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Biomedical subjects

B Lee

Publications and source records attributed to B Lee.

At least 289 records · Page 16Linked to original sources
PubMed1995

The incidence of drug resistant tuberculosis in 1279 Korean patients.

OBJECTIVES: In the past decade, the incidence of tuberculosis has been decreased in Korea and the nationwide survey of tuberculosis from 1965 through 1990 suggested a declining tendency of resistant organisms. But the prevalence rate of multidrug resistance of Mycobacterium tuberculosis is still a serious problem in Korea, and the aim of this study is to check the drug resistance pattern in the patients visiting University Hospital, the 3rd referral center. METHODS: We reviewed 1279 cases (522 female, 757 male, mean age 39.4 +/- 16.7) of bacteriologically proven tuberculosis seen during the period from 1986 to 1992 retrospectively. Of 1093 patients, who were indentified in previous medical history, 454 (41.5%) had a history of prior antituberculous chemotherapy. RESULTS: Resistance rate (resistant to 1 or more drugs) was 33.9%. Eleven percent of patients had resistance to a single drug (INH: 80.6%). Twenty two percent of patients had resistance to 2 or more drugs. Resistance rate is higher (47.4%) in the patients with a history of prior treatment than without a history (25.5%). CONCLUSIONS: These data suggest that the high rate of multidrug resistance in Korea did not show any decreasing tendency. So, mycobacterial culture and sensitivity tests should be recommended at initial treatment of tuberculosis and potent antituberculosis drugs are strongly recommended.

Adult↗
PubMed1995

Breastfeeding.

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Attitude of Health Personnel↗
PubMed1995

Molecular cloning of the E-cadherin cDNAs from rabbit corneal epithelium.

Cadherins are glycoproteins that are members of the superfamily of Ca(2+)-dependent cell adhesion molecules. They are located in the adherens junctions and play an important role in cell-cell interactions that influence cell polarity, cell migration, cell sorting, and morphogenesis. To gain a better understanding of the molecules that mediate cell-cell interactions in corneal epithelium, we wished to characterize the E-cadherin from rabbit corneal epithelium. Using degenerate primers designed against an extracellular region that is highly conserved between the E-cadherin cDNA sequences of mouse and human, reverse transcription-polymerase chain reaction (RT-PCR) was performed on RNA isolated from rabbit corneal epithelial cell cultures. A 381 bp rabbit E-cadherin cDNA fragment that was amplified, was cloned and sequenced. This rabbit E-cadherin cDNA fragment was used as a probe for northern blot analysis of rabbit corneal epithelial cell cultures and subsequently, to isolate rabbit E-cadherin cDNA clones from a rabbit corneal epithelial cDNA library constructed in lambda UniZAP. A 4.3 kb transcript was detected in the northern blots of poly(A) RNA from confluent rabbit corneal epithelial cell cultures. The coding sequence of the 4.3 kb full-length rabbit E-cadherin cDNA was determined from overlapping clones. The deduced 886 amino acid sequence of the rabbit E-cadherin cDNA shows linear organization of the polypeptide into an N-terminal prosequence, five extracellular repeating domains, a transmembrane region, and a cytoplasmic domain at the C-terminus. Thus the rabbit E-cadherin from cornea is a classical member of the cadherin family and shows about 90% amino acid similarity to mouse and human E-cadherins. In the present study, in addition to the characterization of the 4.3 kb rabbit E-cadherin cDNA, a 2.3 kb E-cadherin clone was also isolated from the cDNA library. The 2.3 kb truncated cDNA was found to lack the DNA sequences encoding the transmembrane, the cytoplasmic, and the third, fourth, and fifth extracellular domains that are present in the classical rabbit E-cadherin. Transcripts corresponding to the truncated E-cadherin cDNA were detected by RT-PCR amplification of rabbit corneal epithelial cell culture RNA, but could not be detected by northern analysis, suggesting that message for this putative truncated E-cadherin may be expressed at low levels.

