Biomedical subjects
Publications and source records attributed to B C Lee.
Double pylorus (DP) or duplication of the pylorus is an uncommon condition that is either congenital or acquired. Acquired double pylorus (DP) results from a peptic ulcer eroding through and creating a fistula between the duodenal bulb and the distal stomach. We report a case of an acquired double pylorus in an adult gentleman that resulted from the erosion of a duodenal and prepyloric ulcer.
This study was conducted to establish an effective culture system for supporting in vitro development of cloned bovine embryos and to evaluate whether improved development in the optimal culture system could contribute to enhancing pregnancy and delivery outcomes after transfer. Enucleated oocytes at the metaphase II stage were reconstructed with serum-starved ear fibroblasts and cloned embryos were subsequently cultured for 168 h in vitro. In Experiment 1, cloned embryos were cultured in either modified Charles Rosenkrans 2 amino acid medium (mCR2aa) or modified synthetic oviduct fluid medium (mSOF). More (P < 0.05) 2-cell embryos (78% versus 92%), morulae (51% versus 69%) and blastocysts (2% versus 39%) were obtained after culture in mSOF than after culture in mCR2aa. In Experiment 2, cloned embryos were successively cultured in mSOF supplemented with various macromolecules during different periods of culture. A successive culture of oocytes in BSA-containing medium for 72 h and then in FBS-containing medium for the next 96 h yielded a higher rate of blastocyst formation (49% versus 25-36%) than other combinations (BSA to BSA or PVA to PVA, BSA or FBS). This macromolecule supplementation also significantly increased the number of total blastomeres (117.3 cells/blastocyst) and inner cell mass cells (ICM, 49.7 cells/blastocyst), and the ratio of ICM cells to trophoblast cells (TB, 0.98). In Experiment 3, a total of 85 blastocysts obtained from each 2-step culture were transferred individually to recipient cows at the end of the culture period and 32 pregnancies (38%) were diagnosed on Day 60 after transfer. However, no (P > 0.05) significant differences due to culture were apparent in the pregnancy outcome. Although six calves were produced using the 2-step culture regime of either BSA-BSA or PVA-FBS, no calves were produced using the successive culture of BSA then FBS, which optimized preimplantation development. In conclusion, mSOF has more potential to support the development of clone embryos than mCR2aa, and successive supplementation of BSA and FBS to mSOF further promotes blastocyst formation. However, enhanced development in vitro might not directly contribute to improving pregnancy outcomes.
AIMS/HYPOTHESIS: Genotype could influence vascular function. In some populations, Calpain 10 gene polymorphisms increase susceptibility to diabetes or insulin resistance. Alterations in microvascular function could contribute to insulin resistance. This study investigated whether polymorphisms in the Calpain-10 gene influence microvascular function. METHODS: Skin maximum microvascular hyperaemia to local heating on the dorsum of the foot (30 min at 43 degrees C) was measured by Laser Doppler Fluximetry in 37 healthy volunteers. All were normoglycaemic according to World Health Organisation criteria, normotensive and not on any medication. Four polymorphisms in the calpain-10 gene were typed: SNP-44, SNP-43, SNP-19, SNP-63. The SNP common to all the described high risk haplotypes is the G-allele at SNP-43. This intron 3 polymorphism appears to influence gene expression. Microvascular function was examined in relation to polymorphisms at this site alone as well as the effects of the known extended high risk haplotypes using the SNP's above. RESULTS: Maximum microvascular hyperaemia was increased in the 21 subjects with G/G genotypes at SNP-43 compared to the combined group of subjects ( G/ A genotype at SNP-43 ( n=12) + A/ A genotype at SNP-43 ( n=4)), and the minimum microvascular resistance was reduced 49.4 (39.6-94.2) vs 67.5 (39.1-107.3) mmHg/V, p=0.007). Haplotype analysis of the hyperaemic response revealed no significant differences between haplotypes. The two groups did not differ in terms of anthropometric measures, blood pressure, insulin resistance or glucose. CONCLUSIONS/INTERPRETATION: The polymorphism that confers susceptibility to Type II (non-insulin-dependent) diabetes mellitus in some populations is associated in United Kingdom Caucasians with enhanced microvascular function in the presence of normoglycaemia.
We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.
Between August and September 2000, five 2-7-year-old cows in Korea exhibited neurologic signs and were diagnosed as infected with Akabane virus based on the results of histopathology, immunohistochemistry, serology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss was noted in the brain and the ventral horn gray matter of the spinal cord. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigen was detected in the brain and spinal cord, mainly in degenerating neurons and glial cells. RT-PCR analysis revealed a target band of expected size in four cows. This is the first report on an outbreak of natural Akabane virus infection in adult cattle.
