Biomedical subjects
Publications and source records attributed to C S Lee.
Primary small cell carcinoma of the periampullary region is rare. Only four patients have been reported previously. The authors examined a patient with ampullary small cell carcinoma using a range of immunocytochemical stains. This tumor shows morphologic and neuroendocrine features similar to its pulmonary and extrapulmonary counterparts.
Estrogen receptor (ER) binding has been shown to decrease in breast cancer cell lines exposed to sodium butyrate; however, the underlying mechanisms are unknown. In MCF-7 breast cancer cells, butyrate caused a rapid time- and concentration-dependent decrease in ER mRNA levels, apparent by 3 h at 3 mM butyrate. ER gene transcription rate was decreased and cycloheximide co-treatment did not relieve this inhibitory effect, suggesting that the butyrate effect was not dependent on ongoing protein synthesis. In both MCF-7 and T-47D cells the decrease in ER mRNA was mirrored by an increase in the level of epidermal growth factor receptor (EGF-R) mRNA. A marked inverse relationship exists between ER and EGF-R in human breast cancer biopsies and cell lines, and the reciprocal modulation of these genes by butyrate suggests that the expression of ER and EGF-R may be co-regulated. This relationship was further investigated in lines expressing only one or the other receptor. In the ER-positive EGF-R-negative line, MDA-MB-134-VI, butyrate exposure decreased ER mRNA levels, implying that the regulation of ER mRNA by butyrate is independent of EGF-R expression. However, butyrate decreased EGF-R mRNA in two ER-negative lines, MDA-MB-231 and HBL-100. As this effect differed from that in ER-positive lines, the regulation of EGF-R may depend on the expression of ER. The possibility that ER and EGF-R gene expression are closely linked has implications in the understanding of progression of human breast cancers to a hormone-independent phenotype and for the use of ER and EGF-R levels as independent prognostic indicators.
A concept involving the use of antibodies conjugated with reporter molecules for the direct sensing on the subtle changes in the local electrostatic environment of antigen-antibody complexes due to the antigen binding was proposed and demonstrated. The studies including the effect of solution conditions on the extent and magnitude of the electrostatic potentials around the antigen-antibody complex and the quantitative dependence of the distance between the reporter molecule and the antigen-antibody binding site on the effectiveness of the reporter molecule for sensing the antigen-antibody binding event are presented and discussed. Basic understanding of induced changes in the electrostatic potentials on the antigen-antibody complex is essential for implementing the proposed direct sensing mechanism.
The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.
The sequence-specific binding pattern of mammary nuclear proteins to the 545-base pair (bp) 5'-flanking region of the mouse beta-casein gene was compared between pregnant and lactating mice by a gel mobility shift assay. By using appropriate probes, two complexes were detected only during pregnancy, whereas an additional four complexes were detected during both pregnancy and lactation. The two pregnancy-specific complexes showed identical electrophoretic mobility and were cross-competed with two unlabeled DNA fragments used to generate the probes. Methylation interference experiments indicated that the two binding regions involved guanosine residues at nucleotides -350 and -8, and the sequences around each guanosine residue have a common palindromic sequence, 5'-TGAT/ATCA-3'. This binding factor is termed pregnancy-specific mammary nuclear factor. In gene transfection experiments, expression of the chimeric beta-casein-CAT gene linked to the 545 bp of mouse beta-casein gene promoter region was maximally induced when transfected mammary epithelial cells were cultured with the lactogenic hormones prolactin, hydrocortisone, and insulin, whereas it was very low when insulin alone was added. The addition of progesterone together with the lactogenic hormones inhibited the hormonal induction of the chimeric gene, whereas co-transfection with an oligonucleotide containing the pregnancy-specific mammary nuclear factor binding site substantially overcome the progesterone-mediated repression of transcription. Furthermore, mutation of the pregnancy-specific mammary nuclear factor binding sites of the chimeric beta-casein-CAT gene resulted in attenuation of the inhibitory effect of progesterone without blocking the stimulatory effect of the lactogenic hormones. These results indicate the presence of a pregnancy-specific mammary nuclear factor(s) that bind to two separate sites of the beta-casein gene promoter and suggest that it may serve as a repressor that mediates the inhibitory action of progesterone on beta-casein gene transcription.
DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (D1-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ greater than MeDZQ much greater than D1 (methyl) greater than D3 (n-propyl) greater than D2 (AZQ; ethyl) greater than D5 (n-butyl) greater than D7 (sec-butyl) greater than D4 (isopropyl) D6 greater than (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3' sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.
