Typhlitis in patients with HIV-1 infection.
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Adult↗
Biomedical subjects
N Lee
Publications and source records attributed to N Lee.
Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.
Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi.
Amino Acid Sequence↗
Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.
Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.
Antibodies, Monoclonal↗
Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression.
By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.
Animals↗
Age-induced changes in electrophysiological responses of neostriatal neurons recorded in vitro.
The present studies were undertaken to determine whether the major electrophysiological characteristics of neostriatal neurons are altered during aging. The passive and active membrane properties of 130 neostriatal neurons obtained from young (three to five months, N = 65) and aged (24-26 months, N = 65) Fischer 344 rats were compared using an in vitro slice preparation. The results indicated that in a population of aged neostriatal neurons the majority of the electrophysiological changes that occurred resulted in decreases in cellular excitability. These changes included increased threshold to induce action potentials by intracellular current injection and decreased negativity of membrane potentials at which such action potentials were induced. In addition, there were increases in the amplitude of the action potential afterhyperpolarization and increases in the frequency of occurrence of accommodation when trains of action potentials were induced. These two latter effects can limit the frequency of action potential generation. The thresholds to elicit synaptically evoked depolarizing responses and action potentials were increased. The results also indicated that a number of basic electrophysiological parameters were unchanged by the aging process. These included action potential amplitude, rise time and duration, resting membrane potential, input resistance and time constant. Although thresholds for the induction of synaptic and action potentials by extracellular stimulation were increased, the latency, amplitude and duration of the evoked depolarization remained unchanged. These findings suggest that the ability of neostriatal neurons to integrate spatiotemporal inputs must be severely compromised in this population of aged cells. Furthermore, the present findings, when compared with age-induced electrophysiological alterations in neurons in other brain areas, indicate that age may differentially alter electrophysiological properties of neurons in separate nuclei. Profiles of age-related changes in neurophysiological properties of neurons provide important information that can be related to the contributions of individual neural areas to the behavioral effects of aging.
Action Potentials↗
In vitro stability of EDTA and DTPA immunoconjugates of monoclonal antibody 2G3 labeled with indium-111.
Monoclonal antibody 2G3 directed against a high molecular weight glycoprotein on breast and ovarian cancer cells was conjugated with bicyclic DTPA (or EDTA) anhydride or benzyl isothiocyanate DTPA (benzyl DTPA) and labeled with 111In. DTPA anhydride was more reactive with the antibody than benzyl DTPA, and kinetics of labeling with 111In were more rapid for DTPA substituted 2G3 than for benzyl DTPA substituted 2G3. On the other hand, 111In-2G3 conjugates prepared using DTPA anhydride were subject to more extensive dimerization and higher losses in immunoreactivity than those prepared using benzyl DTPA. On the basis of measurement of transchelation to transferrin, the stability of 111In-2G3 prepared using DTPA anhydride or benzyl DTPA did not differ during incubation in human plasma for 6 days at 37 degrees C. These results suggest that an important advantage of benzyl DTPA over DTPA anhydride for preparing 111In-labeled antibodies is the prevention of intermolecular (and intramolecular) crosslinking during conjugation which ultimately leads to alterations in conformation and losses in immunoreactivity of the radioimmunoconjugate.
Antibodies, Monoclonal↗
Midtubal occlusion: etiology, management, and outcome.
OBJECTIVE: To assess the significance of pathological midtubal occlusion in infertility. DESIGN: Retrospective descriptive case review. SETTING: Tertiary referral center. PATIENTS: Sixteen women, 26 to 38 years of age presenting with infertility and diagnosed as having midtubal occlusion. INTERVENTIONS: Assessment of the etiology of the occlusive disease and yield of microsurgical tubotubal anastomosis. MAIN OUTCOME MEASURES: Histologic examination of occluded tubal segment and fertility outcome subsequent to anastomosis. RESULTS: Occlusion was because of tuberculous salpingitis (n = 6), resolved tubal pregnancy (n = 3), endometriosis (n = 2), chronic inflammatory reaction (n = 1), tubal cyst (n = 1), intratubal adhesions (n = 1), infant hernia repair (n = 1), and calcification (n = 1). Of eight women (including the patient with tuberculous salpingitis) who underwent tubal anastomosis only for midtubal occlusion, three had subsequent term deliveries, two had tubal pregnancies, and three women did not conceive. CONCLUSION: Midtubal occlusion of the tube because of an infectious process is rare. Excluding tuberculous salpingitis, microsurgical anastomosis yields satisfactory fertility, albeit with significant risk of ectopic pregnancy.
Adult↗
Recreational MDMA use in Sydney: a profile of 'Ecstacy' users and their experiences with the drug.
