Biomedical subjects
Publications and source records attributed to L Wen.
The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.
Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.
A cloned Th1 cell line was isolated from pancreatic lymph nodes of NOD mice that carries a T-cell receptor encoding Vbeta14 and proliferates in response to NOD islets, islet supernatant, and crystalline bovine and rat insulin, specifically to a B-chain peptide bound to IA(g7). The response to islet supernatant was reduced by 75% by anti-insulin antibody treatment. The insulin-reactive clone reduced insulitis and totally blocked the development of spontaneous diabetes in NOD mice (n = 8) as well as the adoptive transfer of diabetes into irradiated NOD mice following the injection of splenocytes from diabetic mice (n = 13). Trafficking of the adoptively transferred cells was assessed by labeling the clone or diabetic splenocytes with a fluorescent marker (DiI). The labeled clone was detected in the islet periphery, whereas labeled splenocytes alone invaded the islets by 3 days. In contrast, the protective clone dramatically delayed and reduced the number of labeled diabetic splenocytes infiltrating the islet, although their appearance in the spleen was unaffected. In vitro, the clone as well as supernatant derived from the clone blocked the proliferation of diabetic NOD splenocytes to islets. This inhibitory effect was diminished by anti-transforming growth factor-beta. In conclusion, an insulin-specific Th1 cell was isolated from NOD mice that traffics to the islet and prevents the spontaneous development and the adoptive transfer of diabetes. It appears to act locally by releasing transforming growth factor-beta and/or other factors that inhibit homing to and/or proliferation of diabetic splenocytes within the islet. These findings may provide insights into and suggest mechanisms for the protective effects of insulin therapy against diabetes.
Seventy-eight limb segments with various lower extremity deformity in 60 cases were treated by unilateral axial dynamic fixator (UADF). Bone healing of 77 osteotomy sites was primary union. One site was delayed union. The correction of limb axial line and recovery of joint function were excellent. The incidence of pintrack infection was 0.6%. The advantages of this device were minimum injury, simple procedures, stable fixation and reliable correction of deformity, promotion of callus formation and ossification by elastic fixation, less joint functional effect, and rare complication. The advandage of this method is that limb deformity can be adjusted to an ideal shape by using UADF. The average follow-up for 2.5 years indicated that application of UADF is an alternative method for the treatment of genu varus, genu valgus, and malunion of fracture.
In order to accelerate the development of biological recombinant human prorelaxin in our country, direct cloning of human prorelaxin gene from freshly prepared corpus luteum of Chinese woman by RT-PCR method was conducted. Isolated prorelaxin gene contains B, A chains and connective peptide. The amplified products were cloned and confirmed by DNA sequencing using Sanger Dideoxy method. It was subcloned into LKB2, a prokaryotic expression vector offering promoter LacI control, and highly expressed in E. coli. The results suggest that the PCR amplified DNA fragment shared identical sequence with known prorelaxin gene reported abroad. The SDS-PAGE analysis revealed that the expressed protein accounted for about 30% of the total cell protein, its MW was approximately 20 kD and conformed soluble protein.
To study the human papillomavirus (HPV) infection during pregnancy and transmission of virus to fetus, samples such as, cervical secretion, peripheral blood, amniotic fluid, neonatal umbilical blood from 96 women with normal pregnancy were screened for HPV DNA by polymerase chain reaction (PCR), and HPV DNA in condyloma acuminata tissues, amniotic fluid of 31 pregnant women were detected by PCR as well. The results showed HPV DNA positive rate of condyloma acuminata tissues was 92.32%, of cervical secretion in normal pregnant group, was 35.71%, of peripheral blood was 57.69%. The HPV DNA maternal-fetus transmission rate was 44.44%, the transmission rate via placenta was 60%. It is concluded that HPV DNA can be found not only in condyloma acuminata tissues, but also in genital tract, peripheral blood of pregnant women, Besides the transmission route of genital tract, there is also the blood-placenta route.
