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Biomedical subjects

B Lee

Publications and source records attributed to B Lee.

At least 199 records · Page 11Linked to original sources
PubMed1999

Spatial relationship of motion automated perimetry and optic disc topography in patients with glaucomatous optic neuropathy.

PURPOSE: To compare the spatial relationship of focal motion automated perimetry (MAP) visual field defect with focal defect in optic disc topography. METHODS: Patients (n = 12) with focal MAP visual field loss and focal change in optic disc topography were studied. The MAP visual field was divided into 12 field zones representing retinal nerve fiber layer arcuate bundles. Zones of MAP loss were related to rim area ratio (RAR), which was obtained by dividing the rim area, measured by the Heidelberg Retina Tomograph (HRT; Heidelberg Engineering, Heidelberg, Germany), into 36 10 degrees sectors and then dividing the area of each sector by the total rim area for each subject. Rim area ratio was compared to a normative database (n = 76) to quantify change in optic disc topography. In these same patients, the spatial relationship between standard automated perimetry (SAP) and short-wavelength perimetry (SWAP) and optic disc topography was also assessed. RESULTS: Motion automated perimetry superior visual field zones 14 through 19 were most often associated with a reduction in RAR for inferior sectors 24 through 29, and inferior visual field zones 4 through 7 were most often associated with a reduction in RAR for superior temporal sectors 11 through 16. Similar spatial relationships were found between SWAP and SAP and the RAR. CONCLUSION: Focal MAP visual field loss and focal changes in optic disc topography are spatially related. This relationship is similar to that found between SWAP and SAP with optic disc topography. Focal thinning or notching detected by RAR analysis might be independent of the specific functional test employed.

Glaucoma, Open-Angle↗
PubMed1999

Smart technology improves patient-controlled analgesia: a preliminary report.

UNLABELLED: A new adaptive system has been designed to improve patient-controlled analgesia through the use of a variable bolus dose and a variable background infusion of analgesic. A novel hand set allows patients to rate their own pain on a linear scale of 1 to 10. Data derived from the hand set signals are used by an expert algorithm to repeatedly adapt the drug dosage of the bolus and of the background infusion according to both current pain intensity and the patient's response to previous dosage. To test the system, we performed a small pilot clinical study, using a randomized, double-blinded, cross-over design. The new system was alternated with a conventional system every 12 h. Use of the new system was associated with significantly lower pain scores and fewer bolus requests but more analgesic administration, though without increased adverse effects. It was very well accepted by both patients and clinical staff. IMPLICATIONS: Pain relief after surgery is often best provided by patient-controlled analgesia, which uses an IV infusion pump and a patient-activated switch. We have developed a new computer-controlled or "smart" patient-controlled analgesia that rapidly learns a patient's individual needs and provides continuously tailored pain relief.

Adolescent↗
PubMed1998

Genetic acceleration of AIDS progression by a promoter variant of CCR5.

The CCR5 gene encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for macrophage-tropic (R5) strains of human immunodeficiency virus-type 1 (HIV-1). Genetic association analysis of five cohorts of people with acquired immunodeficiency syndrome (AIDS) revealed that infected individuals homozygous for a multisite haplotype of the CCR5 regulatory region containing the promoter allele, CCR5P1, progress to AIDS more rapidly than those with other CCR5 promoter genotypes, particularly in the early years after infection. Composite genetic epidemiologic analyses of genotypes bearing CCR5P1, CCR5-Delta32, CCR2-64I, and SDF1-3'A affirmed distinct regulatory influences for each gene on AIDS progression. An estimated 10 to 17 percent of patients who develop AIDS within 3.5 years of HIV-1 infection do so because they are homozygous for CCR5P1/P1, and 7 to 13 percent of all people carry this susceptible genotype. The cumulative and interactive influence of these AIDS restriction genes illustrates the multigenic nature of host factors limiting AIDS disease progression.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins.

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell-cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell-cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.

Animals↗
PubMed1998

Improved stability and yield of a Fv-toxin fusion protein by computer design and protein engineering of the Fv.

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 VH and VL with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the VH (T82 and A85) and two in the VL (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs.

Amino Acid Sequence↗
PubMed1998

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus.

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Amino Acid Sequence↗
PubMed1998

Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery.

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.

Adenoviridae↗
PubMed1998

Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: conformational influence of an engineered disulfide bond.

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (BldsFv) crystallizes in space group P6(1)22 with the unit cell parameters a = b = 80.1 A, and c = 138.1 A. The crystal structure of the BldsFv has been determined at 2.1-A resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A and in bond angle of 2.74 degrees. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-A wide and 17-A long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab.

