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Biomedical subjects

A J Watson

Publications and source records attributed to A J Watson.

At least 37 records · Page 2Linked to original sources
PubMed2001

A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori.

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.

Acid Phosphatase↗
PubMed2001

O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells.

OBJECTIVE: Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen. METHODS: Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. RESULTS: Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells. CONCLUSION: These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.

Alkylation↗
PubMed2001

Cyclooxygenase-2 and prostaglandin E(2)(PGE(2)) receptor messenger RNAs are affected by bovine oocyte maturation time and cumulus-oocyte complex quality, and PGE(2) induces moderate expansion of the bovine cumulus in vitro.

Expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) receptor 2 (EP2) are necessary for rodent cumulus expansion in vivo. Prostaglandin E(2) receptor 3 (EP3) has been detected in bovine preovulatory follicles and corpora lutea. The current experiments examined the effect of PGE(2) on bovine cumulus expansion in vitro and expression of COX-2, EP1, EP2, EP3, and EP4 mRNAs in bovine cumulus-oocyte complexes (COCs) at 0, 6, 12, 18, and 24 h time points during maturation in vitro. Concentrations of PGE(2) above 50 ng/ml resulted in moderate cumulus expansion of bovine COCs, but expansion did not occur in the absence of serum. COX-2 mRNA expression increased in bovine COCs at 6 h and 12 h of maturation, then decreased. EP2 mRNA was detectable by reverse transcription-polymerase chain reaction at all time points. EP3 mRNA expression increased in COCs from 0 to 6 h and remained at this higher level through the culture period. Very low levels of EP4 mRNA expression were detectable, but EP1 was not detected in bovine COCs. Because EP receptor mRNAs and COX-2 mRNA are expressed in bovine COCs, there exists the potential for a prostaglandin autocrine/paracrine regulatory pathway during oocyte maturation. Differential expression of the EP3 mRNA among varying COC classes indicates that this gene product may be a useful marker of oocyte competence. Although the PGE(2) pathway is involved in cumulus expansion, serum factors are required to mediate PGE(2)-induced expansion.

Animals↗
PubMed2001

Enhancement of Mycobacterium tuberculosis-induced tumor necrosis factor alpha production from primary human monocytes by an activated T-cell membrane-mediated mechanism.

Mycobacterium tuberculosis alone induces small, donor-variable amounts of tumor necrosis factor alpha (TNF-alpha) from primary human monocytes in vitro. However, TNF-alpha release is increased 5- to 500-fold when fixed activated T cells (FAT) or their isolated, unfixed membranes are added to this system. This FAT-induced synergy was at least as potent as that induced by gamma interferon (IFN-gamma) at 100 U/ml. FAT-enhanced TNF-alpha production is at least in part transcriptionally mediated, as reflected by quantitative changes in TNF-alpha mRNA between 2 and 6 h poststimulation. Unlike IFN-gamma-cocultured cells, FAT-treated monocytes appeared not to have enhanced TNF-alpha message stability, suggesting that de novo transcription may be involved in this effect. Furthermore, M. tuberculosis alone induced only minimal DNA binding of monocyte NF-kappaB, but cells treated with M. tuberculosis and FAT potentiated NF-kappaB activity more effectively. It is therefore possible that one mechanism by which FAT synergize with M. tuberculosis to stimulate TNF-alpha production is via NF-kappaB-enhanced transcription. These data strongly suggest that in the interaction of cells involved in the immune response to M. tuberculosis, T-cell stimulation of monocyte TNF-alpha production involves a surface membrane interaction(s) as well as soluble mediators.

Cell Membrane↗
PubMed2001

Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR.

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Animals↗
PubMed2000

Celecoxib prevents tumor growth in vivo without toxicity to normal gut: lack of correlation between in vitro and in vivo models.