Sequence analysis of the VP7 gene was performed on twenty-one human isolates of serotype 3 related-rotavirus in China and Japan. The five Chinese isolates were found to be not similar to the 16 Japanese isolates and to SA11 (simian rotavirus). The Chinese isolates, especially CHW2 and CH-32, were different from the major serotype 3 human isolates. AU-1 and 02/92 which previously showed a wider spacing between RNA segments 10 and 11 by RNA polyacrylamide gel electrophoretic analysis, were more closely related to each other and could be differentiated from the other Chinese and Japanese isolates. For these reasons, serotype 3 viruses were considered to be intraserotypically more heterologous than serotype 1, 2 and 4 viruses.
Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells.
The crystal structure of the DNA-actinomycin D (AMD) complex and a simple molecular modeling study indicated that AMD analogues derivatized at N-methyl-L-valine residues (fifth amino acid residue in the cyclic depsipeptide of AMD) could bind to DNA as strongly as the parent AMD. The analogues in which N-methyl-L-valine residues were replaced with L- and D-forms of N-methylvalines, N-methylthreonines, N-methylphenylalanies, N-methyltyrosines, and N-methyl-O-methyltyrosines have been totally synthesized. The characteristics of binding of the analogues to various DNAs including DNA-1 [d(TATATATGCATATATA)], DNA-2 [d(TATATACGCGTATATA)], DNA-3 [d(ATATATAGCTATATAT)], and DNA-4 [d(ATATATGGCCATATAT)] have been examined by using visible absorption spectrum methods. The association constants calculated from the absorption spectra indicate that the modifications of the N-methyl-L-valine residues in the AMD molecule do affect the DNA binding characteristics of the analogues. The L-aromatic analogues bind slightly better than the L-aliphatic analogues except for binding to DNA-1 (-TGCA-), whereas the D-aliphatic analogues bind consistently better than the D-aromatic analogues. In the L-form analogues, the L-Tyr analogue has the highest overall association constant, whereas the D-Val analogue has the highest association constant among the D-form analogues. In spite of substitution of bulky aromatic groups, the D-aromatic analogues bind to the DNA-1 quite well. However, D-aromatic analogues have significantly reduced their binding capacities to the other DNAs, indicating that the substitution of the D-aromatic residues creates a unique four-base sequence preference (-TGCA-). The RNA polymerase inhibitory activities of the AMD analogues in vivo have been examined using human cells (HeLa). All AMD analogues except for the L-Thr analogues severely inhibit RNA synthesis at relatively low drug concentrations. The D-Val, L-OMT, L-Phe, and D-Phe analogues inhibit RNA synthesis more strongly than the natural antibiotic (AMD itself).
BACKGROUND: gamma delta T cells, like alpha beta T cells, are components of all well-studied vertebrate immune systems. Yet, the contribution of gamma delta T cells to immune responses is poorly characterized. In particular, it has not been resolved whether gamma delta cells, independent of any other T cells, can help B cells produce immunoglobulin and form germinal centers, anatomical foci of specialized T cell-B cell collaboration. RESULTS: TCR beta-/- mice, which lack all T cells except gamma delta T cells, routinely displayed higher levels of antibody than fully T cell-deficient mice. Repeated parasitic infection of TCR beta-/- mice, but not of T cell-deficient mice, increased antibody levels and induced germinal centers that contained B cells and monoclonal gamma delta cells in close juxtaposition. However, antibody specificities were more commonly against self than against the challenging pathogen. gamma delta T cell-B cell help was not induced by repeated inoculation of TCR beta-/- mice with mycobacterial antigens. CONCLUSIONS: In the absence of any other T cells, gamma delta T cell-B cell collaboration can be significantly enhanced by repeated infection. However, the lack of obvious enrichment for antibodies against the challenging pathogen distinguishes gamma delta T cell help from alpha beta T cell help induced under analogous circumstances. The increased production of generalized antibodies may be particularly relevant to the development of autoimmunity, which commonly occurs in patients suffering from alpha beta T cell deficiencies, such as AIDS.
