Biomedical subjects
Publications and source records attributed to L Lin.
This paper reported the studies on morphological and histological diagnostic characteristics and powder feature the root of Aralia decaisneana Hance. Chemical analysis indicates essential oil from root cortex contains at least 16 constituents which trans-caryophllene, 9,12-octadecaienoic acid, etc. are the major ingredients.
OBJECTIVE: To study the character of non-selective cation channel in human umbilical cord vein endothelial cell line ECV304 and the effect of tumor necrosis factor-alpha (TNF-alpha) on channel activity. METHOD: Patch clamp technique was used to record single channel and whole cell currents. RESULTS: This channel can be activated by calcium, channel conductance was (19.74 +/- 2.35) pS and its Po was 0.260 +/- 0.006 (n = 5). 100 U/ml TNF-alpha inhibited channel activity, showing that conductance reduced to (10.69 +/- 4.68) pS and Po reduced to 0.230 +/- 0.051 (n = 5). Results in whole-cell configuration were consistent with that in cell-attached patches. Currents were obviously blocked by Cl- channel inhibitor anthracene-9-carboxylic acid (A9C). In order to testify the character of this channel further, KCl and NaCl were exchanged by K(+)-Aspartate in pipette solution. Under this condition, the single channel activities as before were recorded again. Its conductance was (18.33 +/- 2.98) pS(n = 8), and TNF-alpha as well as A9C could inhibit channel activity. But this channel activity could not be blocked by another Cl- channel blocker Zn2+. In the condition of K-Asparate replaced by CsCl in solution, identical channel activity as before could not be recorded. It seemed that Cs+ could not pass through this cation channel. CONCLUSION: It is inferred that calcium-activated non-selective-action channel recorded in endothelial cell ECV304, can be inhibited by TNF-alpha and A9C.
OBJECTIVE: To unravel the biological significance of the alternative splicing of de novo methyltransferase 3b, expression of the Dnmt3b gene in various tissues and developmental stages was investigated in postnatal mice. METHODS: RT-PCR and capillary electrophoresis were employed to analyze the alternative splicing pattern of Dnmt3b in tissues of newborn and adult mice. The results had been further reaffirmed by repeating and statistics analysis. Bioinformatics tools were used to predict the structure and hydrophobicity of the Dnmt3b exon10 coding sequence. RESULTS: Isoform with Dnmt3b exon10 was the abundant form in lung of newborn mice and in liver of both newborn and adult mice, while in other tissues of newborn and adult mice, the spliced isoform was presented as the predominant one. Peptide encoded by Dnmt3b exon10 was mainly random coil on the surface of Dnmt3b protein. CONCLUSIONS: The data demonstrate that the specific expression of Dnmt3b exists in tissues and developmental stages of postnatal mice. The alternative splicing of the exon 10 of Dnmt3b is possibly involved in the regulation of Dnmt3b's catalytic function. These results provide an insight into the developmental regulation and physiological function of the alternative splicing of the Dnmt3b gene.
OBJECTIVE: To investigate the effect of angiotensin II (A II) on maxi-conductance calcium-activated potassium channel(BKCa) activity in ECV304 cell membrane, and the possible involvement of G-protein. METHODS: Cell-attached configuration of patch clamp technique was employed to record the current of BKCa in ECV304. RESULTS: 10(-7) mol/L angiotensin II inhibited the BKCa activity. Current amplitude and open probability were decreased, open time was shortened and close time was increased. G-protein activation could eliminate the inhibition of A II. CONCLUSIONS: A II significantly decreased the activity of BKCa in ECV304, which might depolarize the membrane, and the stability of membrane might change. Thus endothelial cell dysfunction might be induced. G-protein activation was involved in the regulation of the above A II inhibition process.
OBJECTIVE: Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. METHOD: Using the cell-attached configuration of patch clamp technique. RESULTS: (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. CONCLUSIONS: TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.
Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.
nfkb2 encodes two members of the NF-kappa B/Rel family of proteins: p52 and p100. The p100 polypeptide has been proposed to serve as a precursor of p52, which corresponds to the N-terminal half of p100. While p52 functions as a Rel transcription factor, the larger p100 protein acts as a cytoplasmic inhibitor of select NF-kappa B/Rel transcription factor complexes. Because of their distinct functions, we have studied the biochemical basis for the production of these two nfkb2-derived gene products. Like the p50 product of the nfkb1 gene, p52 is principally generated in a cotranslational manner involving proteolytic processing by the proteasome. The generation of p52 is dependent on a glycine-rich region (GRR) located upstream of the p52 C-terminus, and repositioning of this GRR alters the location of proteasome processing. In most cells, small amounts of p52 are produced relative to the levels of p100, unlike the usually balanced production of nfkb1-derived p50 and p105. Using p100/p105 chimeras containing different segments of the nfkb1 and nfkb2 genes, we have found that diminished p52 processing is a property conferred by peptide sequences located downstream of the GRR, flanking the site of p52 processing.