Amino Acid Sequence↗
PubMed1998

The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains.

Murine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Krüppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abundant in the developing central nervous system, and in the adult mouse expression is restricted largely to testis and brain. Here we show that at the protein level ZFP-37 is detected readily in neurons of the adult central nervous system but hardly in testis. In brain ZFP-37 is associated with nucleoli and appears to contact heterochromatin. Mouse and human ZFP-37 have a basic histone H1-like linker domain, located between KRAB and zinc finger regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a structural protein of the neuronal nucleus which plays a role in the maintenance of specialized chromatin domains.

Amino Acid Sequence↗
PubMed1998

Inhibitory effect of F-1322 on allergic eosinophil infiltration in airways.

The effects of F-1322 (N-[2-[4-(benzhydryloxy)piperidino]ethyl]-3-hydroxy-5-(3-pyridy lmethoxy)-2-naphthamide) on antigen-induced eosinophil infiltration and interleukin-5 production in the airways and on in vitro eosinophil migration were investigated. F-1322 (10-30 mg/kg, p.o.) inhibited antigen-induced eosinophil infiltration and interleukin-5 production in the airways of sensitized mice in a dose-dependent manner. Furthermore, F-1322 (0.1-10 microM) prevented the in vitro migration of eosinophils from guinea-pigs and humans induced by recombinant human interleukin-5, platelet-activating factor, and leukotriene B4 in a concentration-dependent manner. These results indicate that F-1322 has an inhibitory effect on allergic eosinophil infiltration of the airways by preventing both eosinophil migration and interleukin-5 production. These pharmacological profiles suggest that F-1322 will be a useful therapeutic for allergic diseases, especially asthma.

Animals↗
PubMed1998

Sensitive liquid chromatographic technique to measure isoniazid in alveolar cells, bronchoalveolar lavage and plasma in HIV-infected patients.

The need to monitor the effectiveness of antimicrobial drugs in treating opportunistic infections such as tuberculosis in HIV-infected patients requires the development of sensitive assays. A suitable HPLC method was developed to measure the concentration of isoniazid (INH) in plasma 1 h after a standard 300 mg dose and to detect the low levels typically found in alveolar cells obtained by bronchoalveolar lavage of subjects maintained on a standard regimen of the drug. Following extraction with a chloroform-butanol mixture, the INH was back-extracted into dilute acid which was subsequently analyzed by HPLC using a CN reversed-phase column and an acetonitrile-isopropanol based mobile phase. Another HPLC method was developed using direct injection and a polymer based column to measure minute concentrations of INH in the cell-free lavage. In both systems, detection of the drug was accomplished with a sealed coulometric detector (+0.6 V) capable of giving a consistent daily response without adjustment. When saline, cellular extracts and plasma from untreated subjects were spiked with various amounts of INH and analyzed, the lowest level of quantitation was 10, 25 and 100 ng/ml, respectively. Calibration curves showed good linearity when spiked concentrations were compared to peak areas (r=0.991, 0.993 and 0.998, respectively). Alveolar cell extracts and cell-free bronchoalveolar fluid from HIV-positive patients maintained on a standard INH regimen had detectable levels of INH 4 h after a 300 mg oral dose. The plasma INH at 1 h had a range of 0.3-7.1 microg/ml (n = 50). Precision studies with plasma spiked at 0.1, 0.5, 1.0 and 5.0 microg/ml revealed within-run coefficients of variation (C.V.s) of 8.9, 7.2, 4.2 and 4.9%, respectively and analytical recoveries of 97, 108, 108 and 98%, respectively. The day-to-day C.V.s for the plasma method were 7.6, 4.9 and 3.8% at concentrations of 0.5, 1.0 and 3.0 microg/ml, respectively. The results suggest that this rugged HPLC technique can quantitate INH in 1 h plasma with good precision and can be used to estimate the very low INH concentrations found in alveolar cells and cell-free lavage recovered from patients undergoing anti-tuberculosis therapy.

Administration, Oral↗
PubMed1998

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis.

A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.

Cloning, Molecular↗
PubMed1998

An intricate Web: chemokine receptors, HIV-1 and hematopoiesis.