The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus. Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1). When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E. coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E. coli PE. In addition, when the PE of E. coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40%. The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E. coli PE/DOG > E. coli PE > DOPC. These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes.
The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.
The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.
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This study was conducted to examine whether preimplantation development of bovine ("HanWoo," Bos Taurus corcanae) oocytes reconstituted with ear fibroblasts could be improved by a modified procedure of fusion and activation. In Experiment 1, enucleated oocytes were reconstituted with ear fibroblast by a combined procedure of electric fusion and activation at either 24 or 28 hours after IVM. The 28 hours reconstitution yielded more blastocysts (4% vs. 21%, P = 0.0025) and higher ratio of blastocysts per 2-cell embryos (0.05 vs. 0.25, P = 0.003) than the 24 hours. In Experiment 2, enucleated oocytes were reconstituted by one of the three fusion and activation protocols; 1) a combined procedure of electric fusion and activation at 28 hours after IVM, 2) a combined procedure of electric fusion and chemical activation at 28 hours, and 3) a separate procedure of electric fusion at 24 hours and chemical activation at 28 hours. When compared two combined procedures, chemical activation with 5 microM ionomycin for 4 minutes did not promote embryo development and significantly reduced the fusion rate (42% vs. 53%, P = 0.0395). However, significant (P < 0.0113) increases in the development to 2-cell (90% vs. 70 to 74%) and blastocyst (47% vs. 7 to 13%) stages and in the ratio of blastocysts per 2-cell embryos (0.52 vs. 0.11 to 0.18) were obtained by a separate procedure of electric fusion and chemical activation than by the combined procedures. This separate protocol did not reduce the fusion rate compared with the combined procedures (58%). In conclusion, improved development of oocytes reconstituted with ear fibroblasts was achieved by undergoing a separate procedure of electric fusion and chemical activation 4 hours apart.
Biological signaling generally involves the activation of a receptor protein by an external stimulus followed by protein-protein interactions between the activated receptor and its downstream signal transducer. The current paradigm for the relay of signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins. However, recent results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the nature of structure-function relationships in signaling. Here we study the structural changes that occur upon activation of the blue light receptor photoactive yellow protein (PYP). Activation greatly reduces the tertiary structure of PYP but leaves the level secondary structure largely unperturbed. In addition, activated PYP exposes previously buried hydrophobic patches and allows significant solvent penetration into the core of the protein. These traits are the distinguishing hallmarks of molten globule states, which have been intensively studied for their role in protein folding. Our results show that receptor activation by light converts PYP to a molten globule and indicate stimulus-induced unfolding to a partially unstructured molten globule as a novel theme in signaling.
Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner-nuclear structures, splicing factor-rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF(65) and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread-like structure. The splicing factor-rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF-rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL-60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor-rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell.
A wavelet interpolation filter (WIF) is designed for the removal of motion artifacts in the ST-segment of stress ECGs. The WIF consists of two parts. One part is a wavelet transform that decomposes the stress ECG signal into several frequency bands using a Haar wavelet. The other part is an interpolation method, such as the spline technique, that is used to enhance the reconstruction performance of the signal decomposed by the wavelet transform. To evaluate the performance of the WIF, three indices are used: signal-to-noise ratio (SNR), reconstruction square error (RSE) and standard deviation (SD). The MIT/BIH arrhythmia database, the European ST-T database and the triangular wave are used for evaluation. A noisy ECG signal, corrupted by motion artifacts, is simulated by the addition of two types of random noise to the original ECG signal. For comparison, three indices for the other methods are also computed: mean, median and hard thresholding. The performance of the WIF shows that RSE, SNR and SD are 392.7, 18.3dB and 2.6, respectively, in the case of a noisy signal with an SNR of 7.1 dB. This result is much better than those for the other methods.
Our purpose of this study was to demonstrate the clinical potential and spatial resolution of a new MRI technique: high-resolution blood oxygen-level dependent venography (HRBV), in well-known intracranial vascular lesions, such as cavernous and venous angiomas, and venous sinus thrombosis. HRBV provides unique high-resolution information on veins without administration of contrast medium. The data are independent of conventional findings on MRI and potentially useful in characterising and demonstrating the architecture of vascular lesions of the brain.
