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Biomedical subjects

B Lee

Publications and source records attributed to B Lee.

At least 109 records · Page 6Linked to original sources
PubMed2000

Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity.

Chemical modification of proteins with polyethylene glycol (PEGylation) can increase plasma half-lives, stability, and therapeutic potency. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of anti-Tac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor alpha subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then site-specifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37 degrees C in mouse serum, a 5- to 8-fold increase in plasma half-lives in mice, and a 3- to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.

Animals↗
PubMed2000

In vivo urea cycle flux distinguishes and correlates with phenotypic severity in disorders of the urea cycle.

Urea cycle disorders are a group of inborn errors of hepatic metabolism that result in often life-threatening hyperammonemia and hyperglutaminemia. Clinical and laboratory diagnosis of partial deficiencies during asymptomatic periods is difficult, and correlation of phenotypic severity with either genotype and/or in vitro enzyme activity is often imprecise. We hypothesized that stable isotopically determined in vivo rates of total body urea synthesis and urea cycle-specific nitrogen flux would correlate with both phenotypic severity and carrier status in patients with a variety of different enzymatic deficiencies of the urea cycle. We studied control subjects, patients, and their relatives with different enzymatic deficiencies affecting the urea cycle while consuming a low protein diet. On a separate occasion the subjects either received a higher protein intake or were treated with an alternative route medication sodium phenylacetate/benzoate (Ucephan), or oral arginine supplementation. Total urea synthesis from all nitrogen sources was determined from [(18)O]urea labeling, and the utilization of peripheral nitrogen was estimated from the relative isotopic enrichments of [(15)N]urea and [(15)N]glutamine during i.v. co-infusions of [5-(amide)(15)N]glutamine and [(18)O]urea. The ratio of the isotopic enrichments of (15)N-urea/(15)N-glutamine distinguished normal control subjects (ratio = 0.42 +/- 0.06) from urea cycle patients with late (0.17 +/- 0.03) and neonatal (0.003 +/- 0.007) presentations irrespective of enzymatic deficiency. This index of urea cycle activity also distinguished asymptomatic heterozygous carriers of argininosuccinate synthetase deficiency (0. 22 +/- 0.03), argininosuccinate lyase deficiency (0.35 +/- 0.11), and partial ornithine transcarbamylase deficiency (0.26 +/- 0.06) from normal controls. Administration of Ucephan lowered, and arginine increased, urea synthesis to the degree predicted from their respective rates of metabolism. The (15)N-urea/(15)N-glutamine ratio is a sensitive index of in vivo urea cycle activity and correlates with clinical severity. Urea synthesis is altered by alternative route medications and arginine supplementation to the degree that is to be expected from theory. This stable isotope protocol provides a sensitive tool for evaluating the efficacy of therapeutic modalities and acts as an aid to the diagnosis and management of urea cycle patients.

Adolescent↗
PubMed2000

Expression and coreceptor activity of STRL33/Bonzo on primary peripheral blood lymphocytes.

CCR5 and CXCR4 are the major coreceptors that mediate human immunodeficiency virus 1 (HIV-1) infection, while most simian immunodeficiency virus (SIV) isolates use CCR5. A number of alternative coreceptors can also mediate infection of some virus strains in vitro, although little is known about their in vivo relevance. Therefore, we characterized the expression pattern and coreceptor activity of one of these alternative coreceptors, STRL33/Bonzo, using a newly developed monoclonal antibody. In addition to being highly expressed (approximately 1000-7000 STRL33 ABS [antibody binding sites]) on specific subsets of natural killer cells (CD3(-)/CD16(-/low)/CD56(+) and CD3(-)/CD16(low)/CD56(-)) and CD19(+) B lymphocytes (approximately 300-5000 STRL33 ABS), STRL33 was expressed at levels sufficient to support virus infection on freshly isolated, truly naive CD4(+)/CD45RA(+)/CD62L(+) cells (6000-11 000 ABS). STRL33 expression on peripheral blood mononuclear cells (PBMCs) was increased by mitogenic stimulation (OKT3/IL-2 [interleukin-2] had a greater effect than phytohemaglutinin (PHA)/IL-2), but it was dramatically decreased upon Ficoll purification. Infection of CCR5(-) human peripheral blood lymphocytes (PBLs) showed that 2 different SIV envelope (Env) proteins mediated entry into STRL33(+) cells. More importantly, the preferential infection of STRL33(+) cells in CCR5(-) PBLs by an R5/X4/STRL33 HIV-1 maternal isolate in the presence of a potent CXCR4 antagonist (AMD3100) suggests that STRL33 can be used as a coreceptor by HIV-1 on primary cells. Rhesus macaque (rh) STRL33 was used less efficiently than human STRL33 by the majority of SIV Env proteins tested despite similar levels of expression, thereby making it less likely that STRL33 is a relevant coreceptor in the rhesus macaque system. In summary, the expression pattern and coreceptor activity of STRL33 suggest its involvement in trafficking of tumor-infiltrating lymphocytes and indicate that STRL33 may be a relevant coreceptor in vivo.