The solution structure and internal dynamics of the reactive-site (Lys44-Asp45 peptide bond) hydrolyzed form of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional 1H-15N NMR methods to the uniformly 15N-labeled protein. The 1H-15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V [Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J. J., Huang, J.-K., & Krishnamoorthi, R. (1996) Biochemistry 35, 1516-1524] and CMTI-V* [Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R. (1995) Biochemistry 34, 12087-12094] for which three-dimensional solution structures have been determined. It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V*. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and ¿1H¿-15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan N epsilon H groups in rCMTI-V*, and the model-free parameters [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570] were computed. Most of the backbone of rCMTI-V* is found to be highly constrained (S2 = 0.85), including the N-terminal residues 3-6 (S2 = 0.77). Residues 39-44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (S2 = 0.51); however, the N-terminal segment (residues 46-48) retains rigidity as in the intact form (S2 = 0.83). The S2 values, 0.78 and 0.59, respectively, of Arg50 and Arg52 side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them. This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants [Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S. P., Huang, J.-K., Liu, J., & Krishnamoorthi, R. (1996) Biochemistry 35, 4784-4794]. The dynamical results suggest the mainchain oxygen atom of Asp45 as the hydrogen bond acceptor of Arg50. Residues Trp9 and Trp54, which interact with many others in the protein scaffold and the binding loop region, respectively, remain rigid in the cleaved inhibitor with the S2 values of 0.84 and 0.71 determined for their respective N epsilon Hs. The internal dynamics of rCMTI-V* was compared with that of the noncovalent complex formed between the two fragments of reactive-site-hydrolyzed chymotrypsin inhibitor-2 from barley seeds [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233], another potato I family inhibitor that lacks the Cys3-Cys48 disulfide present in rCMTI-V*.
Signaling molecules are essential for vertebrate embryonic development. Here, two Xenopus homologs of the Drosophila gene fringe, lunatic Fringe (lFng) and radical Fringe (rFng), were identified and the protein product of lFng further characterized. The messenger RNA of lFng is supplied as a maternal message. Its product is a precursor protein consisting of pre-, pro-, and mature regions. The mature lunatic Fringe protein is secreted extracellularly, and it induced mesodermal tissue formation in animal cap assays. These results indicate that secreted lunatic Fringe can induce mesoderm and reveal that the Fringe proteins are a family of vertebrate signaling molecules.
To investigate the possibility that non-alpha beta T cell-dependent mechanisms can induce systemic autoimmune disease, and to address the roles of alpha beta T cells in murine lupus, we analyzed lupus-prone MRL mice congenitally deficient in alpha beta T cells. Surprisingly, TCR-alpha-/- MRL mice developed several characteristics of human systemic lupus erythematosus, including hypergammaglobulinemia, autoantibodies against DNA and small nuclear ribonucleoproteins, and immune deposits in kidneys. These results, which contrast with past studies concluding that MRL autoimmunity requires CD4+ alpha beta T cells, demonstrate that non-alpha beta T cell-dependent mechanisms are capable of inducing lupus phenomena, and further suggest that MRL disease may consist of both alpha beta T cell-independent and alpha beta T cell-dependent mechanisms.
