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Biomedical subjects

C S Hahn

Publications and source records attributed to C S Hahn.

18 recordsLinked to original sources
PubMed1992

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope.

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202-221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site.

Amino Acid Sequence↗
PubMed1992

Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.

Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.

Amino Acid Sequence↗
PubMed1992

The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus.

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

Amino Acid Sequence↗
PubMed1992

Free vascularized fibula grafting for the treatment of osteonecrosis of the femoral head.

A variety of joint-preserving operations have been devised to preserve the necrotic femoral head with varying success. Since 1979, the authors studied the effectiveness of vascularized fibula grafting in the treatment of osteonecrosis of the femoral head (ONFH) for joint preservation. Eighty-one of 121 hips with a minimum follow-up period ranged from three years to ten years eight months (mean, five years two months). All patients were evaluated clinically and roentgenographically on the basis of the causes and the stages of the disease. In the clinical assessment, 60 (74%) of 81 hips were rated excellent, 14 (17%) were rated good, six (7%) were rated fair, and one (2%) was rated poor. Overall satisfactory results, including excellent and good, were seen in 74 hips (91%). In the roentgenographic assessment, 57 hips (71%) had improved radiologically, 15 (18%) were unchanged, and nine (11%) were worse. Seventy-two hips (89%) showed roentgenographic improvement or unchange. Roentgenographic results had no significant correlation with the etiologic factors. Vascularized fibula grafting is one of the better alternatives for treating ONFH. It is highly expected that vascularized fibula grafting can prevent the necrotic femoral head from progressing to collapse and promote directly restored vascularization and new bone formation.

Adult↗
PubMed1991

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.

Amino Acid Sequence↗
PubMed1991

Evaluation of congenital colour vision deficiencies.

Three hundred patients who have congenital colour vision deficiencies were examined at the author's eye clinic for 3 years (1987-1990) using 5 types of colour vision tests: Hahn's, TMC's, Okuma's (new), H-R-R's colour vision tests and Double 15 Hue Test (Hahn). The results obtained from each test were quite different in type and grade, and the summarized results were considered to be the best: Type: protan 23.3%, deutan 76.0%, unclassified 0.7% Grade: mild 20.3%, medium 25.3%, strong 54.4% The frequency of coincidence both in type and grade between the summarized results and those of each test were compared, and the highest was 62.3% in Double 15 Hue Test. The efficiency of the author's colour vision test and Double 15 Hue Test were evaluated with the data in this clinical trial, and they were found to be useful for classifying the type and estimating the grade of the congenital and also acquired colour vision deficiencies.

Adolescent↗
PubMed1990

The effect of UVA irradiation on the epidermal melanocyte after 8-MOP and TMP administration.

Morphological and numerical changes in the epidermal melanocytes of black C57BL mice after phototoxic drug administration followed by ultraviolet A irradiation were studied to compare the effects of photochemotherapy on the epidermal melanocytes using 8-methoxypsoralen and trimethylpsoralen. One hour after intraperitoneal injection of the phototoxic drugs, 1.5 mg/kg in the small dose group and 6.0 mg/kg in the large dose group, the mice were exposed to UVA irradiation. This procedure was performed twice a week for 8 weeks at the small dose group and for 5 weeks in the large dose group. Skin biopsies were taken before irradiation in both groups, and follow up biopsies were done at each week. The number and size of the melanocytes were observed in a split-DOPA preparation. In the drug treated groups, there was an increase in the size of the perikaryon, and the number, length, width, and arborization of dendrites. Such changes were more clearly seen in the group treated with trimethylpsoralen compared with the 8-methoxypsoralen treated group. Therefore, trimethylpsoralen is more effective than 8-methoxypsoralen in the increase of the perikaryon size, and the number, length, width, and arborization of dendrites of melanocytes in the intraperitoneal injection.

Animals↗
PubMed1990

Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease.

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis.

Amino Acid Sequence↗
PubMed1989

Low pH-dependent Sindbis virus-induced fusion of BHK cells: differences between strains correlate with amino acid changes in the E1 glycoprotein.

Expression of alphavirus glycoproteins on the surface of infected cells leads to cell fusion after exposure to acidic pH. Two strains of Sindbis virus, AR339 (SV) and neuroadapted Sindbis virus (NSV), which differ in virulence for weanling mice, were found to differ in pH-dependent fusion. BHK-21 cells infected with SV fused maximally after shifting to pH 5.4, whereas cells infected with NSV required a lower pH, pH 4.8, for maximal fusion. No difference was noted in the optimal pH for agglutination of goose erythrocytes (5.75 for both viruses). To determine the molecular basis for the difference in fusion a series of recombinant viruses was constructed using a cDNA clone of Sindbis virus from which infectious RNA can be transcribed in vitro. Cells infected with a recombinant virus that had the SV E1 and NSV E2 genes had a fusion response curve as a function of pH like SV, while cells infected with recombinant virus with the NSV E1 and SV E2 genes fused like NSV. The E1 glycoproteins of SV and NSV differ at two positions: Val-72 in SV is Ala in NSV (a change near the putative fusion site), and Gly-313 in SV is Asp in NSV. Recombinant viruses which had Val-72 (SV) and Asp-313 (NSV) or Ala-72 (NSV) and Gly-313 (SV) had a lowered pH of fusion like NSV suggesting that both positions participate in determining some aspect of the conformational change in the E1-E2 heterodimer associated with pH-dependent fusion.

Animals↗
PubMed1989

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions.