Based on the use of Scutellaria baicalensis for the treatment of stroke in traditional Oriental medicine, the current study was carried out to evaluate neuroprotective effects of S. baicalensis after transient global ischemia using rat 4-vessel occlusion model. Methanol extracts from the dried roots of S. baicalensis (0.1-10 mg/kg) administered intra-peritoneally significantly protected CA1 neurons against 10 min transient forebrain ischemia as demonstrated by measuring the density of neuronal cells stained with Cresyl violet. Methanol extract of S. baicalensis inhibited microglial tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production, and protected PC12 cells from hydrogen peroxide-induced toxicity in vitro.
1. It has been shown that magnetic fields (MFs) affect a variety of biological effects in animal brains. There have been few experiments on the effects of MFs on sleep. Therefore, we investigated whether extremely low frequency (ELF) MFs affect the sleep induced by clonidine, a central alpha(2)-adrenoceptor agonist. Clonidine produced dose-related increase of the sleeping time and dose-related decrease of the onset time in 2-day-old chicks. 2. Exposure of chicks to MFs (5, 10, 20 G; for 3, 6, 9, 12 h) significantly increased the clonidine-induced sleep time as a direct function of intensity and duration of MF application. Clonidine reduced noradrenaline or tyrosine in the brain, an effect which was not further changed in animals exposed to MF. 3. To determine whether the gamma amino butyric acid A (GABA(A))/benzodiazepine (BZD) receptor system is involved in the decrease in clonidine-induced sleep caused by activation of central alpha(2)-adrenergic systems, we examined exposure of chicks to the effects of the BZD receptor antagonist flumazenil (0.5 mg kg(-1), i.p.) and GABA(A) antagonist bicuculline (0.1 mg kg-1, i.p.) on clonidine-induced sleep. Bicuculline and flumazenil inhibited the increase of clonidine-induced sleep time by MFs. Clonidine or MFs did not change GABA levels in the brain. 4. These results suggest that MFs can increase clonidine-induced sleep via a change of GABA(A) and BZD receptor system irrespective of the concentration of GABA or noradrenaline in the brain of 2-day-old chicks.
1. The putative effects of prenatal exposure to magnetic field (MF) have recently received much interest. In the present study, mice were exposed to a MF of 50 mT during gestation (0-19 days). 2. After the exposure was terminated, mothers and offspring were returned to normal laboratory conditions. We then determined changes in striatal levels of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the offspring. 3. Our results indicate that prenatal exposure to MF increases levels of DA and DOPAC in the striatum at 4, 8 and 12 weeks postnatally.
AIMS: Microvascular dysfunction occurs in Type 2 diabetes and in subjects with fasting hyperglycaemia. It is unclear whether this dysfunction relates to dysglycaemia. This study investigated in normogylcaemic individuals whether a genetic predisposition to diabetes, or indices of insulin resistance including endothelial markers, were associated with impaired microvascular function. METHODS: Maximum microvascular hyperaemia to local heating of the skin was measured using laser Doppler flowmetry in 21 normoglycaemic subjects with no family history of diabetes (Group 1) and 21 normoglycaemic age, sex and body mass index-matched offspring of two parents with Type 2 diabetes (Group 2). RESULTS: Although Group 2 had normal fasting plasma glucose and glucose tolerance tests, the 120-min glucose values were significantly higher at 6.4 (5.3-6.6) mmol/l (median (25th - 75th centile)) than the control group at 4.9 (4.6-5.9) mmol/l (P = 0.005) and the insulinogenic index was lower at 97.1 (60.9-130.8) vs. 124.0 (97.2-177.7) (P = 0.027). Skin maximum microvascular hyperaemia (Group 1: 1.56 (1.39-1.80) vs. Group 2: 1.53 (1.30-1.98) V, P = 0.99) and minimum microvascular resistance which normalizes the hyperaemia data for blood pressure (Group 1: 52.0 (43.2-67.4) vs. Group 2: 56.0 (43.7-69.6) mmHg/V, P = 0.70) did not differ in the two groups. Significant positive associations occurred between minimum microvascular resistance and indices of the insulin resistance syndrome; plasminogen activator inhibitor type 1 (R(s) = 0.46, P = 0.003), t-PA (R(s) = 0.36, P = 0.03), total cholesterol (R(s) = 0.35, P = 0.02), and triglyceride concentration (R(s) = 0.35, P = 0.02), and an inverse association with insulin sensitivity (R(s) = -0.33, P = 0.03). CONCLUSIONS: In normoglycaemic adults cutaneous microvascular vasodilatory capacity is associated with features of insulin resistance syndrome, particularly with plasminogen activator inhibitor type 1. A strong family history of Type 2 diabetes alone does not result in impairment in the maximum hyperaemic response. Diabet. Med. 18, 541-545 (2001)