Animals↗
PubMed2000

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system. In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis.

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.

14-3-3 Proteins↗
PubMed2000

Recombinant polioviruses expressing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes.

T-cell immune response to recombinant poliovirus expressing foreign antigens has not been elucidated well. In order to investigate the potential use of poliovirus as a T-cell vaccine vector, we constructed recombinant polioviruses expressing HBV-derived CTL epitopes, HBsAg(28-39) (L(d)-restricted; IPQSLDSWWTSL) and HBc(93-100) (K(b)-restricted; MGLKFRQL) at the junction between the P2 and P3 regions, designated V3CDs and V3CDc, respectively. The V3CDs and V3CDc recombinant viruses replicated efficiently in HeLa cells and showed a similar infection profile to that of the wild-type Mahoney strain in one-step growth kinetics. Genetic stability analysis showed that V3CDc retained the foreign insert over twelve successive passages examined, whereas V3CDs lost part of the foreign insert after four passages. Our results indicated that the stability of the inserted foreign sequences was rather affected by their nucleotide sequence than by their length when located between the P2 and P3 regions. The junction between these nonstructural protein-coding regions is a novel site for the construction of replication-competent recombinant poliovirus. Immunization of BALB/c (H-2(d)) and C57BL/6 mice (H-2(b)) with V3CDs and V3CDc, respectively, elicited significant antigen-specific CTL responses to HBsAg(28-39) but not to HBcAg(93-100).

Amino Acid Sequence↗
PubMed2000

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Animals↗
PubMed2000

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase.

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

Adenosine Triphosphatases↗
PubMed2000

Viral interferon regulatory factor 1 of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, CREB-binding protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) contains many cellular homologue genes. The K9 open reading frame (ORF) of KSHV encodes a virus-encoded interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction, and as an oncogene to induce cell growth transformation. In addition, KSHV vIRF plays an important role in the regulation of gene expression. From genetic and biochemical analysis, we demonstrate that KSHV vIRF1 binds to a transcriptional coactivator CREB-binding protein (CBP) in vivo and in vitro. KSHV vIRF1 binds to the KIX domain and CH/3 region of CBP. The CH/3 region of CBP coincides with the binding region of adenovirus E1A. We also show that vIRF1 inhibits the transactivational activity of CBP in HeLa cells. These results demonstrate that vIRF1 can modulate gene expression by inhibiting the transactivation function of coactivator CBP.

CREB-Binding Protein↗
PubMed2000

Prompt Optical Observations of Gamma-Ray Bursts.

The Robotic Optical Transient Search Experiment (ROTSE) seeks to measure simultaneous and early afterglow optical emission from gamma-ray bursts (GRBs). A search for optical counterparts to six GRBs with localization errors of 1 deg2 or better produced no detections. The earliest limiting sensitivity is mROTSE>13.1 at 10.85 s (5 s exposure) after the gamma-ray rise, and the best limit is mROTSE>16.0 at 62 minutes (897 s exposure). These are the most stringent limits obtained for the GRB optical counterpart brightness in the first hour after the burst. Consideration of the gamma-ray fluence and peak flux for these bursts and for GRB 990123 indicates that there is not a strong positive correlation between optical flux and gamma-ray emission.

Journal Article↗
PubMed2000

cAMP response element-binding protein-binding protein binds to human papillomavirus E2 protein and activates E2-dependent transcription.

cAMP response element-binding protein-binding protein (CBP) is a eucaryotic transcriptional co-activator that contains multiple protein-protein interaction domains for association with various transcription factors, components of the basal transcriptional apparatus, and other co-activator proteins. Here, we report that CBP is also a co-activator of the human papillomavirus (HPV) E2 protein, which is a sequence-specific transcription/replication factor. We provide biochemical, genetic, and functional evidence that CBP binds directly to HPV E2 in vivo and in vitro and activates E2-dependent transcription. Mutations in an amphipathic helix within HPV-18 E2 abolish its transcriptional activation properties and its ability to bind to CBP. Furthermore, the binding of CBP to E2 was shown to be necessary for E2-dependent transcription. Interestingly, the histone acetyltransferase activity of CBP plays a role in CBP activation of E2-dependent transcription.

