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S Lei

Publications and source records attributed to S Lei.

72 records · Page 4Linked to original sources
PubMed1995

Binding of monoclonal antibodies against the carboxyl terminal segment of the nicotinic receptor delta subunit suggests an unusual transmembrane disposition of this sequence region.

Monoclonal antibodies (mAbs) specific for the carboxyl terminal region of the delta subunit of Torpedo nicotinic acetylcholine receptor (AChR), derived from mice immunized with AChR or a synthetic carboxyl terminal sequence of the delta subunit (C delta-mAbs), were used to determine the transmembrane disposition of their epitope(s) by immunoelectron microscopy, using AChR-rich postsynaptic membrane fragments from Torpedo electroplax. Some C delta-mAbs recognized only the cytoplasmic side of the membranes, some both sides to a similar extent, and others bound mostly, but not exclusively, to the cytoplasmic side. Binding of C delta-mAbs to the membranes was specifically blocked by synthetic peptides containing the carboxyl terminal region of the delta subunit. Control anti-AChR mAbs specific for the alpha or the delta subunits, whose epitopes have known transmembrane topology, uniquely recognized the expected side of the postsynaptic membrane. Residues involved in C delta-mAb binding were identified using single residue substituted peptide analogues of the sequence delta 481-501. All C delta-mAbs recognized epitopes within the same sequence segment, delta 485-493, at the carboxyl terminal of the AChR delta subunit. These results suggest that the delta subunit of the AChR might have alternative conformations, leading to exposure of the same sequence region on the extracellular or the cytoplasmic surface. Several Pro residues are present in this region. The alternative cis or trans conformation of one or more of them might result in different folding patterns of the carboxyl terminal sequence of the delta subunit, as described for a viral protein [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L., & Harrison, S. C. (1991) Nature 354, 278-284.

Amino Acid Sequence↗
PubMed1995

Overexpression of HER2/neu oncogene in pancreatic cancer correlates with shortened survival.

For the purpose of determining the prognostic significance of HER2/neu oncogene in pancreatic and ampullary cancers, 21 pancreatic cancers of ductal origin and six cancers of the ampulla of Vater were studied immunohistochemically using the monoclonal antibody (MAb) CB11, specifically reactive with HER2/neu product. Staining of the epithelium of the normal duct and acini was negative or weakly positive. Moderately and strongly positive reactions indicated the overexpression of this gene, and were found in 10 of 21 (47.6%) pancreatic cancers of ductal origin and in 2 of 6 (33.3%) ampullary adenocarcinomas. Overexpression of HER2/neu was closely and inversely related to the survival of the patients with pancreatic cancer of ductal origin: 19.1 +/- 11.7 mo for those not overexpressing vs 7.3 +/- 3.8 mo for the overexpressors (p < 0.01). Among the pancreatic cancer group, 11 patients underwent cancer resection. The average survival for the 7 with nonoverexpressing cancer was 21.4 +/- 14.3 mo vs 10.5 +/- 3.6 mo for those with overexpressing tumor. Among those not undergoing resection, the average survival for the 4 with nonoverexpressing cancer was 15.0 +/- 3.8 mo as contrasted to 5.2 +/- 2.1 mo for the overexpressors (p < 0.01). Although the number of patients is small, these findings suggest that the overexpression of HER2/neu gene product may be frequently found in pancreatic cancer of ductal origin and may be one of the useful prognostic biomarkers for this cancer.

Adenocarcinoma↗
PubMed1995

Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor.

The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment.

Amino Acid Sequence↗
PubMed1995

[Effect of tetrandrine on pulmonary hypertension induced by monocrotaline in rats].

The effects of Tetrandrine (Tet) on intraacinous pulmonary arteries (IAPA) and bemodynamics were studied by means of a rat pulmonary hypertension model induced by monocrotaline. The results showed that Tet could reduce the contractive pressure of pulmonary artery, right ventricle and right atrium of heart by 28.0%, 28.3%, and 31.5% respectively. It could also decrease the vascular degeneration of endoepithelial cells of IAPA, the endoepithelial subcavity, the collagens of medial membrane and the numbers of smooth muscles of IAPA.

Alkaloids↗
PubMed1994

Pancreatic cancer. Cytologic study of peritoneal washings.

OBJECTIVE: To delineate the frequency and significance of free cancer cells in the peritoneal cavity of patients with pancreatic exocrine adenocarcinoma. DESIGN: Randomly selected patients with pancreatic cancer had peritoneal washings performed at the beginning and conclusion of laparotomy. Results of cytologic studies were correlated with the clinical findings, size, spread, and resectability of the tumor and with the survival time of the patient. All patients were followed up until the present or until their deaths. SETTING: Tertiary care, referral hospitals in Toledo, Ohio. PATIENTS: Only patients with biopsy-proven adenocarcinoma of the pancreas were included. Thirty-six patients, yielding 62 specimens for cytologic study, were included. INTERVENTION: Peritoneal washings were performed at the beginning and completion of laparotomy. Each washing was evaluated independently by two skilled cytologists. If present, ascites was quantitated and studied cytologically. Biopsy specimens were obtained in each patient at the time of the study. MAIN OUTCOME MEASUREMENTS: Presence or absence of malignant cells in peritoneal fluid; maximal diameter, grade, and spread of cancer; presence and volume of ascitic fluid; resectability of cancer; and length of survival of the patient. RESULT: Of the 36 patients studied, three had positive cytologic findings. All three had peritoneal carcinomatosis. Of 11 patients with ascites, only one had positive cytologic findings. CONCLUSIONS: Results of cytologic studies of peritoneal washings or of ascitic fluid are seldom positive with pancreatic exocrine carcinoma. When positive, they denote a very grave prognosis.

