Elasticity and breaking strength of synthetic suture materials incubated in various equine physiological and pathological solutions.

REASONS FOR PERFORMING STUDY: Selection of suture material in equine surgery is often based on costs or subjective factors, such as the surgeon's personal experience, rather than objective facts. The amount of objective data available on durability of suture materials with regard to specific equine physiological conditions is limited. OBJECTIVES: To evaluate the effect of various equine physiological and pathological fluids on the rate of degradation of a number of commonly used suture materials. STUDY DESIGN: In vitro material testing. METHODS: Suture materials were exposed in vitro to physiological fluid, followed by biomechanical analysis. Three absorbable suture materials, glycolide/lactide copolymer, polyglactin 910 and polydioxanone were incubated at 37°C for 7, 14 or 28 days in phosphate-buffered saline, equine serum, equine urine and equine peritoneal fluid from an animal with peritonitis. Five strands of each suture material type were tested to failure in a materials testing machine for each time point and each incubation medium. Yield strength, strain and Young's modulus were calculated, analysed and reported. RESULTS: For all suture types, the incubation time had a significant effect on yield strength, percentage elongation and Young's modulus in all culture media (P<0.0001). Suture type was also shown significantly to influence changes in each of yield strength, percentage elongation and Young's modulus in all culture media (P<0.0001). While the glycolide/lactide copolymer demonstrated the highest Day 0 yield strength, it showed the most rapid degradation in all culture media. For each of the 3 material characteristics tested, polydioxanone showed the least variation across the incubation period in each culture medium. CONCLUSIONS: The duration of incubation and the type of fluid have significant effects on the biomechanical properties of various suture materials. These findings are important for evidence-based selection of suture material in clinical cases.

Which anatomical region determines a positive flexion test of the distal aspect of a forelimb in a nonlame horse?

REASONS FOR PERFORMING STUDY: The flexion test is used routinely as part of lameness and prepurchase examinations. However, little is known about the mechanisms that cause a positive response to a flexion test. OBJECTIVE: To determine which anatomical regions play a role in a positive outcome of a flexion test of the distal aspect of a forelimb in a nonlame horse. METHODS: Eight clinically sound Dutch Warmblood horses were subjected to a standardised flexion test (force 250 N, time 60 s) inducing a consistent lameness. To discriminate between different areas of the distal aspect of a forelimb, effects of various nerve blocks on the outcome of the flexion test were investigated. Low palmar digital, palmar at the abaxial aspects of the base of the proximal sesamoids, high palmar, ulnar and low 4-point nerve blocks were performed. Flexion test induced lameness was scored before and after each nerve block in separate sessions. RESULTS: The low palmar digital nerve blocks and nerve blocks of the palmar nerves at the abaxial aspect of the base of the proximal sesamoid bones had no significant effect on the flexion test induced lameness score. The ulnar, high palmar and, most dramatically, the low 4-point nerve blocks all caused a significant (P<0.05) reduction in the flexion test induced lameness score. CONCLUSIONS: Anatomical structures (soft tissue nor synovial structures) located distal to the metacarpophalangeal joint appear to contribute only minimally to the outcome of a positive flexion test of the distal aspect of a forelimb in a clinically nonlame horse. The structures in the region of, and including, the metacarpophalangeal joint appear to contribute most to a positive flexion test of the distal aspect of a forelimb in a nonlame horse. POTENTIAL RELEVANCE: The flexion test of the distal aspect of a forelimb may be sensitive for investigating the metacarpophalangeal joint region in horses free from lameness, but may be less relevant for structures distal to this region.

Combretastatin A-4 resistance in H460 human lung carcinoma demonstrates distinctive alterations in beta-tubulin isotype expression.

Tubulin isotype distribution may play a role in the development of anti-cancer anti-tubulin drug resistance as well as in drug efficacy and specificity. Stepwise selection was used to establish non-small cell lung carcinoma (NSCLC) H460 cells resistant to combretastatin A-4 (CA4), paclitaxel or vinblastine. The results demonstrated that the rate of CA4 drug resistance development was slower than that for paclitaxel. Western analysis demonstrated alterations in total beta-tubulin and classes I, III and IV tubulin isotypes among the resistant H460 cell lines. Class III beta-tubulin was significantly altered in all resistant cell lines. Cells resistant to paclitaxel, a structural stabilizer of microtubules, exhibited an increased expression while cells resistant to CA-4 and vinblastine, structural destabilizers of tubulin, demonstrated a reduction of the same isotype. To our knowledge, this is the first demonstration of resistance development and of the corresponding tubulin isotype response for the combretastatins.

Recombination between a 3-kilobase tobacco mosaic virus transgene and a homologous viral construct in the restoration of viral and nonviral genes.

Transgenic plants harboring various plant virus sequences have shown resistance to viral infections. An environmental risk associated with the use of these plants is the possibility of forming a novel virus by recombination between challenging viruses and transgenic viral mRNA. Two experiments were designed using tobacco mosaic virus (TMV) vectors and transgenic Nicotiana benthamiana to determine if recombinant viral RNA would be detectable. N. benthamiana was transformed with a nontranslatable portion of a TMV viral vector including part of the replicase gene, the movement protein gene, a gene for green fluorescent protein (GFP), and the coat protein gene. When transformed plants were inoculated with a TMV vector coat protein mutant which could not move efficiently through the host, recombinant RNA was detected in 32% of the infected plants, although virions were not detected. When transformed plants were infected with a TMV vector with a normal coat sequence but three base changes in the GFP sequence, no recombinant RNA or virions were detected. Thus, recombinant RNA between TMV RNA and host mRNA did not accumulate to detectable levels under nonselective conditions, and though recombinant RNA did accumulate in the presence of selective pressure, an encapsidated recombinant viral population did not develop.

Genome evolution of tobacco mosaic virus populations during long-term passaging in a diverse range of hosts.

The effects of host changes on plant virus genome evolution was studied by nucleotide sequencing. A single tobacco (Nicotiana tabacum cv. Xanthi) plant was inoculated with in vitro transcripts from a plasmid clone of tobacco mosaic tobamovirus (TMV). This initial viral population was then transferred 11-12 times in parallel populations in 7 plant host species (1-4 replicates each) over a period of 413-515 days. Virion RNA was then isolated, reverse transcribed, amplified, cloned in bacteria, and sequenced. Portions of the coat protein, movement protein, and replicase genes were sequenced. Fourteen unique mutations were detected from a total of 188 clones (35,607 bases) sequenced, indicating a relatively small overall mutation rate of 3.1 x 10(-4) nucleotide substitutions/base-year. A small Ka/Ks value of 0.09 was also found, indicating selection against amino acid changes. Eighty-five percent of the substitutions were transitions. A G'(ST) value of 0.7 for the coat protein gene suggested that host type affected sequence changes in this region of the genome, but chi(2) analysis did not support this conclusion. This is the first study using sequencing to compare representative sample sections of a plant viral genome following a major selective disturbance such as extended passaging in an alternate host.

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Low level of genetic drift in foreign sequences replicating in an RNA virus in plants.

The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous replication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamiana, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on passaging to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.