A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic agonists stimulates the nonamyloidogenic, alpha-secretase-processing pathway of amyloid precursor protein (APP). To determine whether increased cholinergic neurotransmission can modify the APP processing in vivo, we administered a muscarinic receptor agonist (RS86) to normal or aged rats and rats with severe basal forebrain cholinergic deficits (induced by 192 IgG-saporin). The levels of the cell-associated APP in neocortex, hippocampus, and striatum, as well as the secreted form of APP (APPs) in cerebrospinal fluid, were examined by Western blots. Additionally, we investigated the association between the altered APP levels and behavioral deficits caused by cholinergic lesions. We found that treatment with muscarinic receptor agonist resulted in decreased APP levels in neocortex and hippocampus and increased levels of APPs in cerebrospinal fluid. Regulation of APP processing by the muscarinic agonist treatment occurred not only in normal rats, but also in aged and cholinergic denervated rats that model this aspect of Alzheimer's disease. Interestingly, we found that elevation of APP in neocortex correlated with the cognitive deficits in water-maze testing of rats with cholinergic dysfunction. These data indicate that increased cholinergic neurotransmission can enhance nonamyloidogenic APP processing in intact and lesioned rats and that APP may be involved in cognitive performance.
Narcolepsy is a disabling sleep disorder affecting humans and animals. It is characterized by daytime sleepiness, cataplexy, and striking transitions from wakefulness into rapid eye movement (REM) sleep. In this study, we used positional cloning to identify an autosomal recessive mutation responsible for this sleep disorder in a well-established canine model. We have determined that canine narcolepsy is caused by disruption of the hypocretin (orexin) receptor 2 gene (Hcrtr2). This result identifies hypocretins as major sleep-modulating neurotransmitters and opens novel potential therapeutic approaches for narcoleptic patients.
The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.
Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 10(8) splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 micromol/L S-59 and 2.1 J/cm2 UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 micromol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 10(5.1) murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.
Narcolepsy, a neurological disorder characterized by excessive daytime sleepiness and abnormal REM sleep, is known to be tightly associated with the Human Leukocyte Antigen (HLA) DQ allele DQB1*0602. In this study, we have explored the possibility that normal subjects carrying this HLA allele (25% of the general population) could display subclinical REM sleep abnormalities and increased daytime sleepiness. Data from 525 middle-aged adults enrolled in the Wisconsin Sleep Cohort study were used for this analysis. Nocturnal polysomnography, sleep latency during the multiple sleep latency test (MSLT), and questionnaire items pertaining to excessive daytime sleepiness were compared between DQB1*0602 positive (n = 132) and negative (n = 393) participants. Results indicate shorter REM latency whether or not the latency was adjusted for wake after sleep onset (p = 0.003) and p = 0.02 respectively), increased sleep efficiency (p = 0.06) and decreased percent time spent in stage I sleep (p = 0.02) during nocturnal polysomnography in DQB1*0602 subjects. Data gathered using the Multiple Sleep Latency Test or the Epworth and Stanford sleepiness scales did not differentiate between DQB1*0602 positive and negative subjects. These results support the hypothesis that polymorphisms at the level of HLA DQ modulates sleep tendencies in humans.
PURPOSE: Previous studies have reported an association between bacterial vaginosis (BV) and postoperative fever and infection. This prospective study investigated whether the intermediate or definite stages of BV are risk factors for postoperative infection after major gynecologic surgery. METHODS: Vaginal cultures were obtained preoperatively from 175 women undergoing gynecologic surgery. The diagnostic criteria for BV were based on Nugent's standardized method of Gram stain interpretation. Postoperative fever was defined as at least one temperature equal to 101.0 degrees F or greater, or two or more temperatures more than 6 hours apart equal to 100.4 degrees F or greater. RESULTS: Thirty-six percent of the positive-BV group developed a postoperative fever, compared with 20% of the Lactobacillus-predominant group and 12% of the intermediate-BV group (P = 0.017). The differences between the positive-BV group and the Lactobacillus-predominant group, and between the positive-BV group and the intermediate-BV group, with respect to postoperative fever, were statistically significant (P = 0.045 and P = 0.007, respectively). The difference between the intermediate-BV group and the Lactobacillus-predominant group was not statistically significant (P = 0.28). CONCLUSIONS: Although the association between BV and postoperative febrile morbidity could be a spurious result of confounding with other variables, it may be prudent for the surgeon to identify patients with BV and treat them preoperatively.
Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.
Labeled proteins are used in a variety of applications. This review focuses on methods that utilize genetic engineering to introduce protein kinase recognition sites into proteins. Many protein kinase recognition sites can be introduced into proteins and serve as useful tags for a variety of purposes. The introduction of protein kinase recognition sites into proteins can be achieved without modifying the essential structure or function of the proteins. Because proteins modified by these procedures retain their activity after phosphorylation, they can be used in many applications. The phosphorylatable proteins can be labeled easily to high specific activity with radioisotopes ((32)P, (33)P, or (35)S), or the nonradioactive (31)P can be used. The use of these radioisotopes provides a convenient and safe method for radiolabeling proteins. Moreover, the use of the nonradioactive (31)P with protein tyrosine kinase recognition sites permits the tagging of proteins and their detection with the many anti-phosphotyrosine antibodies available. Overall, the procedure represents a convenient, safe, and efficient method to label proteins for a variety of applications.
OBJECTIVES: The aim of this project was to assess the utility of dobutamine stress echocardiography (DSE) for evaluation of women with suspected ischemic heart disease. BACKGROUND: Most investigations addressing efficacy of diagnosis and treatment of coronary artery disease (CAD) have been performed in predominantly male populations. As part of the Women's Ischemia Syndrome Evaluation (WISE) study, DSE was assessed in women participating at the University of Florida clinical site. METHODS: Women with chest pain or other symptoms suggestive of myocardial ischemia and clinically indicated coronary angiography were eligible for the WISE study. Enrolled subjects underwent DSE using a modified protocol. Coronary stenosis was assessed by core laboratory quantitative coronary angiography (QCA). RESULTS: The 92 women studied ranged in age from 34 to 82 years (mean 57.5). All women had > or = 1 major risk for CAD, and most (89, 97%) had > or = 2 risk factors. In 78 women (85%), left ventricular wall motion was normal at baseline and during peak infusion. The remaining 14 women had wall motion abnormalities during DSE. By QCA, 25 women (27%) had > or = 50% coronary stenosis, including 10 with single-vessel obstruction. Dobutamine stress echocardiography was abnormal in 10 of these 25 women, yielding overall sensitivity of 40%, and 60% for multivessel stenosis. Exclusion of women with inadequate heart rate response yielded overall sensitivity of 50%, and 81.8% for multivessel stenosis. Dobutamine stress echocardiography was normal in 54 of the 67 women with < 50% coronary narrowing, specificity 80.6%. CONCLUSIONS: Dobutamine stress echocardiography reliably detects multivessel stenosis in women with suspected CAD. However, DSE is usually negative in women with single-vessel stenosis, and in the larger subset without coronary stenosis. Ongoing protocols of the WISE study are expected to improve diagnostic accuracy in women with single-vessel disease, as well as provide important data in the substantial number of women with chest pain but without epicardial coronary artery stenosis.
This paper reports a new species and two new records of the Axinidae collected from fishes of the South China Sea. Loxuroides fungilliformis n. sp. was obtained from the gills of Hemirhamphus guoyi collected at Zhapo (21 degrees 34'N, 111 degrees 49'E). The new species is similar to L. sasikala (Unnithan, 1957) Price, 1962 in the structure of the cirrus and genital atrium, but differs from the latter in the shape and number of the spines of its genital atrium and cirrus. Both L. sasikala and Axine tripathii Price, 1962 are recorded for the first time off China.
In previous work, a 33 kDa cysteine proteinase was found in callus initiated from maize (Zea mays L.) resistant to fall armyworm feeding. A callus cDNA library from the maize inbred Mp708 was screened with oligonucleotides derived from the N-terminal amino acid sequence of the 33 kDa proteinase and several cDNA clones were isolated and sequenced. A cDNA clone encoding the 33 kDa cysteine proteinase, mir1, was identified. Two additional clones, mir2 and mir3, encoding putative cysteine proteinases were also identified. mir2 and mir3 are distinct from mir1 and each other, but show a high degree of homology. All of the mir cDNA clones map to distinct sites on the maize genome. Amino acid sequences encoded by the mir clones are similar to other known cysteine proteinases and are most closely related to the oryzain-alpha and -beta precursors. The ERFNIN motif and a 12 amino acid conserved sequence are present in the propeptide region of the putative proteinases encoded by mir clones. mir2 and mir3 appear to have C-terminal extensions. The phylogenetic tree of nucleotide sequences of mir1, mir2, mir3 and other representative cysteine proteinases from protozoa, plants and animals was constructed.