Amino Acid Sequence↗
PubMed1994

Rapid humanization of the Fv of monoclonal antibody B3 by using framework exchange of the recombinant immunotoxin B3(Fv)-PE38.

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of carcinoma-specific antibody B3 is fused to a truncated form of Pseudomonas exotoxin (PE). The efficacy of monoclonal antibody B3 and B3 immunotoxins in cancer therapy and diagnosis may be limited by the human anti-mouse response. Here we describe the humanization of the Fv of B3(Fv)-PE38 by "framework exchange." The variable domains of the heavy (VH) and light (VL) chains were aligned with their best human homologs to identify framework residues that differ. Initially, 11 framework residues in VH and six in VL were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled single-chain Fv expression cassette. Six VH and five VL residues that differ were not changed because they were buried, in the interdomain interface, or previously found to result in decreased affinity when mutated. This basic design resulted in some 20-fold loss of activity. Changing VL residues at the interdomain interfacial position 100 and at the buried position 104 to the human sequence increased the activity 8-fold. Changing VH residue at position 82b from the human sequence back to that of the mouse restored the activity 2- to 3-fold to the full binding and cytotoxic activity of the mouse sequence. Humanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.

ADP Ribose Transferases↗
PubMed1994

Alignment of beta-barrels in (beta/alpha)8 proteins using hydrogen-bonding pattern.

A multiple alignment procedure for aligning the beta-sheet residues of the (beta/alpha)8-barrel structures is described. It uses a two-dimensional numbering scheme which is based on the covalent and hydrogen-bonding pattern of the beta-sheet. Two different scoring functions were used: one measured the sequence and topological similarity and the other the root-mean-square deviation of the coordinates of the matched residues. The procedure was applied to obtain multiple alignments of the beta-barrels of ten (beta/alpha)8-barrel proteins of known structure. Two kinds of alignments were derived: one in which the beta-strand numbering was preserved and another in which the beta-strands were allowed to be cyclically permuted. It is shown that-preservation of the beta-strand numbering corresponds to aligning only the layer structure of the beta-barrels. In order to obtain the optimal rotational alignment of the barrels as well, the beta-strands must be allowed to be renumbered. Although the 2-fold or 4-fold rotational symmetry of the beta-barrels makes it difficult to obtain unique rotational alignment of the barrels, the results of the alignment indicate that the beta-strands in the beta-barrel of enolase, xylose isomerase, taka-amylase, and possibly fructose biphosphate aldolase, must be cyclically permuted in order to be optimally aligned to those of the other proteins, which include triose phosphate isomerase, the alpha-subunit of tryptophan synthetase, flavocytochrome b2, ribulose-1, 5-biphosphate carboxylase/oxygenase, and glycolate oxidase.

Amino Acid Sequence↗
PubMed1994

Site-specific conjugation to interleukin 4 containing mutated cysteine residues produces interleukin 4-toxin conjugates with improved binding and activity.

Fusion of a ligand to another protein frequently impairs the binding of the ligand. Recombinant toxins composed of mutants of Pseudomonas exotoxin (PE) fused to the C-terminus of human interleukin 4 (IL4) are cytotoxic to IL4 receptor- (IL4R-) bearing tumor cells but bind to the IL4R with only 1% the affinity of IL4. We have developed a method to connect a toxin to a ligand which allows the junction to be moved to a location on the ligand which would minimize the binding impairment. We designed mutants of IL4 in which residue 28, 38, 68, 70, 97, or 105 was substituted with cysteine. All purified mutants bound to the IL4R with 60-100% the affinity of IL4, indicating that the IL4 structure was essentially unchanged. The IL4 mutants were then each conjugated through a disulfide bond to PE35, a truncated form of PE which contains a single cysteine. IL4 conjugated to PE35 at residue 28, 38, or 105 of IL4 bound with 10-fold improved affinity and was 10-fold more cytotoxic than the recombinant IL4-toxin in which PE is fused to position 129 at the C-terminus of IL4. IL4 containing PE35 conjugated at position 68, 70, or 97 had lower binding affinity and cytotoxic activity. These results indicate that the location of the ligand-protein junction can be selectively moved to enhance conjugate effectiveness, and implications could be made regarding which regions of IL4 are important for binding.