Acetyltransferases↗
PubMed2000

LP-BM5 infection impairs spatial working memory in C57BL/6 mice in the Morris water maze.

Previous studies show that the LP-BM5 murine leukemia virus causes an acquired immunodeficiency syndrome in C57BL/6 mice (MAIDS) and impairs learning and memory without gross motor impairment. To assess spatial working memory impairment after LP-BM5 infection and the time course of this impairment, we tested mice in a modified working-memory version of the Morris water maze. Twenty mice were inoculated with LP-BM5; controls received medium (Minimum Essential Medium). In the test procedure, animals had two 1-min training sessions to learn the position of a randomly placed hidden platform. Thirty seconds after the second training session, animals were placed in the maze without the platform, and time and pathlength spent in each quadrant of the maze were measured. For 9 weeks after LP-BM5 infection, both groups showed preference for the target quadrant compared to the opposite quadrant. At 10 and 11 weeks after infection, the LP-BM5 virus infected mice lost this target quadrant preference. We conclude that LP-BM5 infection impaired spatial working memory in a modified working-memory version of the Morris water maze test in C57BL/6 mice at 10 and 11 weeks after virus infection.

Animals↗
PubMed2000

IL-13 and TNF-alpha inhibit dual-tropic HIV-1 in primary macrophages by reduction of surface expression of CD4, chemokine receptors CCR5, CXCR4 and post-entry viral gene expression.

We show that IL-13 in the presence of TNF-alpha effected an equal or greater antiviral activity against a dual-tropic HIV-1 (R5X4) in macrophages. A temporary or continued exposure of macrophages to both cytokines significantly decreased the infection and replication of R5X4 HIV-1(89.6) (median, 128-fold, n = 9, p = 0.024) in macrophages as compared to untreated controls when analyzed over six decreasing multiplicities of infection. A quantitative flow cytometric assay revealed that IL-13 induced a significant (approximately 50 %) reduction in the number of CD4 and CC chemokine receptor 5 (CCR5) antibody binding sites while completely abrogating surface expression of CXC chemokine receptor 4 (CXCR4). In the presence of IL-13 and TNF-alpha, expression of CCR5 was completely abrogated while the expression of CD4 and CXCR4 remained significantly reduced as compared to untreated controls. A reduction in CD4 and HIV-1 coreceptors was associated with a decrease in reverse-transcribed viral DNA at 24 h post-infection. Quantification of viral gene expression using amphotropic MLV Env pseudotyped luciferase reporter viruses suggested that IL-13 inhibited HIV-1 gene expression within 24 h by up to 90 % in the presence or absence of TNF-alpha. In conclusion, our data suggest that IL-13 is a powerful counter-regulatory agent against TNF-alpha-induced HIV-1 expression while also acting with TNF-alpha in inhibiting de novo infection of macrophages.

CD4 Antigens↗
PubMed2000

Acid and base catalyzed intramolecular cyclizations of N-benzoylthiocarbamoyl-acetals.

Acid and base catalyzed intramolecular cyclizations of N-benzoylthioureidoacetal, containing four functional groups adjacent to thiourea such as benzocarbamoyl, acetal, thioure and amide, were investigated. The condensation reaction of N-benzoyl thiocarbamoylglycine amide in the presence of 10% aqueous NaOH provided 1-(2,2-dimethoxy)ethyl-imidazolidine-2-thione exclusively. In the presence of pyridine, it was transformed to 2-thiohydantoin. N-Benzoyl thiocarbamoyl glycine amide was completely transformed to an iminothiazolidine exclusively in the presence of Lewis acid such as borontrifluoride etherate or trimethylsilyl iodide. 1-(2,2-Dimethoxy)ethyl-imidazolidine-2-thione was transformed to imidazole[2,1-b]thiazole and pyrazino[5,1-a]imidazole in the presence of BF3.Et2O and formic acid, respectively.

Acids↗
PubMed2000

Optic disk topography after medical treatment to reduce intraocular pressure.