The production of class-switched antibodies, particularly immunoglobulin (Ig) G1 and IgE, occurs efficiently in T cell receptor (TCR) alpha-/- mice that are congenitally devoid of alpha/beta T cells. This finding runs counter to a wealth of data indicating that IgG1 and IgE synthesis are largely dependent on the collaboration between B and alpha/beta T cells. Furthermore, many of the antibodies synthesized in TCR alpha-/- mice are reactive to a similar spectrum of self-antigens as that targeted by autoantibodies characterizing human systemic lupus erythematosus (SLE). SLE, too, is most commonly regarded as an alpha/beta T cell-mediated condition. To distinguish whether the development of autoantibodies in TCR alpha-/- mice is due to an intrinsic de-regulation of B cells, or to a heretofore poorly characterized collaboration between B and "non-alpha/beta T" cells, the phenotype has been reconstituted by transfer of various populations of B and non-alpha/beta T cells including cloned gamma/delta T cells derived from TCR alpha-/- mice, to severe combined immunodeficient (SCID) mice. The results establish that the reproducible production of IgG1 (including autoantibodies) is a product of non-alpha/beta T cell help that can be provided by gamma/delta T cells. This type of B-T collaboration sustains the production of germinal centers, lymphoid follicles that ordinarily are anatomical signatures of alpha/beta T-B cell collaboration. Thus, non-alpha/beta T cell help may drive Ig synthesis and autoreactivity under various circumstances, especially in cases of alpha/beta T cell immunodeficiency.
The side chains of Arg50 and Arg52 iin Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J.L-F., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys44-Asp45 peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CmTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on a reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate that Arg50 contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg50 and Arg52 were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: 15N NMR longitudinal and transverse relaxation rates and 15N-1H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in 15N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-59; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58 and 66 are found to be highly mobile, as the caluculated general order parameters, S2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg50 amd Arg52 are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hydrogen bonded. At pH 2.5, these hydrogen bonds are still retained with Arg50 appearing to be more restrained (S2 = 0.71) than Arg52 (S2 = 0.56). This is consistent a greater contribution by Arg50 to the conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the Arg50 and Arg52 side chains are not hydrogen-bonded to carboxylate groups, which would be protonated at pH 2.5 and, hence, unavailable for hydrogen-bonding interactions. The overall folding of rCMTI-V appears not to be significantly affected by the pH change, as indicated by comparisons of 1H and 15N chemical shifts, sequential NOE cross-peaks, and S2 values of the backbone atoms, and the conserved side-chain dynamics of Trp9 and Trp54--residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.
The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.
T cells play an important role in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse. CD8 cytotoxic T cell lines and clones were generated from the lymphocytic infiltrate in the islets of Langerhans of young (7-wk-old). NOD mice by growing them on (NOD x B6-RIP-B7-1)F1 islets. These cells proliferate specifically to NOD islets and kill NOD islets in vitro. The cells are restricted by H-2Kd, and all bear T cell antigen receptor encoded by V beta 6. When these CD8 T cell lines and clones are adoptively transferred to irradiated female NOD, young NOD-SCID, and CB17-SCID mice, diabetes occurs very rapidly, within 10 d of transfer and without CD4 T cells.
OBJECTIVE: To study the early effects of fetal development and remote sequalae of mental development of infants after intrauterine human cytomegalovirus (HCMV) infection. METHODS: Enzyme-linked immunoabsorbant assay (ELISA) were used for screening HCMV antibodies in the serum of 1419 pregnant women. HCMV DNA were examined by PCR in maternal blood, amniotic fluid and/ or umbilical blood for those with serum HCMV-IgM positive and some with HCMV negative. According to the results, group A (n = 53) included women with positive serum HCMV-IgM and HCMV DNA, and also positive HCMV DNA in amniotic fluid and/or umbilical blood: group B (n = 87) were women with positive serum HCMV-IgM and HCMV DNA but negative HCMV DNA in amniotic fluid and/or umbilical blood: group C (n = 34) served as control with all negative. The above 174 cases were carefully monitored for fetal development. Baley scales of infant development (BSID) were used to test both mental development index (MDI) and psychomotor development index (PDI) at 2-30 months after birth for babies in group A and C. RESULTS: The occurrences of fetal intrauterine death, congenital malformation, fetal growth retardation and neonatal asphyxia were significantly higher in group A than that in group B and C (P < 0.05) and the average birth weight and height were also significantly lower in group A (P < 0.05). The mental development of infants in group A were remarkably slower than that of group C (P < 0.01). CONCLUSION: Active HCMV intrauterine infection may cause congential infection, birth defects and mental retardation in childhood.