ADP Ribose Transferases↗
PubMed1994

Methylation- and mutation-dependent stimulation of Alu transcription in vitro.

Alu genes are GC-rich, highly repetitive genetic elements whose functions remain unknown. Members of this family are readily transcribed in vitro by RNA polymerase III, but RNA corresponding to only a small sub-set of Alu elements has been found in vivo. Based on the hypothesis that methylation of Alu elements affects their transcription, the transcriptional activity of unmethylated and methylated template DNA was assessed in vitro. It was found that methylation of a single CG site just 5' to Alu functions to stimulate transcription; the base composition in this region also affects transcriptional activity. These results indicate that the methylation state and sequence of DNA flanking Alu elements influence its transcription rate.

Base Sequence↗
PubMed1994

Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment.

e23(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of anti-erbB2 monoclonal antibody e23 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the inherently unstable Fv heterodimer (composed of VH and VL) is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have now found that e23(dsFv)-PE38KDEL is considerably more cytotoxic to antigen-positive cell lines than the corresponding single-chain immunotoxin. The basis for the enhanced cytotoxic activity is that the e23 dsFv-immunotoxin binds to erbB2 with greater affinity than the single-chain counterpart. The dsFv-immunotoxin had 4-fold increased binding compared to the scFv and almost identical to the binding affinity of e23 Fab. e23(dsFv)-PE38KDEL was also considerably more stable at 37 degrees C than the single-chain immunotoxin. The therapeutic potential of the disulfide-stabilized immunotoxin was compared with its single-chain counterpart using two animal models of immunodeficient mice bearing subcutaneous tumor xenografts of human gastric tumor N87 cells or human A431 epidermoid carcinoma cells. The antitumor effect of e23(dsFv)-PE38KDEL was significantly better than that of the single-chain immunotoxin. e23(dsFv)-PE38KDEL caused complete regression of tumors at doses which caused no toxic effects in mice, whereas the single-chain immunotoxin did not cause complete regressions at the same doses.

ADP Ribose Transferases↗
PubMed1994

Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions.

Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗
PubMed1994

A comparison of in vitro skin-penetration cells.

A new low-volume flow-through diffusion cell (LVFC) was designed to provide accurate determinations of penetrant flux across skin while minimizing the dilution of penetrant in receptor fluid and eliminating the need for magnetic stirring. The performance of the 0.3-mL LVFC was compared to a magnetically stirred, 4.3-mL high-volume flow cell (HVFC) and to a magnetically stirred, manually sampled 7.5-mL static cell (SC) with hydrophilic and lipophilic penetrants. The clearance of 14C-labeled benzoic acid from the LVFC and HVFC followed an exponential profile expected for complete mixing when the LVFC and HVFC were run at flow rates of 0.4-0.9 and 4.0-5.2 mL/h, respectively. The in vitro dispositions of 14C-labeled benzoic acid and estradiol were determined in the LVFC and HVFC by applying the compounds to split-thickness pig skin at a 4 micrograms/cm2 dose. Additionally, the effects of receptor fluid flow rate (1.2 vs 3.5 cell volumes/h) and method of skin attachment (O-ring vs compression) were determined on disposition in the HVFC. The percutaneous penetration of benzoic acid and the residue of estradiol within skin did not differ between the LVFC and HVFC. However, the percutaneous penetration of benzoic acid increased significantly (p < 0.05) using the O-ring attachment as compared to compression at a flow rate of 1.2 cell volumes/h. The in vitro permeation of benzoic acid-saturated water and 17 beta-estradiol-saturated propylene glycol monolaurate through human epidermis was compared between the LVFC, HVFC, and SC. The LVFC and HVFC had flow rates of 0.9-1.0 mL/h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3.

