Biomedical subjects
Publications and source records attributed to T J King.
Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.
Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
OBJECTIVE: The aim of this study was to evaluate the flexure strength under static and cyclic loading and the fracture toughness under static loading of six restorative ceramic materials. Specifically the intent was to compare four leucite (K2O.Al2O(3).4SiO2) strengthened feldspathic (pressable) porcelains (OA, OI, E, and FP) to a low fusing feldspathic porcelain (F) and an experimental (EC) lithium disilicate containing ceramic. METHODS: All materials were tested as a control in air and distilled water (without aging) and after three months aging in air or distilled water to determine flexure strength and fracture toughness. A staircase approach was used to determine the cyclic flexure strength. EDS and SEM analysis were conducted on the polished and fracture surfaces. RESULTS: The mean flexure strength for the controls in air and water (without aging or cyclic loading) ranged from 67 to 99 MPa, except the experimental ceramic that was twice as strong with a mean flexure strength of 191-205 MPa. For the mean fracture toughness, the range was 1.1-1.9 MPa/m0.5 with the experimental ceramic being 2.7 MPa/m0.5. The effect of testing in water and aging for three months caused a moderate reduction in the mean flexure strength (6-17%), and a moderate to severe reduction in the mean fracture toughness (5-39%). The largest decrease (15-60%) in mean flexure strength was observed when the samples were subjected to cyclic loading. SIGNIFICANCE: The lithium disilicate containing ceramic had a significantly higher flexure strength and fracture toughness when compared to the four pressable leucite strengthened ceramics and the low fusing conventional porcelain. All of the leucite containing pressable ceramics did provide an increase in mean flexure strength (17-19%) and mean fracture toughness (3-64%) over the conventional feldspathic porcelain. Further, the influence of testing environment and loading conditions implies that these ceramic materials in the oral cavity might be susceptible to cyclic fatigue, resulting in a significant decrease in the survival time of all-ceramic restorations.
A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.
A 4-year-old, entire female, German Shepherd Dog was referred with a 3-month history of right foreleg lameness that partially responded to nonsteroidal anti-inflammatory and antimicrobial therapy. The bitch lost weight, was polydipsic and had reduced exercise tolerance. On referral, the animal was in poor condition, pyrexic and exhibited moderate pain on full extension of the right shoulder. Blood, urine and joint fluid were obtained and radiographs were taken of the right shoulder and chest. The bitch was lymphopaenic, hyperfibrinogenaemic, hyperglobulinaemic, mildly azotaemic, mildly proteinuric and isosthenuric. Branching fungal hyphae were present in the urine. On radiography, the thorax contained a large ventral mediastinal mass and the humeral head had extensive areas of radiolucency. An aspirate from the right humeroscapular joint exhibited branched fungal hyphae and numerous neutrophils and macrophages. A diagnosis of disseminated mycosis was made and euthanasia was performed. At necropsy, numerous caseating granulomas were present, especially in the kidneys, adrenal glands, heart and lymph nodes. Extensive osteomyelitis involved the head of the right humerus, the sternebrae and the fifth intervertebral disc. Fungal hyphae were detected in sections of granulomas in all affected organs and a diagnosis of disseminated fungal granulomatosis was made. Aspergillus deflectus was readily isolated from affected lymph nodes, but confirming its identity as A deflectus using standard procedures proved difficult. The identity of the fungus was finally confirmed by sequencing part of the 185 rRNA of the isolate. This is the first report in Australia of a disseminated mycosis caused by A deflectus. Previously, the involvement of A deflectus as a cause of disseminated mycosis was limited to 5 cases from the West Coast of the USA, four of which occurred in German Shepherd Dogs.
9-cis beta-Carotene was extracted from a commercial extract of the algae Dunaliella salina (Betatene), and its actions on proliferation and gene expression were examined in murine 10T1/2 cells and human HaCaT keratinocytes. The 9-cis isomer was less active than all-trans beta-carotene in reducing proliferation and in upregulating expression of connexin 43 in 10T1/2 cells. However, it had comparable ability to suppress carcinogen-induced neoplastic transformation. When tested in HaCaT cells in organotypic culture, it was less active in inducing connexin 43 expression and suppressing expression of keratin K1. In this assay the all-trans isomer was highly active at 10(-6) M, whereas 10(-5) M 9-cis beta-carotene was required to produce a comparable effect. Only small reductions in expression of the basal keratin 5 were seen. All-trans and 9-cis retinoic acids, potential metabolites of beta-carotene isomers, were studied in the same systems. In contrast to the carotenoids, the 9-cis isomer of retinoic acid was approximately 10-fold more active in suppressing neoplastic transformation and inducing connexin 43 expression in both cell types than the all-trans isomer. The retinoic acid isomers were about equipotent in suppressing K1 expression. Cellular levels of 9-cis beta-carotene were approximately 3.5-fold lower than levels of all-trans beta-carotene, suggesting that part, but not all, of this decreased activity of the 9-cis isomer was due to decreased cell uptake. Thus 9-cis beta-carotene is consistently less active than the all-trans isomer; that 9-cis retinoic acid is, in general, much more potent than the all-trans isomer suggests little or no conversion from the carotenoid to the retinoid under these culture conditions.
Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.
The intent of this study was to determine pullout (shear) strength of carbon and stainless steel serrated posts from teeth. The tooth-posts groups were controls, thermal-cycled, load-cycled (load applied at either a 45 degrees angle or along the long axis of the post), and thermal- and load-cycled. Human extracted third molars were embedded in denture acrylic. Post space was prepared and the posts cemented with a resin cement according to manufacturer's instructions. The testing was in tension at a loading rate of 2 mm/min. The pullout (shear) strengths (MPa+/-SD) for the carbon posts were: controls 6.1+/-3.3; load-cycled 4.3+/-2.2; and thermal-cycled 4.2+/-2.2. The thermal-cycled and then load-cycled carbon posts all fractured during testing and were unable to be evaluated for shear strength. The pullout (shear) strengths (MPa+/-SD) for the stainless steel posts were: controls 6.6+/-2.9; load-cycled 7.1+/-3.2; thermal-cycled 4.8+/-2.0; and thermal load-cycled 6.8+/-3.5. The statistical analysis indicated no significant difference in the pullout (shear) strength between any of the post groups tested. Two different operators then prepared specimens using carbon posts abraded with 50 microm alumina and non-abraded carbon posts. The alumina abrasion caused a decrease in the post bond strength with the alumina particles impinging on the carbon fibers resulting in their fracture. The significant difference between bond strengths was attributed to each operator's years of clinical experience.
Explore the source record for details and available documents.
Our goal in this study was to assess the feasibility and safety of performing an intracorporeal laparoscopic jejunal harvest. The initial technique was developed and refined in a pig and then a dog model. In the animal studies, careful dissection of the jejunal flap with its feeding vessel was accomplished along with an intracorporeal anastomosis. The laparoscopic dissection was facilitated by temporarily anchoring the jejunal flap to the anterior abdominal wall and transilluminating the mesentery. We present the first case report of a patient who underwent a laparoscopic jejunal harvest, intracorporeal small bowel anastomosis, and a microvascular anastomosis in the neck for reconstruction of the laryngopharynx.
Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.
A phenotypic analysis on synovial fluid cells from the carpal and tarsal joints of sheep with visna virus-induced inflammatory synovitis was performed. The results showed increased representation of cells bearing lymphocyte, macrophage, and dendritic cell markers compared to equivalent synovial fluid cells from normal uninfected age-matched controls. In infected sheep, CD8+ T cells tended to predominate over CD4+ cells, while the numbers of gamma delta T cells varied from being absent in some samples to constituting the major T cell subset in others. B cells were found in relatively smaller numbers. Analysis of the large mononuclear cells showed that they stained with monoclonal antibodies that recognize macrophages and afferent lymph dendritic cells. Major histocompatibility complex (MHC) class II+ macrophage/dendritic cells were found in normal joints, but significantly elevated proportions of such cells were present in the carpal joints of infected sheep. The intensity of MHC class II staining was also significantly elevated in infected animals compared to control animals. A high proportion of these cells also stained for CD1 in both normal and infected animals, but were significantly elevated in number in the carpal joints of infected sheep. The elevated proportion of cells expressing molecules associated with accessary cell function and the increase in the numbers of accessory molecules per cell suggests an enhanced capacity for presenting antigen to a variety of T cell subsets within the joints of infected sheep, which could initiate or perpetuate potentially damaging local synovial inflammatory responses.
The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.
Explore the source record for details and available documents.
X-ray crystallographic studies on synthetic DL-quisqualic acid and the corresponding carbon analogue have revealed pyramidal and almost planar geometries respectively for the ring nitrogen atoms carrying the alkyl side chain. Semi-empirical molecular orbital calculations on methyl-substituted model systems predict ring geometries in close agreement with experimentally observed data. The calculated energy barriers between the two enantiomeric forms (invertomers) of the oxadiazolidine systems along with some physical data would suggest that such forms are rapidly interconverting.