'Ecstasy' (3,4-methylenedioxymethamphetamine or MDMA) is a recreational drug that is gaining popularity world wide. There is a paucity of research regarding the ways in which Ecstasy is used and the nature of its effects. A 'snowball' peer network technique was used to recruit 100 users who completed anonymous questionnaires. The research revealed that Ecstasy is primarily used by infrequent recreational drug users for 'fun' at dance parties and social gatherings. The primary reported effects of Ecstasy were a 'positive mood state' and feelings of intimacy and closeness to others. The secondary effects of Ecstasy were the stimulant effects of energy and activation, and the psychedelic effects of insight and perceptual and sensual enhancement. Ecstasy was reported to share the properties of both amphetamines and hallucinogens in the nature of its side effects and residual effects which were no more severe than those of the latter two classes of drug. It appeared Ecstasy was not conductive to regular and frequent use, because tolerance was reported to develop to the positive effects of Ecstasy, while negative effects increased with use. Although few problems associated with the recreational use of Ecstasy have surfaced to date, animal research has shown it to be neurotoxic to serotonergic nerve terminals. Caution must be observed until further research can determine the level of hazard in humans.
3,4-Methylenedioxyamphetamine↗
Thrombotic thrombocytopenic purpura in pregnancy.
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Adult↗
Do we require initiatives to reduce ophthalmic outpatient waiting lists?
This paper describes a five week initiative, which aimed to reduce the waiting list for new referrals to an ophthalmic department. A cohort of 317 patients was examined qualitatively to help determine the future direction of ophthalmic resources. Cataract was the most frequent diagnosis, and one in five new patients were referred for surgery. A third of all referral letters contained inaccurate diagnoses, suggesting that grading waiting times on the basis of referral letters is unsatisfactory. Ten per cent of referrals suffered from conditions that could potentially lead to irreversible visual loss. There was no significant difference between the non-attenders (19%) and attenders in relation to their diagnoses or subsequent management. This initiative effectively reduced the outpatient waiting time from 28 weeks to two weeks, thereby maintaining the outpatient waiting time at eight weeks over the following year.
Appointments and Schedules↗
[Limits of duration of chest tube drainage in the first episode of spontaneous pneumothorax].
We treated three patients with spontaneous left pneumothorax who underwent a long period of chest tube drainage before surgery. Case 1 was a 61-year-old man. On day 13 after drainage started, much air leakage occurred and surgery was done the next day. Case 2 was a 57-year-old man. Drainage failed to allow the air leakage to seal. The patient did not consent to surgery for about a month, but on day 38 after drainage started, he underwent surgery. Case 3 was a 19-year-old man. First, chest tube drainage was successful and he was extubated on day 16. However, pneumothorax recurred the next day. Liver dysfunction delayed surgical treatment, and the patient underwent surgery on day 54 after the first drainage. Postoperatively, he developed wound infection of the chest tube route and aseptic pleurisy of unknown origin. Limits of duration of unsuccessful chest tube drainage for the first episode of spontaneous pneumothorax are controversial. These cases suggest that when two weeks of chest tube drainage is unsuccessful, surgery should be undertaken in view of postoperative complication and social indications.
Adult↗
Effect of terfenadine in asthmatic patients.
The bronchodilator effect of terfenadine, 60-mg or 120-mg single dose, and 1 week twice daily dosing, was evaluated in 12 allergic asthmatic patients. When compared with baseline, FEV1 rose significantly for single dose 120 mg terfenadine at one, one and one-half, three, five and one-half, and six hours and for 60 mg terfenadine at three, five and one-half, six, and eight hours postdose. Variations in patient response were observed. At steady state, 120 mg terfenadine b.i.d. showed consistent improvement over placebo from three to 12 hours postdosing but no improvement in FEV1 was noted for terfenadine, 60 mg b.i.d. There no longer was a statistically significant difference in mean FEV1 or percent change from baseline. Thus, terfenadine proved to be a safe and a mild bronchodilator; however, tachyphylaxis might develop to the bronchodilator effect after 1 week of continuous b.i.d. dosing.
Asthma↗
Regulation by glutathione of interleukin-4 activity on cytotoxic T cells.
We have previously shown that cellular glutathione (GSH) regulates the T-cell proliferative activity of interleukin-2 (IL-2). Here, we examined whether and how GSH affects the activity of interleukin-4 (IL-4) on murine cytotoxic T cells. CT.4R, a T-cell line that is responsive to both IL-4 and IL-2, was used as a model. Although GSH alone had little effect on the thymidine incorporation of CT.4R cells, it enhanced the response of CT.4R to IL-4 and increased the level of thymidine incorporation up to more than 60-fold in a concentration-dependent manner. GSH affected the binding of IL-4 to cellular receptors. Scatchard plot analysis showed that GSH treatment did not change the dissociation constant significantly; however, it increased the receptor number from 1173 +/- 126 to 2112 +/- 492 molecules per cell. Internalization and degradation studies of IL-4 showed that the amount of IL-4 internalized and degraded in the GSH-treated cells was about twofold higher than those in the cells without GSH treatment. These results suggest that GSH regulates the binding, internalization, degradation and T-cell proliferative activity of IL-4; alteration of cellular GSH levels may thus affect the growth and replication of cytotoxic T cells through growth stimulating cytokines such as IL-2 and IL-4.
Animals↗
[Endoscopic ultrasonography for preoperative diagnosis of the hilar and mediastinal lymph node metastases in lung cancer].
Fifty patients with lung cancer underwent transesophageal endoscopic ultrasonography (EUS) for preoperative detection of metastases to the hilar and mediastinal lymph nodes. An electronic ultrasonic fiberscope with a linear array (EPB-503-FS, Machida-Toshiba) was used. Later, in surgery, a total of 513 nodes that could have been detected by EUS were removed. Of these, 54 nodes were found to be metastatic histologically, and 459 were non-metastatic. The rate of detection by EUS was 65% (35/54) for the metastatic nodes; the rate was 41% (186/459) for the non-metastatic nodes (p less than 0.01). Metastatic nodes were detected at high rates in every lymph node site. Non-metastatic nodes were detected at low rates in sites 1, 2, and 4, and at the highest rate in site 7. Metastatic nodes had characteristic internal echoes, affected by the extent of tumor and necrosis present in a node, and were detected more easily than non-metastatic nodes. For larger or rounder nodes, metastasis was more common (p less than 0.01). Lymph nodes that could be detected were classified into six types by their internal echo patterns; three of these types were rarely metastatic, and were called 'negative'; the other three were often metastatic, and were called 'positive'. In histological examinations, of the 'negative' nodes found in fact to be metastatic histologically, invasion by the tumor tended to be diffuse and necrosis was minute. The 'positive' nodes that were in fact metastatic tended to have one of two internal echo patterns (depending on the amount of necrosis) when invasion was diffuse, and a third pattern when invasion was localized.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex.
The poly-N-acetyllactosamines on neutrophils and monocytes have been shown to serve as ligands for various selectins present on endothelial cells and platelets. We have previously shown that only a limited number of glycoproteins contain poly-N-acetyllactosamine and found that lysosomal membrane glycoproteins (lamps) are the major glycoproteins carrying poly-N-acetyllactosamine. In order to understand the reason why only certain glycoproteins can be modified by poly-N-acetyllactosamine, we have utilized 21 degrees C incubation conditions, which were previously shown to cause the accumulation of glycoproteins at the trans-Golgi. HL-60 cells were labeled with [3H]galactose at 21 or 37 degrees C for 6 or 24 h, and lamp-1 and lamp-2 were immunoprecipitated. Upon examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each lamp from HL-60 cells incubated at 21 degrees C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37 degrees C. The number of N-glycans containing poly-N-acetyllactosamine, estimated by their binding to tomato lectin column, increased approximately 30-50% after incubation at 21 degrees C than incubation at 37 degrees C. The analysis of oligosaccharides released by endo-beta-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21 degrees C, and methylation analysis confirmed these results. The same analysis and the results obtained by ion-exchange chromatography also provided evidence that the N-glycans of lamps are sialylated at 21 degrees C as much as at 37 degrees C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21 degrees C than at 37 degrees C. These results therefore indicate that incubation at 21 degrees C causes the lamps to reside longer within the Golgi complex, and such longer residence allows lamps to acquire more polylactosaminoglycan. These results also suggest that the time necessary for moving through the Golgi complex is a critical factor for poly-N-acetyllactosamine formation.
Carbohydrate Sequence↗
Design, chemical synthesis, and expression of genes for the three human color vision pigments.
Color vision in humans is mediated by three pigments from retinal cone photoreceptor cells: blue, green, and red. We have designed and chemically synthesized genes for each of these three pigments. The genes were expressed in COS cells, reconstituted with 11-cis-retinal chromophore, and purified to homogeneity using an immunoaffinity procedure. To facilitate the immunoaffinity purification, each pigment was modified at the carboxy terminus to contain an additional eight amino acid epitope for a monoclonal antibody previously used to purify bovine rhodopsin. The spectra for the isolated pigments had maxima of 424, 530, and 560 nm, respectively, for the blue, green, and red pigments. These maxima are in excellent agreement with the maxima previously observed by microspectrophotometry of individual human cone cells. The spectra are the first to be obtained from isolated human color vision pigments. They confirm the original identification of the three color vision genes, which was based on genetic evidence [Nathans, J., Thomas, D., & Hogness, D.S. (1986) Science 232, 193].
Amino Acid Sequence↗
Efficient transformation and regeneration of rice small cell groups.
Transgenic plants have been regenerated from small cell groups of rice using a simpler, faster, and more efficient method than used previously (e.g., protoplast transformation and regeneration methods). Small cell groups of 50-100 cells were prepared from established suspension cultures of rice cell lines. Transformation of small cell groups with intact cell walls was carried out with a plasmid harboring the beta-glucuronidase gene and was mediated by polyethylene glycol. Assay of beta-glucuronidase activity indicated that the frequency of transformation was about 7%. beta-Glucuronidase activity was detected in the roots and leaves of plants regenerated from transformed calli. One or two copies of the beta-glucuronidase gene per cell were determined to be integrated into the rice chromosomal DNA isolated from transformed calli. This method of transformation and regeneration is widely applicable to both dicotyledonous and monocotyledonous plants, especially those varieties that are resistant to regeneration from protoplasts.
Journal Article↗
Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.
A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.
Base Sequence↗