The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S. DHHS, NIH publication No. 91-3242, 1991). This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.

ADP Ribose Transferases↗
PubMed1994

Effects of early and late treatment with anti-CD4 monoclonal antibody on autoimmune disease in MRL/MP-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation, with target organ inflammatory lesions composed largely of CD4+ (helper) T cells. Previous reports have demonstrated that anti-CD4 monoclonal antibody (mAb) treatment of MRL/lpr mice from 1 to 5 months of age resulted in a dramatic reduction in both the frequency and the severity of autoimmune disease. In order to investigate the effects of early, short-course and late, short-course anti-CD4 mAb therapy on the autoimmune disease in MRL/lpr mice, groups of 12 to 15 animals were treated with weekly intraperitoneal injections according to one of four regimens: (i) anti-CD4 mAb from age 1 to 5 months (continuous treatment); (ii) anti-CD4 mAb from age 1 to 3 months (early treatment); (iii) anti-CD4 mAb from age 3 to 5 months (late treatment); and (iv) either normal saline or rat immunoglobulin (control treatment). Continuous treatment resulted in a dramatic reduction of both frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Early treatment also resulted in a significant reduction in autoimmune disease, while late treatment had little effect. Glomerulonephritis was detected in none of the animals in the continuously treated group (P < 0.05), 38% of those in the early-treated group (P = < 0.05), 92% of the late-treated group, and 100% of controls. The titer of antinuclear antibodies, of anti-dsDNA antibodies, and total immunoglobulin levels were all significantly reduced in the continuous-treatment and early-treatment groups, but not in the late-treatment group. Murine antibodies to rat anti-CD4 mAb were present in the late-treatment group. These results indicate that early short-course anti-CD4 mAb treatment of MRL/lpr mice is effective in ameliorating the autoimmune disease in this model, while late-treatment is ineffective, probably due to the induction of antibody directed against anti-CD4 mAb itself.

Animals↗
PubMed1994

Sex-related differences in iodothyronine metabolism in the rat: evidence for differential regulation among various tissues.

Various aspects of thyroid hormone metabolism were examined in vitro in age-matched (experiment I) and weight-matched (experiment II) male and female Sprague-Dawley rats; unless specified otherwise, results were similar in both experiments. The activity and content of iodothyronine 5'-monodeiodinase (type I-MD) in the liver of the female rat were markedly reduced, but there was no sex-related difference in these parameters in the kidney. The activity of the brain type III-MD was also not significantly influenced by the sex of the rat. Hepatic triiodothyronine (T3) sulfation activity in the females was only about 20% of that of the males. However, kidney and brain did not show this decrease in T3 sulfation. Similarly, hepatic T3 sulfate (T3S) desulfation activity was significantly reduced in the liver of the female rat (P < .001), whereas the activity in the kidney was either similar to (experiment I) or higher than (experiment II) that in the male, and the activity in the brain was similar in the two sexes. The mean serum T3S concentration in the female rat was no greater than 25% of the corresponding value measured in the male rat. The mean serum thyroxine (T4) concentration in female rats was similar to that in age-matched males (experiment I), whereas it was somewhat lower than that in weight-matched males (P < .05, experiment II). No significant difference in the mean serum T3 concentration was observed in rats of female and male sex. However, the mean serum thyrotropin (TSH) concentration in the female rat was significantly lower than that in the male.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Relation between volume correction and the standard state.

The suggestion by Sharp et al. (Biochemistry 30 (1991) 9686) that a volume correction should be made to the standard chemical potential of a non-polar solute molecule in water is examined by re-deriving their results using an exact statistical mechanical procedure and comparing them with those of a couple of other similar schemes. It is shown that their suggestion is equivalent to choosing a standard state based on the ideal gas behavior.

Chemical Phenomena↗