Forty patients with cervical myelopathy due to OPLL (Ossification of the Posterior Longitudinal Ligament) of the cervical spine were studied. According to Abe's or Yamamoto's classification, 12 of them had a 50% decrease in the cross-sectional area of the spinal canal. Subtotal vertebrectomy was carried out in 8 of these patients and the remaining 4 patients received posterior decompression. We concluded that anterior decompression, if possible, is the treatment of choice and posterior decompression is recommended only for the longitudinal type involving more than 3 segments.
Picibanil (OK432), an extract from streptococci, has been widely utilized to treat malignant ascites and pleural effusions. The antitumor mechanism is believed to include complement-mediated neutrophil activation. Employing a flow-cytometric analysis of actin polymerization as an indicator of cell activation as well as a tumor proliferation assay, we have found that monocyte-derived neutrophil-activating factors were involved in OK432-induced neutrophil activation as well as antitumor activity. OK432-stimulated (0.1 KE/ml; 0.01 mg/ml) monocyte supernatants (OKMS) induced neutrophil actin polymerization and chemotaxis. OKMS were responsible for neutrophil-mediated inhibition of human leukemic (CEM) cell proliferation and stimulated neutrophils to produce superoxide in the presence of CEM leukemic cells at an effector/target ratio higher than 20/1. In contrast, OK432 alone, OK432-stimulated lymphocyte supernatants, or OK432-stimulated neutrophil supernatants had no effect on neutrophil activation or suppression of tumor cell proliferation. OK432 in combination with mononuclear cells also had no effect on the inhibition of CEM cell proliferation. Pretreatment of OKMS at 56 degrees C for 30 min did not affect its ability to activate neutrophils, implying that complement activation is not responsible for the neutrophil activation. Supernatants from OK432-stimulated mononuclear cells, as determined by enzyme-linked immunosorbent assays and radioimmunoassays, contained high levels of interleukin-8 (IL-8; 1567 +/- 145 pg/ml) and tumor necrosis factor (TNF alpha; 2105 +/- 152 pg/ml), low levels of leukotriene B4 (800 +/- 45 pg/ml) and IL-1 beta (180 +/- 22 pg/ml), but interferon gamma was not detectable. IL-1 beta, IL-8, and TNF alpha transcripts, undetectable in untreated monocytes, increased significantly after 30-60 min exposure to OK432. These results suggest that neutrophil-activating factors from monocytes or resident macrophages may play an important role in the OK432-induced neutrophil activation and antitumor activity.
We have studied the influence of hydrocortisone (HC) on the neuromuscular junctions (NMJs) established on cultured human muscle fibers that had been innervated by fetal rat spinal cord neurons. Treatment with HC was begun 4 weeks after innervation and continued for 1-28 days. Four weeks of treatment significantly increased (a) size of acetylcholinesterase (AChE)-positive sites, indicative of NMJs; (b) intensity of AChE staining; (c) A12-AChE (junctional) molecular fraction; and (d) organization of junctional postsynaptic folds. The effect of HC depended on the dose and duration of treatment. These effects on the molecular properties of the postsynaptic component of the human neuromuscular junction could be through an action of HC directly on the muscle fiber or indirectly by affecting the motor neuron. Because the increased organization of the postsynaptic folds and the increased AChE seem to be salutory effects on the NMJ of prolonged HC treatment, these changes of the NMJ itself might contribute to the long-term beneficial effect of prednisone, another glucocorticoid, in myasthenia gravis patients.
A substantial degree of purification, up to 3200-fold, with recoveries of 8-11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.
Disseminated infection with the fungus Scedosporium inflatum in a neutropenic patient with non-Hodgkins lymphoma presented with the triad of muscle tenderness, papular skin lesions and fever, and progressed rapidly to a fatal outcome. This represents the first reported instance of fatal widely disseminated infection with this organism, and demonstrates that the triad of presenting clinical features, formerly reported to be pathognomic of systemic candidiasis, can no longer be regarded as specific for infection with a particular species of yeast or fungus.
The mammary glands of pregnant and non-pregnant sheep were stimulated by infusion of killed Staphylococcus aureus, and the lymphoid cell response delineated with a panel of monoclonal antibodies. Seven days after antigen infusion, the mammary glands of both pregnant and non-pregnant sheep displayed a striking feature, characterised by the presence of numerous CD45R+ MHC class II+ B cells in the periductal connective tissues. These cells were seen to be clustering around blood capillaries with very prominent endothelial cell lining. Some CD5+ CD4+ lymphocytes were scattered among the B-cell clusters, whereas a few CD8+ lymphocytes were seen mainly at the periphery of the B-cell clusters. Fourteen days after antigen infusion, numerous plasma cells were observed, most of them being of the IgA isotype. Seven days after parturition (approximately 40 days after antigen infusion) the number of lymphocytes and plasma cells in the infused glands had declined dramatically. These data indicate that B cell and helper T-cell interaction can take place at the local sites of antigen stimulation in the mammary gland.