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.

Animals↗
PubMed1988

Western equine encephalitis virus is a recombinant virus.

The alphaviruses are a group of 26 mosquito-borne viruses that cause a variety of human diseases. Many of the New World alphaviruses cause encephalitis, whereas the Old World viruses more typically cause fever, rash, and arthralgia. The genome is a single-stranded nonsegmented RNA molecule of + polarity; it is about 11,700 nucleotides in length. Several alphavirus genomes have been sequenced in whole or in part, and these sequences demonstrate that alpha-viruses have descended from a common ancestor by divergent evolution. We have now obtained the sequence of the 3'-terminal 4288 nucleotides of the RNA of the New World Alphavirus western equine encephalitis virus (WEEV). Comparisons of the nucleotide and amino acid sequences of WEEV with those of other alphaviruses clearly show that WEEV is recombinant. The sequences of the capsid protein and of the (untranslated) 3'-terminal 80 nucleotides of WEEV are closely related to the corresponding sequences of the New World Alphavirus eastern equine encephalitis virus (EEEV), whereas the sequences of glycoproteins E2 and E1 of WEEV are more closely related to those of an Old World virus, Sindbis virus. Thus, WEEV appears to have arisen by recombination between an EEEV-like virus and a Sindbis-like virus to give rise to a new virus with the encephalogenic properties of EEEV but the antigenic specificity of Sindbis virus. There has been speculation that recombination might play an important role in the evolution of RNA viruses. The current finding that a widespread and successful RNA virus is recombinant provides support for such an hypothesis.

Amino Acid Sequence↗
PubMed1988

Molecular basis of Sindbis virus neurovirulence in mice.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.

Age Factors↗
PubMed1987

Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences.

We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).

Base Sequence↗
PubMed1987

Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it.

We have sequenced the virulent Asibi strain of yellow fever virus and compared this sequence to that of the 17D vaccine strain, which was derived from it. These two strains of viruses differ by more than 240 passages. We found that the two RNAs, 10,862 nucleotides long, differ at 68 nucleotide positions; these changes result in 32 amino acid differences. Overall, this corresponds to 0.63% nucleotide sequence divergence, and the changes are scattered throughout the genome. The overall divergence at the level of amino acid substitution is 0.94%, but these changes are not randomly distributed among the virus protein. The capsid protein is unchanged, while proteins NS1, NS3, and NS5 contain 0.5% amino acid substitutions, and proteins ns4a and ns4b average 0.8% substitutions. In contrast, proteins ns2a and ns2b have 3.0 and 2.3% amino acid divergence, respectively. The envelope protein also has a relatively high rate of amino acid change of 2.4% (a total of 12 amino acid substitutions). The large number of changes in ns2a and ns2b, which are largely conservative in nature, may result from lowered selective pressure against alteration in this region; among flaviviruses, these polypeptides are much less highly conserved than NS1, NS3, and NS5. However, many of the amino acid substitutions in the E protein are not conservative. It seems likely that at least some of the difference in virulence between the two strains of yellow fever virus results from changes in the envelope protein that affect virus binding to host receptors. Such differences in receptor binding could result in the reduced neurotropism and vicerotropism exhibited by the vaccine strain.

Amino Acid Sequence↗
PubMed1985

Gianotti-Crosti syndrome associated with hepatitis B surface antigen (subtype adr).

The Gianotti-Crosti syndrome, papular acrodermatitis of childhood (PAC), is an infrequently recognized disorder with distinctive characteristics. At present hepatitis B virus is thought to be an etiologic agent. The disease is very rare in Korea, in spite of the high frequency of hepatitis B surface antigen (HBsAg) in the general population. It is known that subtype ayw of the HBsAg may influence the pathogenesis of PAC, but subtype analysis of HBsAg in these patients disclosed adr. Therefore, our studies reconfirmed that PAC may in addition be associated with subtype adr of HBsAg. We believe that the lower incidence of PAC in Korea as compared with the high incidence of PAC in other parts of the world, such as in the Mediterranean area, may be due to the fact that a higher predisposition to PAC is conferred by subtype ayw of HBsAg.

Acrodermatitis↗
PubMed1985

Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease.

We have cloned and sequenced the cDNA made to the region of RNA encoding the structural proteins of three complementation group C mutants of Sindbis virus, ts2, ts5, and ts13, and of their revertants. These mutants possess defects in the posttranslational processing of their structural proteins at the nonpermissive temperature. Comparison of the deduced amino acid sequences of the mutants with those of the revertants and with the parental HR strain of virus showed all three mutants to have single amino acid substitutions in the highly conserved COOH-terminal half of the capsid protein that give rise to temperature sensitivity. ts2 and ts5 were found to have the same lesion and thus represent independent isolations of the same mutant, whereas ts13 possessed a different change. Reversion to temperature insensitivity in all three mutants occurred by reversion of the mutated nucleotide to the parental nucleotide, restoring the original amino acid. It has been previously postulated that the capsid protein possesses an autoproteolytic activity that cleaves the capsid protein from the nascent polyprotein during translation. Comparison of the amino acid sequence of the capsid protein with that of serine proteases leads us to hypothesize that histidine-141, aspartate-147, and serine-215 of the Sindbis capsid protein form the catalytic triad of a serine protease. This hypothesis is supported by the finding that all three temperature-sensitive lesions mapped occur near these residues: ts2 and ts5 change proline-218 to serine and in ts13 lysine-138 has been replaced by isoleucine.

Animals↗