Biomedical subjects

J Muth

Publications and source records attributed to J Muth.

12 recordsLinked to original sources

PubMed2002

Accelerated aging studies of UHMWPE. I. Effect of resin, processing, and radiation environment on resistance to mechanical degradation.

The resin and processing route have been identified as potential variables influencing the mechanical behavior, and hence the clinical performance, of ultra-high molecular weight polyethylene (UHMWPE) orthopedic components. Researchers have reported that components fabricated from 1900 resin may oxidize to a lesser extent than components fabricated from GUR resin during shelf aging after gamma sterilization in air. Conflicting reports on the oxidation resistance for 1900 raise the question of whether resin or manufacturing method, or an interaction between resin and manufacturing method, influences the mechanical behavior of UHMWPE. We conducted a series of accelerated aging studies (no aging, aging in oxygen or in nitrogen) to systematically examine the influence of resin (GUR or 1900), manufacturing method (bulk compression molding or extrusion), and sterilization method (none, in air, or in nitrogen) on the mechanical behavior of UHMWPE. The small punch testing technique was used to evaluate the mechanical behavior of the materials, and Fourier transform infrared spectroscopy was used to characterize the oxidation in selected samples. Our study showed that the sterilization environment, aging condition, and specimen location (surface or subsurface) significantly affected the mechanical behavior of UHMWPE. Each of the three polyethylenes evaluated seem to degrade according to a similar pathway after artificial aging in oxygen and gamma irradiation in air. The initial ability of the materials to exhibit post-yield strain hardening was significantly compromised by degradation. In general, there were only minor differences in the aging behavior of molded and extruded GUR 1050, whereas the molded 1900 material seemed to degrade slightly faster than either of the 1050 materials.

Biocompatible Materials↗

PubMed2002

Accelerated aging studies of UHMWPE. II. Virgin UHMWPE is not immune to oxidative degradation.

In Part I of this series, we showed that aging at elevated oxygen pressure is more successful at increasing the depth to which degradation occurs although it, too, generally causes greater degradation at the surface than at the subsurface. Therefore we hypothesized that thermal degradation alone, in the absence of free radicals, could be sufficient to artificially age UHMWPE in a manner analogous to natural aging. In the present study, virgin and air-irradiated UHMWPE (extruded GUR 1050 and compression-molded 1900) were aged up to 4 weeks at elevated oxygen pressure, and the mechanical behavior at the surface and subsurface was examined. All the materials were substantially degraded following 4 weeks of aging, but the spatial variations in the nonirradiated materials more closely mimicked the previously observed subsurface peak of degradation seen in naturally aged UHMWPE following irradiation in air. This aged material could provide a more realistic model for subsurface mechanical degradation, making it suitable for further mechanical testing in venues such as wear simulation.

Biocompatible Materials↗

PubMed2001

A transcript encoding a nucleic acid-binding protein specifically expressed in maize seeds.

A cDNA clone has been obtained for a low-abundance, seed-specific mRNA that encodes a polypeptide which defines a novel family of plant proteins with some similarities to the DnaJ class of molecular chaperones. The MEM1 (Maize Endosperm Motif binding protein) protein is capable of binding to the endosperm motif and activating transcription in the yeast one-hybrid system. Recombinant MEM1 was shown to bind in vitro to nucleic acids, with a preference for RNA over DNA. MEM1 is capable of forming homodimers, a property that is dependent on a domain close to the C-terminus of the protein. The protein is expressed in mid- to late-term endosperm cells. Subcellular fractionation and size fractionation under non-denaturing conditions indicate that the protein is present in the cytosol of endosperm cells. Possible roles of MEM1 in endosperm and protein body development are discussed.

Amino Acid Sequence↗

PubMed1997

Capillary electrophoresis for the detection of known point mutations by single-nucleotide primer extension and laser-induced fluorescence detection.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was used to detect known point mutations using the method of single-nucleotide primer extension (SNuPE). Three different point mutations in human mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) were detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled dideoxy terminator complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild type could be simultaneously detected. The advantages of using CE-LIF for detecting SNuPE reactions include speed and ease of analysis, absence of radioactivity, and potential for automation.

