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T T Wu

Publications and source records attributed to T T Wu.

At least 19 recordsLinked to original sources
PubMed1992

Subgroups of Tcr alpha chains and correlation with T-cell function.

T-cell receptor (Tcr) alpha chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93-103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, and 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr alpha chains seems possible.

Amino Acid Sequence↗
PubMed1992

Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis.

We have examined the consequences on duck hepatitis B virus DNA synthesis of deleting the 5' and 3' copies of the 12 base sequence, DR1, from the viral pregenome. With the wild-type virus, reverse transcription initiates at nt 2537 within the 3' copy of DR1. When this sequence was deleted, initiation of reverse transcription was found at two other sites located closer to the 3' end of the pregenome (nt 2576 and nt 2644). The 3-base motif UUA was the only sequence common to these sites as well as the wild-type initiation site in DR1. Deletion of the 5' copy of DR1 did not alter minus strand synthesis, but led to aberrant priming of plus strand synthesis to generate predominantly linear rather than relaxed circular, double-stranded viral DNA, in agreement with the recent report by Loeb et al. (EMBO J. 10, 3533-3540, 1991). A mutant lacking only the 3' copy of DR1 rapidly converted to wild type in transfected cells. This apparently occurred as a consequence of conversion of newly synthesized relaxed circular to covalently closed circular (CCC) DNA, which might then serve as a template for the synthesis of wild-type viral RNAs. A mutant lacking only the 5' copy of DR1 did not exhibit this behavior. These results support the conclusion that amplified CCC DNA serves as transcriptional template.

Animals↗
PubMed1992

Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies.

We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.

Amino Acid Sequence↗
PubMed1992

Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.

Age Factors↗
PubMed1991

Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites.

By examining a large database of amino acid sequences of antibodies of various specificities, we have found that many antibodies of distinctly different specificities assemble identical VL domains with different VH domains. In contrast, rarely is the same VH domain found in sets of antibodies of different specificities. We identified additional sets of antibodies of different specificities and identical sequences covering amino acid residues VH 1 to 94 and VL 1 to 95. In addition, there were segments of additional antibodies for which complete sequences were not available, but identities were seen in VL CDR1, VL CDR2, and VL CDR3 up to the VL-JL junction. The finding that there are many identical VL 1 to 95 segments with different VH 1 to 94 sequences, and vice versa, raises important questions as to the role of VH in influencing the conformation of VL and, conversely, the role of VL in influencing the conformation of VH. Evidence is also cited indicating that a single amino acid change may seriously disrupt site structure and in some instances abolish binding. Our findings suggest that it will be important in the future to investigate further conformational effects on antibody structure by using X-ray crystallography, nuclear magnetic resonance spectroscopy, or other methods, to obtain a better understanding of the functions and topography of antibody-combining sites.

Amino Acid Sequence↗
PubMed1991

Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis.

Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.

Base Sequence↗
PubMed1991

Autosomal dominant polycystic kidney disease: an unusual presentation as unilateral renal mass in the infant.

The onset of autosomal dominant polycystic kidney disease in infants and children is unusual, and renal involvement is typically bilateral. The presentation of a unilateral renal mass in such a disorder is extremely rare. We report a 2-month-old infant with autosomal dominant polycystic kidney disease presenting with unilateral renal involvement; the literature concerning this entity is reviewed.

Female↗
PubMed1990

Antigenic and functional analysis of a neutralization site of HSV-1 glycoprotein D.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies (MAbs) against this protein can be arranged in groups, on the basis of a number of biological and biochemical properties. Group I antibodies are type-common, have high complement-independent neutralization titers, recognize discontinuous (conformational) epitopes, and block each other in a binding assay. The sum of their epitopes constitutes antigenic site I of gD. Using a panel of neutralization-resistant mutants, we previously found that group I MAbs can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies, and vice versa. Antigenic site I therefore consists of two parts, Ia and Ib. We have now identified the point mutations which prevent neutralization. Two Ib MAbs (DL11 and 4S) selected a Ser to Asn change at residue 140; this alteration creates a new N-linked glycosylation site, which is used. A third Ib MAb (D2) selected a Gln to Leu change at 132. The mutation selected by the Ia MAb HD1 (Ser to Asn at residue 216) is identical to that selected by MAb LP2, another Ia antibody. By using oligonucleotide-directed mutagenesis, we have produced gD genes with combinations of the above mutations. Attempts to recombine these genes into the virus genome were unsuccessful, suggesting that the combinations are lethal. This was confirmed by a complementation assay which measures the ability of gD transiently expressed in transfected Vero cells to rescue the production of infectious virus by the gD-minus mutant F-gD beta.

Base Sequence↗
PubMed1990

In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway.

