrence of epidural infiltration or paravertebral abscesses.     

Molecular features of an alcohol binding site in a neuronal potassium channel

Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K(+) channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 micros) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K(+) channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only approximately 2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K(+) channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the alpha-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.     

Detection of recombinant P-glycoprotein in multidrug resistant cultured cells

The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells. Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer. Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers. Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents. P-glycoprotein was found to be overexpressed in many of these models. Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells. Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions. Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes. This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells. Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein. Strategies to visualize P-glycoprotein include metabolic labeling using ³⁵S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.     

Length-dependent formation of parallel-stranded DNA in alternating AT segments

Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.     

The concept of fillet flaps: classification, indications, and analysis of their clinical value

Tissue of amputated or nonsalvageable limbs may be used for reconstruction of complex defects resulting from tumor and trauma. This is the "spare parts" concept.By definition, fillet flaps are axial-pattern flaps that can function as composite-tissue transfers. They can be used as pedicled or free flaps and are a beneficial reconstruction strategy for major defects, provided there is tissue available adjacent to these defects.From 1988 to 1999, 104 fillet flap procedures were performed on 94 patients (50 pedicled finger and toe fillets, 36 pedicled limb fillets, and 18 free microsurgical fillet flaps).Nineteen pedicled finger fillets were used for defects of the dorsum or volar aspect of the hand, and 14 digital defects and 11 defects of the forefoot were covered with pedicled fillets from adjacent toes and fingers. The average size of the defects was 23 cm2. Fourteen fingers were salvaged. Eleven ray amputations, two extended procedures for coverage of the hand, and nine forefoot amputations were prevented. In four cases, a partial or total necrosis of a fillet flap occurred (one patient with diabetic vascular disease, one with Dupuytren's contracture, and two with high-voltage electrical injuries).Thirty-six pedicled limb fillet flaps were used in 35 cases. In 12 cases, salvage of above-knee or below-knee amputated stumps was achieved with a plantar neurovascular island pedicled flap. In seven other cases, sacral, pelvic, groin, hip, abdominal wall, or lumbar defects were reconstructed with fillet-of-thigh or entire-limb fillet flaps. In five cases, defects of shoulder, head, neck, and thoracic wall were covered with upper-arm fillet flaps. In nine cases, defects of the forefoot were covered by adjacent dorsal or plantar fillet flaps. In two other cases, defects of the upper arm or the proximal forearm were reconstructed with a forearm fillet. The average size of these defects was 512 cm2. Thirteen major joints were salvaged, three stumps were lengthened, and nine foot or forefoot amputations were prevented. One partial flap necrosis occurred in a patient with a fillet-of-sole flap. In another case, wound infection required revision and above-knee amputation with removal of the flap.Nine free plantar fillet flaps were performed-five for coverage of amputation stumps and four for sacral pressure sores. Seven free forearm fillet flaps, one free flap of forearm and hand, and one forearm and distal upper-arm fillet flap were performed for defect coverage of the shoulder and neck area. The average size of these defects was 432 cm2. Four knee joints were salvaged and one above-knee stump was lengthened. No flap necrosis was observed. One patient died of acute respiratory distress syndrome 6 days after surgery.Major complications were predominantly encountered in small finger and toe fillet flaps. Overall complication rate, including wound dehiscence and secondary grafting, was 18 percent. This complication rate seems acceptable. Major complications such as flap loss, flap revision, or severe infection occurred in only 7.5 percent of cases. The majority of our cases resulted from severe trauma with infected and necrotic soft tissues, disseminated tumor disease, or ulcers in elderly, multimorbid patients.On the basis of these data, a classification was developed that facilitates multicenter comparison of procedures and their clinical success. Fillet flaps facilitate reconstruction in difficult and complex cases. The spare part concept should be integrated into each trauma algorithm to avoid additional donor-site morbidity and facilitate stump-length preservation or limb salvage.