Nonsteroidal anti-inflammatory drugs have potential for use in the prevention and/or treatment of colorectal cancer. We have studied the cytotoxic effect of a specific COX-2 inhibitor, celecoxib, against LLC, HCA-7, and HCT-15 cells grown in cell culture and have compared these results with its effect on HCA-7 cells grown as xenografts in nude mice. "High-dose" celecoxib (>20 microM) reduced the viability of all three cell lines in vitro as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometric analysis demonstrated that this loss of viability was attributable to the induction of apoptosis. Significantly, concentrations of the drug <10 microM had no effect on cell viability in vitro. The cytotoxic effects of high-dose celecoxib were independent of COX-2 inhibition because similar effects were observed in cox-2 (+/+), cox-2 (+/-) and cox-2 (-/-) fibroblasts. A plasma concentration of 2.3+/-0.7 microM was achieved when celecoxib (1250 mg/kg of chow) was fed to animals ad libitum. Despite a lack of toxicity at 2-3 microM celecoxib in vitro, there was attenuation of HCA-7 xenograft growth in vivo. Celecoxib had no effect on apoptosis, cell division, or the epithelial architecture of the normal gut in treated mice. These results support the need for additional clinical evaluation of celecoxib for treatment and/or prevention of colorectal cancer in humans.

Animals↗
PubMed2000

A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization.

Changes in iron supply to oceanic plankton are thought to have a significant effect on concentrations of atmospheric carbon dioxide by altering rates of carbon sequestration, a theory known as the 'iron hypothesis'. For this reason, it is important to understand the response of pelagic biota to increased iron supply. Here we report the results of a mesoscale iron fertilization experiment in the polar Southern Ocean, where the potential to sequester iron-elevated algal carbon is probably greatest. Increased iron supply led to elevated phytoplankton biomass and rates of photosynthesis in surface waters, causing a large drawdown of carbon dioxide and macronutrients, and elevated dimethyl sulphide levels after 13 days. This drawdown was mostly due to the proliferation of diatom stocks. But downward export of biogenic carbon was not increased. Moreover, satellite observations of this massive bloom 30 days later, suggest that a sufficient proportion of the added iron was retained in surface waters. Our findings demonstrate that iron supply controls phytoplankton growth and community composition during summer in these polar Southern Ocean waters, but the fate of algal carbon remains unknown and depends on the interplay between the processes controlling export, remineralisation and timescales of water mass subduction.

Atmosphere↗
PubMed2000

Effect of iron supply on Southern Ocean CO2 uptake and implications for glacial atmospheric CO2.

Photosynthesis by marine phytoplankton in the Southern Ocean, and the associated uptake of carbon, is thought to be currently limited by the availability of iron. One implication of this limitation is that a larger iron supply to the region in glacial times could have stimulated algal photosynthesis, leading to lower concentrations of atmospheric CO2. Similarly, it has been proposed that artificial iron fertilization of the oceans might increase future carbon sequestration. Here we report data from a whole-ecosystem test of the iron-limitation hypothesis in the Southern Ocean, which show that surface uptake of atmospheric CO2 and uptake ratios of silica to carbon by phytoplankton were strongly influenced by nanomolar increases of iron concentration. We use these results to inform a model of global carbon and ocean nutrients, forced with atmospheric iron fluxes to the region derived from the Vostok ice-core dust record. During glacial periods, predicted magnitudes and timings of atmospheric CO2 changes match ice-core records well. At glacial terminations, the model suggests that forcing of Southern Ocean biota by iron caused the initial approximately 40 p.p.m. of glacial-interglacial CO2 change, but other mechanisms must have accounted for the remaining 40 p.p.m. increase. The experiment also confirms that modest sequestration of atmospheric CO2 by artificial additions of iron to the Southern Ocean is in principle possible, although the period and geographical extent over which sequestration would be effective remain poorly known.

Atmosphere↗
PubMed2000

Differential involvement of Na(+),K(+)-ATPase isozymes in preimplantation development of the mouse.