PURPOSE: We examined changes in optic disk topography using confocal scanning laser ophthalmoscopy after reducing intraocular pressure with administration of latanoprost. METHODS: Twenty-nine patients with glaucoma or ocular hypertension were imaged using the Heidelberg Retina Tomograph before and after the administration of latanoprost to decrease intraocular pressure. Average time between pretreatment and posttreatment imaging was 2.7 +/- 1.8 weeks. Heidelberg Retina Tomograph software-measured parameters were mean height of contour, cup area, cup volume, mean cup depth, maximum cup depth, cup shape, rim area, rim volume, cup-to-disk ratio, and retinal nerve fiber thickness. RESULTS: Average intraocular pressure decreased significantly (mean +/- SD) by 7.2 +/- 5.4 mm Hg (25 +/- 16% decrease). No statistically significant changes in measured topographic parameters were found. When data from patients with decreases in intraocular pressure of 7 mm Hg or greater were analyzed separately (mean intraocular pressure decrease = 10.79 +/- 4.32 mm Hg, 36 +/- 8% decrease), cup area (P =.005), cup volume (P =. 002), and cup-to-disk ratio (P =.005) decreased significantly, and rim area (P =.005) increased significantly. Linear regression analysis of the data from all subjects showed that a change in intraocular pressure after latanoprost administration accounted for 12% or more of the variance in two measured topographic parameters (mean cup depth and cup shape). CONCLUSIONS: These results suggest that, in some patients, moderate decreases in intraocular pressure may affect disk topography, as measured by Heidelberg Retina Tomograph. Intraocular pressure should be considered when analyzing consecutive confocal scanning laser ophthalmoscopy images for glaucomatous progression.

Antihypertensive Agents↗
PubMed2000

Center surround receptive field structure of cone bipolar cells in primate retina.

In non-mammalian vertebrates, retinal bipolar cells show center-surround receptive field organization. In mammals, recordings from bipolar cells are rare and have not revealed a clear surround. Here we report center-surround receptive fields of identified cone bipolar cells in the macaque monkey retina. In the peripheral retina, cone bipolar cell nuclei were labeled in vitro with diamidino-phenylindole (DAPI), targeted for recording under microscopic control, and anatomically identified by intracellular staining. Identified cells included 'diffuse' bipolar cells, which contact multiple cones, and 'midget' bipolar cells, which contact a single cone. Responses to flickering spots and annuli revealed a clear surround: both hyperpolarizing (OFF) and depolarizing (ON) cells responded with reversed polarity to annular stimuli. Center and surround dimensions were calculated for 12 bipolar cells from the spatial frequency response to drifting, sinusoidal luminance modulated gratings. The frequency response was bandpass and well fit by a difference of Gaussians receptive field model. Center diameters were all two to three times larger than known dendritic tree diameters for both diffuse and midget bipolar cells in the retinal periphery. In one instance intracellular staining revealed tracer spread between a recorded cell and its nearest neighbors, suggesting that homotypic electrical coupling may contribute to receptive field center size. Surrounds were around ten times larger in diameter than centers and in most cases the ratio of center to surround strength was approximately 1. We suggest that the center-surround receptive fields of the major primate ganglion cell types are established at the bipolar cell, probably by the circuitry of the outer retina.

Animals↗
PubMed2000

Enhanced proteolysis of pre-mRNA splicing factors in myeloid cells.

OBJECTIVE: Molecular identification and characterization of the bone marrow nuclear protein detected by the B92 monoclonal antibody. MATERIALS AND METHODS: The protein was purified to homogeneity from acute myeloid leukemia cells and was subjected to peptide digestion and amino acid sequencing. Identified sequences were used to screen a bone marrow cDNA library in search of matching transcripts. The protein was further studied in different cells and tissues by examination of protease inhibitors and harsh lytic conditions and during apoptosis in HL-60 cells. RESULTS: We found that the apparent bone marrow specific protein is a 47 kD proteolytic cleavage product of PSF, an essential pre-mRNA splicing factor. PSF is completely cleaved to p47 during lysis of immature myeloid cells due to potent proteolytic activity found in these cells but is rare in other cells and tissues. Furthermore, p47 is abundant in intact normal and tumor myeloid cells while in other cell types it is undetectable. The cleavage of PSF is accompanied by digestion of the PTB splicing regulator but not other proteins tested. In contrast, during apoptosis PTB is degraded while PSF remains intact. CONCLUSIONS: The bone marrow 47 kD protein is a fragment constituting the N-terminal, protease-resistant half of the splicing factor PSF. Proteolytic degradation of PSF specifically occurs in intact myeloid cells and this process is enhanced upon myeloid cell lysis.

Amino Acid Sequence↗
PubMed2000

Solitary hepatic abscess with associated glomerulonephritis in a neonate.

A full-term neonate with a history of umbilical venous catheterization followed by coagulase-negative staphylococcal sepsis is presented. The infant developed a solitary hepatic abscess with saprophytic organisms. Her liver abscess resulted in acute glomerulonephritis characterized by hypertension, proteinuria, oliguria, and azotemia. Surgical drainage and antibiotic treatment of the abscess was associated with resolution of the glomerulonephritis. Glomerulonephritis due to solitary liver abscess in a neonate has not been reported previously. Acute onset of glomerulonephritis should prompt a search for occult sources of infection.

Abscess↗