Adenocarcinoma↗
PubMed1994

Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes.

T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide T alpha 360-378 was co-injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides T alpha 360-378 and T alpha 63-80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide T alpha 360-378. Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.

Amino Acid Sequence↗
PubMed1994

Characterization of the CGRP receptor and mechanisms of action in rat mesenteric small arteries.

Rat alpha-calcitonin gene-related peptide-induced concentration-dependent (100 pM-10 nM) relaxations in rat mesenteric small arteries (i.d. approximately 220 microns) contracted with noradrenaline, prostaglandin F2 alpha or K+, however, the maximal relaxation depended on the precontractile stimulus, being highest (95%) in arteries contracted with PGF2 alpha and lowest (51%) in arteries contracted with 125 mM K+. The relaxation was inhibited between 10 pM and 1 nM by removal of the endothelium, but was not antagonized by glibenclamide (1 microM), tetraethylammonium (30 mM), apamine (0.3 microM) and 4-aminopyridine (3 mM). The concentration-response curve to rat alpha-CGRP and human beta-CGRP was shifted to the right in the presence of 1 microM human alpha-CGRP(8-37) indicating a receptor affinity, -log(KB[M]), equal to 7.2 and 7.0, respectively. It is concluded that the relaxation induced by CGRP depends minimally on the endothelium and K(+)-channel opening is not a principal process in the relaxing effect of CGRP, thus a third mechanism must mediate the relaxation in these vessels. The main CGRP receptor type mediating relaxation in rat mesenteric small arteries belongs to the CGRP1 subtype.

Animals↗
PubMed1993

Homologous kappa-neurotoxins exhibit residue-specific interactions with the alpha 3 subunit of the nicotinic acetylcholine receptor: a comparison of the structural requirements for kappa-bungarotoxin and kappa-flavitoxin binding.

kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M. W., & Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537]. Like the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX binds with high affinity to alpha 3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha 3 subunit involved in formation of the binding site for this family of kappa-neurotoxins, we investigated kappa-FTX binding to overlapping synthetic peptides screening the alpha 3 subunit sequence. A sequence region forming a "prototope" for kappa-FTX was identified within residues alpha 3 (51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha 3 subunit [McLane, K. E., Tang, F., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544] and alpha-bungarotoxin to the Torpedo AChR alpha subunit [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence alpha 3 (51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with kappa-FTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Monoclonal antibodies against synthetic sequences of the nicotinic receptor cross-react fully with the native receptor and reveal the transmembrane disposition of their epitopes.

Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100. mAbs against peptide alpha 304-322, alpha 332-350, and alpha 360-378 cross-reacted with most or all Triton-solubilized TAChR molecules and, in immunoelectron microscopy experiments, bound to the cytoplasmic surface of AChR-rich postsynaptic membrane fragments. Two mAbs specific for the sequence alpha 376-393, proposed to form an amphypathic alpha helix possibly involved in formation of the ion channel, recognized only approximately 35% of Triton-solubilized TAChR molecules and did not react with membrane-bound TAChR. All of these sequence-specific antibodies recognized SDS-denatured TAChR alpha subunit in Western blots. MAbs specific for the amino-terminal sequence region of the alpha subunit, alpha 1-20, and for the sequences alpha 291-308, alpha 346-364, and alpha 390-409 did not recognize native TAChR. A mAb directed against the carboxyl-terminal region, alpha 420-437, recognized with low apparent titer Triton-solubilized TAChR, not membrane-bound TAChR. In conclusion, a complex membrane protein, TAChR, contains several continuous sequence segments exposed on the TAChR surface, because different mAbs raised against certain synthetic sequences recognized most or all native TAChR molecules. By analogy, it should be possible for most proteins of known sequence to raise anti-peptide antibodies fully cross-reactive with the native cognate protein.

Amino Acid Sequence↗
PubMed1993

Relaxing actions of corticotropin-releasing factor on rat resistance arteries.

1. Although it well established that corticotropin-releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150-300 microns). 2. Initial in vivo experiments confirmed that CRF (1.5 nmol.kg-1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9-41. 3. For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre-contracted with one of 3 agonists (prostaglandin F2 alpha, arginine vasopressin or noradrenaline) or with a high-potassium solution (K+). 4. With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nM, and near complete relaxation with 100 nM. Femoral vessels pre-constricted with agonists and all vessels pre-constricted with K+ were less affected by CRF. In the mesenteric vessels, with sub-maximal levels of pre-constriction, CRF caused substantial relaxation at 1 nM and could cause complete relaxation at 10 nM. 5. The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nM CRF9-41. Neither tetraethyl ammonium (30 mM) nor glibenclamide (3 microM) antagonized the relaxant effect of CRF. 6. The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium. 7. The results indicate that CRF causes an endothelial-independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.

Animals↗
PubMed1992

Epitope mapping of monoclonal antibodies to Torpedo acetylcholine receptor gamma subunits, which specifically recognize the epsilon subunit of mammalian muscle acetylcholine receptor.

Epitopes for four monoclonal antibodies (mAbs) to the gamma subunit of Torpedo nicotinic acetylcholine receptor (AChR), and one mAb crossreactive with the gamma and delta subunits of Torpedo AChR were mapped using overlapping synthetic peptides corresponding to the complete amino acid sequence of Torpedo gamma subunit. The epitopes for all mAbs were within a 50 residue sequence region, on the cytoplasmic surface of the AChR. Three mAbs crossreacted with mammalian muscle AChRs. Two of them specifically recognized the epsilon subunit of AChRs at adult neuromuscular junction. The epsilon-specific mAbs were used, in conjunction with mAbs specific for the alpha and beta subunits and anti-peptide antisera specific for the epsilon, gamma and delta subunits, to identify in Western blots the subunit complement of embryonic and adult bovine muscle AChRs.

Amino Acid Sequence↗
PubMed1992

T helper function of CD4+ cells specific for defined epitopes on the acetylcholine receptor in congenic mouse strains.

We previously identified sequence segments of Torpedo acetylcholine receptor (TAChR) alpha subunit recognized by CD4+ cells of congenic mouse strains of different H-2 haplotypes, susceptible to experimental autoimmune myasthenia gravis. CD4+ cells from BALB/c and CB17 mice (H-2d) recognized the peptide sequences alpha 1-20 and alpha 304-322, while C57BL/6 and BALB/b mice (H-2b) recognized alpha 150-169 and alpha 360-378. C57BL/6 mice recognized to a lesser extent also peptide alpha 181-200. In the present study we demonstrate that CD4+ cells which recognize these epitopes have T-helper function. CD4+ cells from TAChR immunized mice, stimulated in vitro with synthetic epitope peptides, induced proliferation in vitro of B cells via soluble factors which were not strain specific, and induced secretion in vitro of anti-AChR antibodies. Upon in vitro stimulation with T-epitope peptides, they secreted interleukin-2. Immunization of mice with synthetic T-epitope peptides caused sensitization of CD4+ cells, which responded in vitro both to the immunizing peptides and to TAChR, and appearance of anti-AChR antibodies in vivo, further identifying the epitope-specific CD4+ cells as AChR-specific T-helper cells.

Animals↗
PubMed1992

Spinal interactions between opioid and noradrenergic agonists in mice: multiplicativity involves delta and alpha-2 receptors.

The nature of the interaction between spinally administered opioid and alpha-2 agonists was investigated using the substance P behavioral test in mice. Morphine and agonists which more selectively activate mu or delta opioid receptors were co-administered intrathecally with direct and indirect acting adrenergic agonists norepinephrine, cocaine or clonidine and the behavioral responses to intrathecally coadministered substance P were evaluated. The ED50 values for agonists administered separately and concurrently were computed and drug interactions were evaluated using isobolographic analyses. After separate administration, all the opioid and adrenergic agonists inhibited the substance P-induced behavioral responses. Upon coadministration of opioid and adrenergic agonists, a multiplicative interaction was observed between morphine or the delta agonist D-Pen2-D-Pen-5-enkephalin and the adrenergic agonists. Additive or antagonistic interactions were found between the mu agonist Tyr-D-Ala-NMe-Phe-Gly(ol) and the same adrenergic agonists. The opioid antagonist naloxone and the alpha-2 adrenergic antagonist idazoxan were given as intrathecal pretreatments at doses chosen to shift the dose-response curves of their corresponding agonist (given alone) 4- to 10-fold to the right; this always resulted in a smaller, but significant (2- to 4-fold) shift in the dose-response curve of the other agonist given alone. Intrathecal pretreatment with naloxone or idazoxan altered some interactions between the opioids and clonidine. Although naloxone blocked completely the multiplicative interaction between morphine and clonidine, idazoxan did not. Both naloxone and idazoxan changed the antagonistic interaction between Tyr-D-Ala-NMe-Phe-Gly(ol) and clonidine to a multiplicative interaction. Neither antagonist blocked the multiplicative interaction between D-Pen2-D-Pen5-enkephalin and clonidine. These results suggest that: 1) interactions between opioid and adrenergic agonists in mouse spinal cord are mediated by delta and alpha-2 receptor subtypes; 2) the synergistic interaction between morphine and alpha-2 adrenergic agonists may involve action at delta opioid receptors; and 3) antagonist action on these drug interactions is complex.

Adrenergic alpha-Antagonists↗
PubMed1991

Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the alpha subunits of Torpedo californica and murine acetylcholine receptors.

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.

Amino Acid Sequence↗