Na(+),K(+)-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the alpha1 and beta1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional alpha subunit isoform (alpha3) and the remaining two beta subunit isoforms are also present in preimplantation embryos. Whereas alpha3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for beta2 and beta3 mRNAs. Immunoblot analyses confirmed that these subunits are present in cavitating embryos. Using confocal immunofluorescence microscopy we found that alpha1 and beta1 subunits are concentrated in the basolateral membranes of the trophectoderm while being equally distributed in plasma membranes of the inner cell mass. In contrast, alpha3, beta2, and beta3 subunits were not detected in plasma membranes. Our current assessment, therefore, is that as many as six isozymes of Na(+),K(+)-ATPase could be involved in preimplantation development although it is primarily the alpha1beta1 isozyme that is responsible for blastocoel formation. Our findings imply that the regulation of sodium transport within the preimplantation mouse embryo is more complex than had been appreciated.

Animals↗
PubMed2000

Characterization of a bovine cDNA encoding citrate synthase, and presence of citrate synthase mRNA during bovine pre-attachment development.

Citrate synthase is a key regulatory metabolic enzyme that catalyzes the first step in the tricarboxylic acid (TCA) cycle, the synthesis of citrate from acetyl coenzyme A and oxaloacetate. Aerobic metabolism via the TCA cycle is high in bovine embryos at the 4-cell stage then decreases until the compact morula stage before increasing at the expanded blastocyst stage. This study characterizes the presence of citrate synthase mRNA in bovine pre-attachment embryos to determine if a variation in mRNA transcript expression patterns is associated with previous reports of the patterns of TCA cycle activity. The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect citrate synthase mRNA from the 1-cell to blastocyst stage of bovine embryo development, and in embryos cultured under either an atmosphere of 5% CO(2) in air or 5% CO(2)/5% O(2)/90%N(2). The nucleotide sequence encoding citrate synthase was determined from bovine heart cDNA by the rapid amplification of cDNA ends (RACE) technique. This 1455-bp nucleotide fragment contained an open reading frame that encoded a deduced protein of 466 amino acids. The bovine nucleotide sequence was 92.1% and 93.8% identical to the human and porcine coding sequence, respectively. The amino acid sequence predicted from the bovine sequence is 95.1% identical to the human sequence and 96.3% identical to the porcine sequence. The porcine sequence contains a stop codon that results in a peptide truncated by 2 amino acids. The detection of citrate synthase transcripts from the 1-cell to blastocyst stage demonstrates that the decrease in TCA cycle activity observed following the 4-cell stage is not associated with an absence of citrate synthase mRNA.

Amino Acid Sequence↗
PubMed2000

Assessment by differential display-RT-PCR of mRNA transcript transitions and alpha-amanitin sensitivity during bovine preattachment development.

The objectives of this study were to compare patterns of mRNA expression, investigate the onset of transcription, and isolate stage-specific and alpha-amanitin-sensitive mRNAs during early bovine development by differential-display-reverse transcription-polymerase chain reaction (DD-RT-PCR). Embryos representing a preattachment developmental series from the 1-cell to the expanded/hatched blastocyst stage were cultured in synthetic oviduct fluid medium + citrate and amino acids (cSOFMaa) with and without alpha-amanitin (100 microg/mL) for 4 and 12 hr. mRNA profiles were displayed by DD-RT-PCR using 5' primers A and N. Total conserved cDNA banding patterns varied according to embryo stage with cDNA band numbers declining during early cleavage stages compared to oocyte values and then increasing in total number from the 6-8-cell stage through to the blastocyst stage. A cDNA banding pattern was established at the 8-16-cell stage that was largely unchanged through to the blastocyst stage. These findings with respect to cDNA banding patterns were conserved between oligo primer sets and experimental replicates. alpha-Amanitin sensitivity was first detected at the 2-5-cell stage but became predominant following the 6-8-cell stage of development to eventually affect the appearance of up to 40% of all cDNA bands by the blastocyst stage. A 12 hr alpha-amanitin treatment was required to effectively block (3)H-uridine incorporation into mRNA in blastocyst stage embryos. Several stage-specific and alpha-amanitin-sensitive cDNAs were isolated and they will be a focus for future studies. In conclusion, DD-RT-PCR is an effective tool for contrasting gene expression patterns and isolating uncharacterized mRNA transcripts during bovine early development. Mol. Reprod. Dev. 55:152-163, 2000.