Cellular infection by the human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with CD4 and at least one additional cell surface molecule, termed a "cofactor" or "coreceptor." Recent discoveries have determined that macrophage-tropic strains of HIV-1 which are largely responsible for sexual transmission require the beta-chemokine receptor CCR5 in addition to CD4, while the T cell tropic viruses that emerge later after infection use the alpha-chemokine receptor CXCR4. Thus, both CD4 and the appropriate chemokine receptor must be expressed on the cell surface in order for HIV-1 to enter the cell and establish an infection. The in vivo importance of CCR5 for HIV-1 is demonstrated by the finding that individuals homozygous for a 32 bp deletion (delta 32) in the CCR5 gene that renders them effectively CCR5-negative are highly resistant to virus infection. In this review, the structure-function correlates of the chemokine receptors that serve as major coreceptors for HIV-1 and simian immunodeficiency virus entry will be reviewed. Since certain chemokines have been implicated as stem cell inhibitory factors, the biological consequences of chemokine receptor expression as it relates to HIV-1-associated hematodyspoiesis will also be discussed.

Animals↗
PubMed1998

Genomic organization, expression of the human CBFA1 gene, and evidence for an alternative splicing event affecting protein function.

The Cbfa1 gene, which encodes the transcription factor Osf2/Cbfa1 required for osteoblast differentiation in mouse and human, is mutated in cleidocranial dysplasia, a skeletal dysplasia. We describe here the isolation of the full-length human OSF2/CBFA1 cDNAs, the genomic organization of the entire CBFA1 gene, its expression, and the existence of an alternative splicing event. Nucleotide sequence analysis of the human and mouse OSF2/CBFA1 cDNAs showed a 98% homology in the coding sequence and 96% in the 5' untranslated (UTR) sequence. Analysis of CBFA1 genomic clones revealed that the 5' UTR sequence of the human OSF2/CBFA1 cDNA lies 75 kb upstream of the originally described 5' end of the gene. The existence of two OSF2/CBFA1 cDNAs is due to an alternative splicing event around exon 8 that affects the transcriptional activity of the protein. Northern blot analysis demonstrates that the expression of the human OSF2/CBFA1 gene is restricted to osteoblastic cells.

Alternative Splicing↗
PubMed1998

Cloning, characterization, and chromosomal assignment of the human ortholog of murine Zfp-37, a candidate gene for Nager syndrome.

In an effort to identify putative transcription factors involved in chondrocyte differentiation during human endochondral bone formation, a human fetal cartilage-specific cDNA library was screened with a degenerate oligonucleotide probe corresponding to a conserved stretch of eight amino acids from the zinc finger region of the Drosophila Krüppel gene family of DNA-binding proteins. Using this strategy, we have identified a novel zinc finger gene ZFP-37. ZFP-37 corresponds to a putative transcription factor containing 12 tandemly repeated zinc finger motifs and a Krüppel-associated box (KRAB) domain. The KRAB domain has been reported to function as a transcriptional repressor and is located in the amino terminus, while the zinc finger repeats are positioned at the carboxy-terminal end of ZFP-37. Gene mapping with a somatic cell hybrid panel and fluorescence in situ hybridization (FISH) localized ZFP-37 to human Chr 9q32. The gene is expressed at low level as a 3.2-kb mRNA in several tissues including fetal human cartilage. Sequence comparison revealed that ZFP-37 may represent the human homolog of the mouse gene Zfp-37. The map location and expression pattern suggest ZFP-37 as a candidate gene for a craniofacial-limb malformation, Nager syndrome (acrofacial dysostosis).

Amino Acid Sequence↗
PubMed1998

A new procedure for constructing peptides into a given Calpha chain.

BACKGROUND: In ab initio protein folding studies, it is often advantageous to build the Calpha chain first and then to construct the full structure by filling in the peptide groups and the sidechains. Many algorithms have been reported for constructing peptide groups on the Calpha chain, but most are unsuitable for use in such studies; some are too slow for screening a large number of trial Calpha chains and others use only the local geometry and ignore the effects of specific non-neighbor interactions, which can be crucial for proper folding. We needed a fast procedure for constructing the peptide groups that does not ignore the effects of long-range, specific interactions. RESULTS: We first found rich correlations between the peptide orientation angle and both the local Calpha-chain geometry and the type of the flanking amino acid residues. These correlations can be used to greatly limit the range of possible peptide orientation angles. We devised a simple peptide construction procedure in which all orientations within this reduced range are systematically examined and the orientation is selected that minimizes a suitable energy function that includes long-range, specific interactions. When tested on known structures, the method is found to be among the fastest of known methods and attains an accuracy comparable with or better than most methods. CONCLUSIONS: The new method is fast and takes into account both the local and non-local specific interactions. It therefore appears to be suitable for use in ab initio protein folding studies, wherein a large number of Calpha chains are screened.

Algorithms↗