Base Sequence↗

PubMed1997

Synthesis and binding properties of oligodeoxynucleotides containing phenylphosphon(othio)ate linkages.

A method for the synthesis of chimeric oligodeoxynucleotides comprised of phosphodiester and phenylphosphonate [3'-O-P(= O)(C6H5)-O-5'] or phenylphosphono-thioate [3'-O-P(= S)(C6H5)-O-5'] linkages has been developed. Synthesis was performed using suitably protected nucleoside phenylphosphonamidites as building blocks following an adjusted solid-phase phosphoramidite synthesis protocol. The new oligodeoxy-nucleotide analogues were characterized by electrospray ionization- and matrix-assisted laser desorption mass spectrometry, as well as by 31P NMR spectroscopy. Additionally, their binding properties to complementary oligodeoxynucleotides has been studied.

Chromatography, High Pressure Liquid↗

PubMed1996

DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices.

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.

Automation↗

PubMed1996

Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.

High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.

Base Sequence↗

PubMed1995

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients.

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted from delta TCS-1-positive T lymphocytes. At concentrations of 1.25-10 microM, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, of delta TCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.

Antigens, CD↗

PubMed1992

Antisense oligodeoxynucleotide: inhibitor of splicing of mRNA of human immunodeficiency virus.

Antisense oligodeoxynucleotide (ODN), which are directed against the splice acceptor site of exon II of the regulatory gene tat of the human immunodeficiency virus type 1 (HIV-1), have been described. These 20-mer ODN's displayed moderate anti-HIV activity in vitro. Using the same antisense ODN (termed ODN-2), which was additionally modified and protected both at the 3'- and the 5'-terminus by two phosphorothioate internucleotide linkages, a strong anti-HIV activity (EC50: 2.7 micrograms/ml) could be measured in the HIV-1/CEM- and HIV-1/HeLa-T4+ cell system. The analogous ODNs which were protected only at one end were either inactive (up to 10 micrograms/ml) or displayed a low antiviral activity. Time kinetic studies revealed that the antisense ODN-2 reduced the release of HIV-1 already after an incubation time of 1 h. By applying S1 nuclease protection procedures, it could be established that the antisense ODN-2 inhibited splicing of high molecular weight transcript to the 2-kb tat mRNA in HIV-1-infected CEM cells. Transfection experiments with pU3R-III chloramphenicol acetyltransferase expression vector in HeLa-T4+ cells revealed that the antisense ODN-2 blocked the Tat protein-mediated transactivation process. In co-transfection experiments using pSV2tat72 or scrape loading studies with purified Tat, the transactivation was restored. These data indicate that the selected antisense ODN-2 displays its anti-HIV effect by blocking the splicing process leading to the functional 2-kb tat mRNA.

Antiviral Agents↗

PubMed1991

Backbone modified oligodeoxynucleotides: stereoselective synthesis of methylphosphonates and HIV inhibitory capacity of phosphorothioates.

Oligonucleotides bearing phosphorothioate linkages of the HIV tatIII splice acceptor site were synthesized by automated solid phase synthesis. Especially 5'- and 3'-end capped thioates of the sequence 5'-ACACCCAATTCTGAAAATGG-3' show microM inhibition of HIV replication. ODN-methylphosphonates of defined stereochemistry were obtained with suitably modified proline phosphonamidite derivatives as monomeric building blocks. Asymmetric induction for nucleosidephosphonamidates up to 5:1 (Rp:Sp rsp. Sp:Rp depending on configuration of proline-moiety) could be reached. The intermediate phosphonamidite can be further reacted to dinucleoside methylphosphonates of enriched diastereomeric excess.

Antiviral Agents↗

PubMed1988

[14C]acetate incorporation by cultured normal, familial hypercholesterolemia and Down's syndrome fibroblasts.

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [14C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [14C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [14C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 micrograms low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C27 sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [14C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.

Acetates↗

PubMed1985

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue.

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.

Affinity Labels↗