During the productive phase of chronic hepadnaviral infections, virion DNA synthesis occurs in the cytoplasm of the infected hepatocyte, but viral RNA is synthesized in the nucleus, apparently from a covalently closed, circular (CCC) viral DNA. J. Tuttleman, C. Pourcel, and J. Summers (1986a, Cell 47, 451-460) have shown that the intracellular levels of CCC DNA can increase during initiation of infection of duck hepatocytes in vitro with duck hepatitis B virus and during long term culture of infected duck hepatocytes in vitro. This amplification of CCC DNA occurs through the reverse transcription pathway. To distinguish between an entirely intracellular process of amplification and amplification due to multiple infections by extracellular virus in the virus producing cultures, suramin was added to the infected cultures to block superinfection. We found that CCC DNA amplification occurred at least as efficiently in the presence of suramin as in its absence. First, there was a net increase in the total amount of CCC DNA in the cultures both in the presence and in the absence of suramin. Second, synthesis of CCC DNA in the presence and absence of suramin was observed by density labeling of this viral DNA by growth of the cultures in medium containing BUdR. Amplification was also demonstrable in the presence of neutralizing duck antibodies. These results support the hypothesis of Tuttleman et al. (1986a) that CCC DNA amplification in chronically infected cultures and, by inference, the mechanism of persistent infection involves primarily intracellular regulatory mechanisms.

Animals↗
PubMed1990

Efficient duck hepatitis B virus production by an avian liver tumor cell line.

Duck hepatitis B virus (DHBV) is produced in small amounts following transfection of human hepatoma or hepatoblastoma cell lines with cloned viral DNA. In a search for better hosts for DHBV replication, two avian liver cell lines were investigated. One of these cell lines, LMH, produced 5 to 10 times more DNA replicative intermediates and 10 to 20 times more infectious DHBV than did either of the two human cell lines, HuH-7 and Hep G2. Utilization of cell lines in genetic analyses of virus replication is often dependent upon obtaining efficient complementation between cotransfected viral genomes. We assayed transcomplementation of a viral polymerase (pol) gene mutant, which is rather inefficient in transfected human cells, and found that viral DNA synthesis was at least 20 times more efficient following cotransfection of LMH cells than in similarly transfected HuH-7 cells. Recombination, a potential interpretation problem in complementation assays, occurred at low levels in the cotransfected cultures but was substantially reduced or eliminated by creation of an LMH subline stably expressing the viral polymerase. This cell line, pol-7, supported the replication of DHBV pol mutants at ca. 10 to 15% of the level of virus replication obtained following transfection with wild-type viral DNA. By transcomplementation of a pol gene mutant in LMH cells, we were able to produce sufficient virus with the mutant genome to investigate the role of polymerase in covalently closed circular DNA amplification. Our results substantiate the hypothesis that covalently closed circular DNA is synthesized by the viral reverse transcriptase.

Animals↗
PubMed1979

Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

Amino Acid Sequence↗
PubMed1979

Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.

A comparison between the cloned mouse DNA segments that were found to code for the lambda and kappa light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNA's of phage phi X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the mini-gene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.

Amino Acid Sequence↗
PubMed1978

Variable region genes for the immunoglobulin framework are assembled from small segments of DNA--a hypothesis.

Sequences of each of the four framework segments FR1, FR2, FR3, and FR4 of the variable regions (V-regions) of light and heavy chains of immunoglobulins were grouped into sets with identical sequences. Sets contained from 1 to 18 members. When each V-region was traced from one FR to the next, it was seen that members of the same set in FR1 could be associated with different sets in FR2, FR3, and FR4. This suggests that the framework for the light and heavy chain V-regions is assembled during embryonic development from sets of minigenes for each FR segment. FR4 from three sets of human V(k)I chains also contained members of V(k)II, V(k)III, and V(k)IV subgroups; one FR2 set contained eight rabbit V(k), one human V(k)IV, and four mouse V(k) and an FR4 set contained two human V(H)III and one mouse V(H)III, indicating substantial evolutionary preservation of these sequences and suggesting that the sets of minigenes are highly conserved in the germ line. The clone of Tonegawa et al. [Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, O. & Gilbert, W. (1978) Proc. Natl. Acad. Sci. USA 75, 1485-1489] could be a hybrid FR1 and FR3 coming from minigenes of MOPC 315 (a V(lambda)II) whereas FR2 would come from MOPC 104E (a V(lambda)I). That FR4 is not joined to the rest of the V-region in 12-day-old mouse embryo DNA is also in accord with this hypothesis. Mouse sperm DNA should be examined to establish whether the hypothesized minigenes are separated by intervening sequences and whether the complementarity-determining (hypervariable) regions or segments of the V-region are separated from the framework in genomic DNA. Sperm DNA from rabbits or other species could be used to search for minigene segments whose sequences are identical in several species.

Animals↗
PubMed1978

Velocity sedimentation in isotonic saline and sucrose buffer as a possible method of fractionating normal erythrocytes.

On velocity sedimentation in isotonic saline and sucrose buffer, normal human erythrocytes could be fractionated into many different populations. The fastest sedimenting cells consisted entirely of normal discocytes, had a tendency to form clusters of eight to ten cells, were less fragile, and seemed to be separable into two distinct groups of different buoyant densities. The slowest sedimenting cells were echinocytes, did not clump, showed extreme fragility, and were of similar density.

Cell Separation↗