A modified inside-to-outside technique of meniscal repair is presented. The technique employs a 14 gauge needle that acts as the receiving conduit for a standard 10 in meniscal repair needle. The repair needle is placed into the joint through a repair cannula in a routine manner. Twenty-four meniscal repairs with an average follow-up of 26 months are presented with a clinical success rate of 92% and no complications. The technique provides a particularly effective and safe approach to the placement of meniscal repair sutures at the posterior extremes of medial and lateral meniscal tears.
The control of human breast cancer cell proliferation in vitro is known to involve complex interactions between steroid hormones, peptide hormones and growth factors. Little is known, however, of the mechanisms by which these factors, alone or in combination, control cell cycle progression and the expression of specific genes involved in cell cycle control. A pre-requisite for such studies is a cellular system in which non-proliferating or slowly proliferating cells can be maintained in a defined environment and stimulated to progress through the cell cycle by addition of hormones and growth factors. Such a system has been developed for T-47D human breast cancer cells: quiescent or slowly proliferating cells maintained in a serum-free medium can be stimulated to increase their rate of cell cycle progression upon a single addition of insulin, IGF-I, EGF, TGF alpha or bFGF. Oestradiol alone was ineffective but caused a significant increase in % S phase cells when added in the presence of insulin. Progestins, in the presence of absence of insulin, had a biphasic effect with an initial increase in cell cycle progression followed by cell cycle arrest. Both antioestrogens and the antiprogestin, RU 486, in the absence of oestrogen or progestin, were potent inhibitors of insulin-induced proliferation. Increases in cell cycle progression were invariably accompanied by acute increases in c-fos and c-myc mRNA levels. Induction of c-myc by oestrogen and progestin was inhibited by antioestrogens and RU 486, respectively. These data illustrate that the culture of breast cancer cells in a serum-free, chemically defined environment provides an excellent model in which to define the role of individual factors involved in breast cancer growth control. The biological data derived from this system provide a basis for identifying and characterizing genes involved in the control of cell cycle progression in human breast cancer.
Forty patients suspected to have ectopic pregnancy by transvaginal ultrasonography had surgery. 77.5% were confirmed to have ectopic pregnancy and 10% had other abnormal pelvic findings. The transvaginal approach appears to enable us to diagnose ectopic pregnancy at an earlier date, with the earliest diagnosis made at 4 weeks 0 days of amenorrhoea. In cases of confirmed ectopic pregnancy, the presence of a complex adnexal mass was the most common feature seen on transvaginal ultrasound and its predictability of ectopic pregnancy is enhanced by a concomitant finding of an empty uterus (95%) or free fluid in the Pouch of Douglas (94%) in the presence of a positive hCG test. The significance of transvaginal ultrasound features, and the advantages of transvaginal over transabdominal ultrasound, are discussed.
PROBLEM: Previous studies have revealed the presence of a unique population of CD45R+ granulated cells in the sheep uterine epithelium. In the present study, dramatic changes in this cell population and in the nongranulated lymphocytes in the uterine and endometrial glandular epithelium of non-cycling, cycling, pregnant, and postparturient sheep are described. In noncycling and cycling sheep, the granules in the granulated intraepithelial cells were small. From days 55 to 134 of pregnancy, the granules in these cells were large, and there was a significant increase (P < 0.01) in the proportion of this cell population in the uterine epithelium but not in the endometrial glandular epithelium located in the deeper region of the stroma. The number of these cells declined dramatically (P < 0.01) from 2 to 15 days after parturition. Both the tissue distribution and the time of activation of these cells suggests they are different from the granulated lymphocytes described in placentae of mice and man. CONCLUSIONS: It is concluded that this unique population of granulated cells is derived from lymphocytes, and that these cells become metabolically active from mid- to late-pregnancy and may play a physiological role during pregnancy or birth. In contrast, the number of nongranulated intraepithelial lymphocytes were suppressed throughout pregnancy and they probably do not play a role in pregnancy.
We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 10(7) T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37 degrees C for 30 min. With a multichamber chemotactic assay, we found that there were 112 +/- 15 migrated neutrophils (mean +/- standard deviation, n = 7) for STV and 11 +/- 4 for HBSS per high-power field (x 400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100 degrees C or deproteinization by treatment with proteinase K at 65 degrees C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (< 20 pg/ml) or prostaglandin E2 (< 8 pg/ml) was found in the STV by radioimmunoassay. Production of LTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 microM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells.