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Biomedical subjects

S Yang

Publications and source records attributed to S Yang.

At least 19 recordsLinked to original sources

The receptor-binding site of human relaxin II. A dual prong-binding mechanism.

Recent structure/function studies on human relaxin II have led to the conclusion that the arginines B13 and/or B17 are important for biological activity. These studies have been confirmed and extended with the help of chemically synthesized derivatives, i.e. dicitrulline (B13, B17), two monocitrulline (B13 and B17), a dilysine (B13, 17), and alanine (B17) relaxins. The CD spectra of synthetic human relaxin and of the derivatives are indistinguishable. Yet, only the native human relaxin II is biologically active and binds strongly to relaxin receptor preparations in vitro. The inactivation is strictly due to side chain functions, in particular the replacement of either or both arginines in the positions B13 or B17. Binding is mediated by a two-prong electrostatic and hydrogen-binding interaction via arginines B13 and B17. Neither B13 nor B17 alone are sufficient and a positive charge equidistant from the B chain helix is equally insufficient. This binding mechanism appears to be unique, as concerns hormone receptor interaction.

Amino Acid Sequence

Comparison of CNS homing pattern among murine TH cell lines responsive to myelin basic protein.

A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).

Animals

Human colon cancer cells express ICAM-1 in vivo and support LFA-1-dependent lymphocyte adhesion in vitro.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface adhesion glycoprotein that mediates leukocyte adhesion through interaction with the leukocyte CD11/CD18 adhesion complex. The aim of this study was to determine whether ICAM-1 is expressed by normal or neoplastic colonic epithelial cells. Immunohistochemical studies on human colonic tissue demonstrated focal ICAM-1 expression by colonic carcinomas but not by normal colonic epithelium. ICAM-1 expression by colonic carcinomas showed a positive correlation with the presence of a peritumoral inflammatory infiltrate. Surface expression of ICAM-1 was also observed in HT-29 cultured human colon cancer cells by both immunohistochemistry and enzyme immunoassay. Interferon-gamma and interleukin-1 beta significantly increased ICAM-1 surface expression by HT-29 cells in a dose-dependent manner. Upregulation of ICAM-1 surface expression became evident some hours after cytokine stimulation and was inhibited by both actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis. HT-29 monolayers supported adhesion of human lymphocytes as determined by a quantitative 111In-labeled leukocyte adhesion assay. Adhesion was mediated in part via interaction of ICAM-1 on HT-29 cells with lymphocyte function-associated antigen-1 (CD11a/CD18) on lymphocytes, as defined by using blocking monoclonal antibodies. Expression of ICAM-1 and/or other leukocyte adhesion receptors by neoplastic epithelial cells may play a role in directing leukocyte trafficking and leukocyte-epithelial cell interactions in colonic carcinoma.

Carcinoma

Relaxin receptors in mice: demonstration of ligand binding in symphyseal tissues and uterine membrane fragments.

A monocomponent, high specific activity, carrier-free porcine relaxin tracer (125I) has made it possible for us to demonstrate relaxin receptors in the symphysis pubis, uterus, and ovary via autoradiography. The receptors are concentrated in the symphyseal ligament and the peripheral layers of uterus and ovary. Specific relaxin binding was observed in crude membrane preparations of uteri, ovaries, and brain, whereas crude membranes of leg muscle and kidney showed only nonspecific binding. Uterine membranes prepared from estrogen-primed mice showed tracer binding, which could be significantly inhibited by porcine relaxin in a dose-dependent manner, but not by insulin. A linear Scatchard plot suggested the presence of only one kind of receptor and a dissociation constant of 5 x 10(-10) M, which is commensurate with an electrostatic double ion pair binding mechanism.

Animals

Cellular distribution of a colonic adenoma-associated antigen as defined by monoclonal antibody Adnab-9.

Adenomatous colonic polyps constitute a precursor for colorectal cancer. Antibodies to these precancerous lesions might identify specific early tumor antigens. Adnab-9 is a murine monoclonal antibody raised against membranes of colonic adenomas. Adnab-9 binding in colonic washings (effluent) correlates with the presence of colorectal cancer. Immunohistochemical staining with Adnab-9 shows cytoplasmic reactivity in scattered cells in 4 of 31 adenomatous tissue sections, 0 of 14 sections of colorectal cancer cells, and 1 of 8 normal-appearing colonic mucosa specimens examined. Adnab-9 recognized a dominant M(r) 87,000 protein species in tissue extracts in the membrane-bound fraction of effluent by Western blotting. Adnab-9 binding by enzyme-linked immunosorbent assay in adenomatous extracts is higher than cancer or normal tissue, is membrane-bound, and is absent from established colorectal cancer cell lines. This distribution and nature of immunostaining suggest that Adnab-9 recognizes a determinant associated with the membrane component of a subpopulation of adenoma cells which may have a role in early colorectal neoplasia.

Adenoma

Regulatory mode of the pac gene expression.

1.65kb HindIII-BgIII DNA fragment containing pac operator was cloned from plasmid pPA4 to pBR322 at HindIII-BamHI sites, the resultant plasmid pPA41 was transformed into E. coli D816 carrying an intact pac operon on its chromosome, then the effect of operator titration was estimated. It was found that the penicillin acylase activity in E. coli D816 (pPA41) cells was higher than that of in E. coli D816 cells. The operators on high copy number plasmid pPA41 competed for the regulatory proteins with the single copy operator on the chromosomal pac operon, thus the expression of the pac gene was enhanced, because the free regulatory proteins were decreased by operator titration. The results of RNA-DNA hybridization showed that the cellular pac mRNA concentration was parallel to the penicillin acylase activity, and the pac mRNA in E. coli D816 (pPA41) cells was much higher than that of in E. coli D816 cells. These results indicated that the expression of pac gene was negatively controlled at transcriptional level.

Biotechnology

Cloning of par region and the effect of par region on the stability of pUC9.

The par region of pSC101 was cloned into pUC9, and pEC302 was obtained. The stability of pEC302 and pUC9 in E. coli HB101 was tested on M9 medium to determine the effect of par region on the stability of plasmid. After 40 generations, pUC9 was almost completely lost, while pEC302 was 100% maintained in host cells.

Biotechnology

Location and trans-action of the pac regulatory gene.

The study on the localization of the pac gene indicated that the regulatory and structural genes were located in a 3.5 kb HindIII-EcoRI fragment. In this paper we report that a series of deletion derivatives of plasmid pPA6 was constructed and the effect of deletions on the pac expression was determined. The results suggested that the pac regulatory gene was located in the SphI-PvuII fragment within the pac structural gene. Then the SphI-PvuII fragment was cloned to the plasmid pTZ18U at the SphI-SmaI sites and the resultant plasmid pPA57 was transformed into the penicillin acylase producing strain E. coli D816 to observe the effect of SphI-PvuII fragment on the chromosomal pac expression. The results demonstrated that the regulatory gene within the SphI-PvuII fragment regulated the pac expression in trans. Computer analysis indicated that there were two ORFs in the SphI-PvuII fragment as possible candidates coding for the regulatory protein. The study on precise localization of the pac regulatory gene is in progress.

Amino Acid Sequence

[Refractive error and amblyopia in children].

The refractive status of 3,099 children was analyzed. The result showed that the incidence and degree of hyperopia decreased gradually and those of myopia increased along with the growing up of children in ametropia. In binocular refractive amblyopia, high and medium hyperopia and myopia in severe and medium amblyopia were significantly more than those in mild amblyopia. In monocular refractive amblyopia, high and medium hyperopia and high myopia in the amblyopic eyes were more than those in the nonamblyopic eyes. The refractive status of binocular esotropic amblyopia had no significant difference in various ages and degrees of amblyopia. There was also no significant difference between the refractive status of the amblyopic and nonamblyopic eyes in monocular esotropic amblyopia. It was considered that refractive amblyopia was closely related to high ametropia and the deviation of the eye might be the main cause of strabismic amblyopia.

Adolescent

[Measuring the threshold of stereopsis for 111 children with amblyopia].

The threshold of stereopsis was measured by static stereopter for 111 children with amblyopia aged from 4 to 13 years. They were divided into three groups: 38 cases ranging in age from 4 to 5 years, 43 cases 6 to 7 and 30 cases 8 to 13. Fifty six cases were unilateral and 55 bilateral amblyopia. The average threshold of stereopsis was 149.64 second angle for the 4-5 group, 66.69 for the 6-7 and 48.48 for the 8-13. The average threshold for unilateral amblyopia was 65.3, in which mild type was 38.6 and under moderate type was 88.84, whereas the average threshold for bilateral cases was 115.81, in which mild type was 44.09 and under moderate type was 175.44. The distribution of stereopsis for different age groups, the threshold of stereopsis influenced by different types of amblyopia and the comparison of amblyopia between bilateral and unilateral are also discussed.

Adolescent

[Chondronecrosis induced in rhesus monkeys fed with grains and water of Kaschin-Beck's disease endemic area].

Immature Rhesus monkeys were fed with grains and/or water of Kashin-Beck disease (KBD) endemic area for 6 or 18 months. Multifocal and zonal chondronecrosis and a series of secondary reactions following necrosis were found in articular cartilage and growth plate in most of the monkeys. These changes are very similar to the characteristics of human KBD. The results suggest that growing Rhesus monkey should be the susceptible animal for reproduction of chondronecrosis model of KBD and the articular cartilage of greater trochanter proximal to femur is a portion predisposing to cartilage lesions. The experiment also indicated that pathogenic factor resulting in chondronecrosis is still remaining in water and grains of endemic area, though the area is no longer actively endemic for KBD.

Animals

[Purification and characterization of alpha-glucosidase from an extreme thermophile, Thermus thermophilus HB 8].

A highly thermostable alpha-glucosidase (E C.3.2.1.20) from an extreme thermophile, Thermus thermophilus HB 8, was purified to homogeneous by ammonium sulfate fractionation, DEAE-cellulose chromatography and preparative slab gel electrophoresis. The enzyme was purified 17 fold with 21% recovery of activity. The enzyme had a molecular weight of 67000 by SDS-PAGE. The isoelectric point was pH4.5 by IEF on PAG. The enzyme hydrolized p-nitrophenyl-alpha-glucoside (PN-PG), sucrose and maltose, but not cellobiose, melibiose and soluble starch. The km value for PNPG was 0.4mmol/L, the Vmax was 0.29 mumol.min-1.mg-1. The enzyme exhibited high thermostability. After incubation at 90 degrees C for 10 h in 50 mmol/L acetate buffer pH 5.8, the enzyme retained 90% of its original activity. The half-live (t1/2) at 95 degrees C was 108 min. The enzyme was activated by Mg2+, Mn2+, Ca2+, Ba2+ and strongly inhibited by Hg2+, Cu2+. Modification of the enzyme by EDC or DEPC led to complete loss of activity, which suggests that carboxyl group(s) and histidine residue(s) are essential for activity of alpha-glucosidase.

Thermus thermophilus

Altered T cell subpopulations and lymphocytes expressing natural killer cell phenotypes in patients with progressive systemic sclerosis.

Scleroderma (progressive systemic sclerosis [PSS]) is known to be associated with abnormal T cell immunoregulation. In the present study, we evaluated lymphocyte phenotypes in patients with PSS and normal control subjects by flow cytometry and monoclonal antibodies for total T (CD3), T suppressor (CD8), T helper (CD4), T helper-inducer (CDw29), T suppressor-inducer (CD45R), human leukocyte antigen, DR+B (CD19), DR+T, and natural killer subsets, HNK-1 (CD57) and NKH-1 (CD56) cells. Patients with PSS compared to normal subjects had significantly lower percentages of CD3+ (p less than 0.005) and CD8+ (p less than 0.05) (similar to several patients with rheumatoid arthritis also evaluated), as well as CD45R (p less than 0.05), T+DR+ (p less than 0.05), and NKH-1 (CD56) (p less than 0.0005) cells. Patients with PSS with late-limited or generalized disease had lower percentages of CD8+, CD19, NKH-1+, and CDw29, but higher percentages of CD4+, HNK-1, and CD45R cells compared to patients with early stage disease, but these results were not statistically significant. These unique alterations in patients with PSS may prove to be useful in monitoring the stage of disease activity for therapy and further define immunologic defects.

Adult

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Antigens, Viral

An experimental study on the laryngeal electromyography and visual observations in varying types of surgical injuries to the unilateral recurrent laryngeal nerve in the neck.

A series of varying types of surgical injuries to the recurrent laryngeal nerve, including half section, double crush, suture ligation, and complete section of the nerve, was investigated in dogs by correlating clinical and electromyographic findings. The state of recovery from palsy was evaluated by recording the electromyography from the affected laryngeal muscles as well as by observing the movements of the vocal cords. Six electromyography patterns were recorded from the affected muscles following injuries to the recurrent laryngeal nerve. There is a close relationship between the electromyography patterns and the types of nerve injuries. Within 3 months after injuries of the recurrent laryngeal nerve, the electromyography motor unit potentials in the involved intrinsic laryngeal muscles and vocal cord movements on the side of the nerve injury were back to normal in the groups of dogs in which the recurrent laryngeal nerves were partially sectioned and doubly crushed; the electromyography potentials and vocal cord motion on the affected side did not return to normal in the group of dogs in which the recurrent laryngeal nerves were permanently ligated with suture. In the group of dogs which had had a complete section of the recurrent laryngeal nerve, no recovery of both electromyography and vocal cord function on the affected side was observed 6 months after injury of the recurrent laryngeal nerve. This study showed that the frequency of the regenerated potentials recorded from the affected muscles is related to the types and degrees of injuries to the recurrent laryngeal nerve. Recovery from recurrent laryngeal nerve palsy is complete within 3 months after nerve impairment if over half of the nerve fibers of the impaired nerve are maintained intact without degeneration. Moreover, the period from onset to complete recovery from palsy was remarkably short, less than 2 months. The most appropriate time for the electromyography examination to evaluate the degrees of the nerve injury and to anticipate the prognosis of recurrent laryngeal nerve palsy was proposed.

Animals