Biomechanical analysis of multilevel fixation methods in the lumbar spine.

Study Design: The authors measured and compared the stiffness of cadaveric lumbar spines stabilized with several anterior interbody fusion devices. The information obtained provides a foundation for determining how methods of anterior lumbar fixation can maximize rigidity and promote development of bony fusion.

Objectives: To compare the utility of three anterior spinal instrumentation systems for stabilizing the lumbar spine.

Genetic instability induced by the tumor microenvironment.

The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. To determine the genetic consequences of growth under these conditions, we used a tumorigenic cell line carrying a recoverable, chromosomally based lambda phage shuttle vector designed to report mutations without the need for genetic selection of mutant cells. The cells were grown in parallel either in culture or as tumors in nude mice.

Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene.

Surgical exposure of the dorsal proximal third of the radius: how vulnerable is the posterior interosseous nerve?

This study determined the distance between the posterior interosseous nerve and the bicipital tuberosity of the radius in cadavers. Both elbows of 15 preserved cadavers were dissected (eight male cadavers, seven female cadavers). The most prominent point of the radial tuberosity was located, and the shortest distance from this area to the posterior interosseous nerve was measured.

Biomechanical analysis of lumbosacral fixation.

Study Design: The authors sought to measure the stiffness at the lumbosacral junction when it has been immobilized by means of two different posterior fixation systems in conjunction with three different anterior interbody fixation techniques. The information obtained provides a foundation for determining how methods of lumbosacral spinal fixation can maximize rigidity and improve fusion rates at this clinically important anatomic site.

Objectives: To determine which spinal instrumentation systems, alone or in combination, provide the most stability at the lumbosacral junction.

Rec-A protein-mediated irreversible fixation of an oligodeoxyribonucleotide to specific site in DNA.

RecA protein can polymerize on an oligodeoxyribonucleotide to form a filament that finds its homologous sequence in double-stranded DNA. When such an oligonucleotide is linked to psoralen, a photoactivatable DNA intercalator, it irreversibly binds to the homologous site in double stranded DNA as a result of psoralen photoadduct formation at thymidines. The relative efficiency of specific vs.

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells.

The use of bone allografts in the spine.

Bone allografts are commonly used in spinal surgery. Structural allografts placed anteriorly in the spine may be used as interbody grafts or as strut grafts spanning multiple segments. Posterior allografts are used to supplement autologous bone for spinal fusions in patients who lack sufficient host bone and to avoid significant donor site morbidity.

Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair.

When mammalian cells were treated with triplex-forming oligonucleotides of sufficient binding affinity, mutations were specifically induced in a simian virus 40 vector contained within the cells. Triplex-induced mutagenesis was not detected in xeroderma pigmentosum group A cells nor in Cockayne's syndrome group B cells, indicating a requirement for excision repair and for transcription-coupled repair, respectively, in the process. Triplex formation was also found to stimulate DNA repair synthesis in human cell extracts, in a pattern correlating with the inhibition of transcription in such extracts.

Triplex-mediated, in vitro targeting of psoralen photoadducts within the genome of a transgenic mouse.

Light-activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double-stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes.

Tissue specificity of spontaneous point mutations in lambda supF transgenic mice.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 x 10(-5)), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.

Frequent spontaneous deletions at a shuttle vector locus in transgenic mice.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from several different lines of transgenic mice were analyzed. One mouse line, 1139, was identified in which the frequency of spontaneous mutations was unusually high (3.

Pediatric spinal infections.

A high index of suspicion for spinal infection and an appropriate and prompt diagnosis are essential for the treatment of pediatric spinal infections. A 3-week course of antibiotics and supportive therapy is effective in the majority of cases of discitis, the most common pediatric spinal infection. Patients who are not toxic may be treated with bracing, or with casting alone in many cases.

p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells.

To study the pathways associated with genomic instability in cancer, we examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-risk E6 and E7 bind and functionally inactivate tumor suppressor proteins p53 and Rb, respectively, and both disrupt the G1 arrest in response to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower affinity than high-risk E6 and fail to mediate p53 degradation or to disrupt the G1 checkpoint.

Induction of p53 in mouse cells decreases mutagenesis by UV radiation.

The tumor suppressor protein, p53, is proposed to have a critical role in maintaining the integrity of the genetic material. It has been established that p53 induces a cell cycle block in the G1 phase upon cellular DNA damage. Recent evidence also indicates the involvement of p53 directly and indirectly in nucleotide excision repair (NER).

Altered repair of targeted psoralen photoadducts in the context of an oligonucleotide-mediated triple helix.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplex-directed lesion.

Mutagenesis by 8-methoxypsoralen and 5-methylangelicin photoadducts in mouse fibroblasts: mutations at cross-linkable sites induced by offoadducts as well as cross-links.

Psoralens are used clinically in the treatment of several skin diseases, including psoriasis, vitiligo, and cutaneous T cell lymphoma. However, psoralen treatment has been associated with an increased risk of squamous cell carcinoma of the skin. To elucidate molecular events that may play a role in the psoralen-related carcinogenesis, we examined psoralen-induced mutagenesis in a mouse fibroblast cell line carrying a recoverable, chromosomally integrated lambda phage shuttle vector.

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site.

Frequent T:A-->G:C transversions in X-irradiated mouse cells.

Ionizing radiation is a known carcinogen and teratogen. However, the point mutations produced by ionizing radiation in mammalian cells have not been fully characterized. Determination of a characteristic spectrum of X-ray-induced mutations in mammalian cells could provide clues to cellular repair processes and could serve as a marker of individual exposure to radiation.

Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations.

Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide.

Triple-helical DNA can be formed by oligonucleotides that bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-forming oligonucleotides have been used to inhibit gene expression by blocking transcription factor access to promoter sites in transient expression assays. In an alternative approach to genetic manipulation using triplex DNA, we show that triplex-forming oligonucleotides can be used to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and expression.

Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site.