Amanitins↗
PubMed2000

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei.

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.

Animals↗
PubMed2000

mRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

Animals↗
PubMed2000

Pyrrolidinedithiocarbamate increases the therapeutic index of 5-fluorouracil in a mouse model.

BACKGROUND & AIMS: The thiol-containing antioxidant pyrrolidinedithiocarbamate (PDTC) enhances the cytotoxic efficacy of 5-fluorouracil (5-FU) against human colorectal cancer cell lines in vitro and in vivo. This process appears to be mediated by a sustained increase in p21 expression, independent of p53 function, resulting in growth arrest and apoptosis. We determined whether PDTC augmented 5-FU intestinal toxicity in non-tumor-bearing mice. METHODS: Apoptotic and mitotic indices were measured in the small and large intestine on a cell positional basis at intervals throughout the 72-hour period after administration of 5-FU (40 mg/kg) and PDTC (250 mg/kg). The proportion of crypts regenerating after 5-FU (600-1200 mg/kg) and PDTC (500 mg/kg) was also measured. RESULTS: 5-FU therapy induces substantial apoptotic cell death with simultaneous inhibition of mitotic activity within the small and large intestinal epithelium. PDTC reduces 5-FU-induced apoptotic events in the colon by 49%, predominantly among clonogenic stem and transit cells while promoting the early recovery of mitotic activity. As a consequence, PDTC increased the proportion of regenerating colonic crypts after 5-FU therapy. PDTC did not, however, significantly modulate 5-FU toxicity in the small intestine. CONCLUSIONS: PDTC does not augment the intestinal toxicity of 5-FU and actually protects the colonic mucosa. These results support further investigation of PDTC and related compounds as treatments for colorectal cancer.

Animals↗
PubMed2000

A prospective study examining the association between the symptoms of anxiety and depression and severity of urinary incontinence.

OBJECTIVE: To investigate the association of the presence or absence of the symptoms of anxiety and depression compared with the 48 h pad test as an objective measure of incontinence. DESIGN: Prospective study. SETTING: Urodynamics clinic in a large teaching hospital. SUBJECTS: All patients with urinary incontinence attending for urodynamic assessment from 23.4.96 to 29.10.96. INTERVENTIONS: 48 h pad test, Hospital Anxiety and Depression scale (HAD scale). MAIN OUTCOME MEASURES: Urodynamic diagnosis of cause of incontinence. Urinary loss over 48 h as measured by weight change in pads. Presence of symptoms of anxiety or depression as defined by HAD scale score of 8 or more. RESULTS: Urodynamic investigation was performed for incontinence on 133 patients. Of these 127 (95.4%) completed the HAD scale questionnaire. Of the 43 patients (32.2%) who returned the pads 18 (41.8%) patients were found to have symptoms of anxiety and six patients (13.9%) had symptoms of depression. Patients with symptoms of anxiety had lower mean measured urinary loss over a 48 h period compared to women with no symptoms of anxiety (median loss 44.2 ml range 6.8-622.4 versus 97.1 ml range 8.2-4384.4 ml) (P=0.05). There was no significant association between symptoms of depression and pad test results. CONCLUSIONS: Patients presenting with incontinence who have symptoms of anxiety are on average less incontinent compared to than those without symptoms of anxiety. It suggests that anxious patients present with a lesser degree of incontinence than nonanxious patients.

Anxiety↗