Biomechanical evaluation of a veterinary suture anchor in the canine cadaver pelvis and femur.

A commercially available veterinary suture anchor was tested in the acetabula and femurs of canine cadavers. Size #2 suture anchor constructs were compared to a traditional screw and Teflon spiked washer constructs in a model of coxofemoral luxation repair. The screw/washer constructs failed at a higher maximum load than the #2 anchor constructs. In the acetabulum, significant differences in strength were also found in the position of the implant and in the direction of pull. The constructs in a more caudal position, and constructs pulled 90 degrees to the axis of insertion, failed at higher loads. The predominant mode of failure of the constructs was a suture failure. In the femur, size #5 suture anchors were used in a model of cranial cruciate ligament repair and collateral ligament repair. The anchor constructs failed predominantly by anchor pull-out in the distal femur. The constructs pulled 90 degrees to the axis of insertion were stronger than construcs pulled at 0 degrees to the axis of insertion. Varying the location of the implant in the femur did not affect the maximum load to failure.

Glucose tolerance and lipid profiles in dogs fed different fiber diets.

Increased dietary fiber is thought to have beneficial effects on health in humans. In diabetic dogs, a beneficial effect of fiber on glycemia has been suggested, while its effect on lipid profiles in dogs has not been described. The effects of different amounts and types of fiber on glucose tolerance and lipid concentrations were evaluated and compared with those of a standard maintenance ration in 30 healthy dogs. It was concluded that increased fiber intake had no influence on glucose in healthy dogs but it may have modulated lipid homeostasis.

Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.

Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability.

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis -acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.

Regional differences in the compaction of chromatin in human G0/G1 interphase nuclei.

The large-scale structure of chromatin corresponding to G- and R-bands in human G0/G1 interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1-1.5 Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.

DNA methylation associated with repeat-induced point mutation in Neurospora crassa.

Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.

Technetium-99m-nitroimidazole (BMS181321): a positive imaging agent for detecting myocardial ischemia.

UNLABELLED: A new technetium-99m-labeled nitroimidazole (BMS181321) has been proposed for positive imaging of myocardial ischemia. METHODS: An in vivo open-chest canine model of partial coronary occlusion and pacing-induced demand ischemia was used to correlate myocardial retention of BMS181321, following an intravenous injection at peak stress, with regional microsphere blood flow. Postmortem measurements of myocardial BMS181321 activity and flow were correlated with in vivo planar and ex vivo SPECT images. Myocardial and hepatic clearance of BMS181321 was derived from ROI analysis of serial planar images. RESULTS: Anaerobic metabolism was documented in the ischemic region by selective venous and arterial sampling for lactate and oxygen consumption. Normalized myocardial BMS181321 activity (165% +/- 42% nonischemic) in the central ischemic region (flow < 0.3 ml/min/gm) was significantly greater than activity in normal regions (p < 0.05). Quantitative circumferential analysis of SPECT images revealed a comparable increase in myocardial BMS181321 activity in the ischemic region. Sixty minutes after injection of BMS181321, liver activity was 423% of ischemic myocardial activity. CONCLUSION: BMS181321 was preferentially retained in ischemic but viable canine myocardium and was inversely related to regional myocardial blood flow. Although enhanced retention of BMS181321 was detectable by ex vivo SPECT imaging, an unfavorable heart-to-liver ratio was observed with in vivo planar imaging which may limit its use in clinical myocardial imaging.

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

Dense nonsymmetrical DNA methylation resulting from repeat-induced point mutation in Neurospora.

Cytosine methylation has been implicated in epigenetic control of gene expression in animals, plants, and fungi. It has been assumed that all methylation in eukaryotes is at symmetrical sequences such as CpG/GpC, because this can explain perpetuation of methylation states. Here the bisulfite genomic sequencing method was used to examine methylation in DNA from a Neurospora gene exposed to repeat-induced point mutation. 5-Methylcytosine was not limited to symmetrical sites and individual molecules showed different patterns and amounts of modification. The methylation extended beyond the mutated region and even beyond the edge of the duplicated segment.

Recurrence of repeat-induced point mutation (RIP) in Neurospora crassa.

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.

[Autologous fat transplantation in rats]. / Transplante autólogo de gordura em ratos.

The authors perform an experimental study in rats to demonstrate the integration of fat cells transplanted from the inguinal region to the dorsal region of the same animal. Histological studies were performed with the material removed and with the material injected. Final results show the transplanted fat cell integration after 360 days.