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Biomedical subjects

C Lee

Publications and source records attributed to C Lee.

At least 469 records · Page 26Linked to original sources
PubMed1997

Advances in the generation of transgenic pigs via embryo-derived and primordial germ cell-derived cells.

The development of new technologies that would increase the efficiency for generation of transgenic livestock and would overcome some of the problems associated with random insertion of the transgene will greatly benefit animal agriculture. A potential alternative technology to pronuclear injection for the generation of transgenic pigs involves the isolation, culture and genetic manipulation of cell lines that can be reintroduced into the embryo for participation in the formation of the germ cells. We have isolated and cultured pig primordial germ cells (PGC) while maintaining them in an undifferentiated state as determined by morphology and alkaline phosphatase (AP) activity. More importantly, PGC-derived cells were stably transformed with the green fluorescent protein marker driven by the cytomegalovirus promoter. After visual identification of transgenic colonies, the pluripotential characteristics of the transgenic PGC-derived cells were tested by chimaera formation and to date we have identified, by genomic Southern blots, two chimaeric fetuses that contain tissues with the transgene incorporated into their chromosomes. To our knowledge, this is the first report of a chimaeric transgenic pig fetus obtained via a cultured cell line.

Animals↗
PubMed1997

Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer.

Our previous observations in LNCaP cells in vitro demonstrated an association between apoptotic cell death resistance and SGP-2 (Clusterin) overexpression. Accordingly, we hypothesized that high levels of cellular SGP-2 would aid in identifying biologically aggressive prostate cancer cells with unique survival advantages. To test this hypothesis, 40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1-3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior.

Adenocarcinoma↗
PubMed1996

Selective activation of effector pathways by brain-specific G protein beta5.

While multiple G protein beta and gamma subunit isoforms have been identified, the implications of this potential diversity of betagamma heterodimers for signaling through betagamma-regulated effector pathways remains unclear. Furthermore the molecular mechanism(s) by which the betagamma complex modulates diverse mammalian effector molecules is unknown. Effector signaling by the structurally distinct brain-specific beta5 subunit was assessed by transient cotransfection with gamma2 in COS cells and compared with beta1. Transfection of either beta1 or beta5 with gamma2 stimulated the activity of cotransfected phospholipase C-beta2 (PLC-beta2), as previously reported. In contrast, cotransfection of beta1 but not beta5 with gamma2 stimulated the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways even though the expression of beta5 in COS cells was evident by immunoblotting. The G protein beta5 expressed in transfected COS cells was properly folded as its pattern of stable C-terminal proteolytic fragments was identical to that of native brain beta5. The inability of beta5 to activate the MAPK and JNK pathways was not overcome by cotransfection with three additional Ggamma isoforms. These results suggest it is the Gbeta subunit which determines the pattern of downstream signaling by the betagamma complex and imply that the structural features of the betagamma complex mediating effector regulation may differ among effectors.

Animals↗
PubMed1996

Molecular modeling and design of invariant chain peptides with altered dissociation kinetics from class II MHC.

We have used molecular modeling to design substitutions in an invariant chain-derived peptide (CLIP), so as to alter the stability of its complex with class II major histocompatibility complex (MHC) proteins. We sought first to test whether CLIP binds in the same way to different class II MHC proteins. We designed destabilizing substitutions of two residues (Met 91 and Met 99) previously predicted to act as the major anchor residues for binding to all class II MHC and measured their effect on CLIP's dissociation rate from a series of three murine I-A MHC proteins. Even a conservative substitution preserving size and hydrophobicity but reducing flexibility (leucine, a branched residue) caused large accelerations in dissociation rates (up to 25-fold) at either position in all three MHC alleles, supporting the consistent role of these positions as the major anchors for MHC binding. These data also support the view that the special flexibility of the methionine side chains at these positions is essential for binding to diverse MHC molecules. We also used molecular modeling to design allele-specific enhancements of peptide binding. Designed substitutions of CLIP Pro 96 by Ala (for Ad), Glu (Ak), and Tyr (Au) each yielded strong enhancement of binding (up to 128-fold) for their targeted allele and only moderate or destabilizing effects to the other alleles. These results demonstrate the accuracy of the molecular models and the predictive value of this modeling. Moreover, they provide strong evidence for the proposed general model of invariant chain association, indicating that it binds to all class II MHC in the same conformation.

Alleles↗
PubMed1996

Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, may be the infectious cause of KS. Its prevalence in the general population, on the basis of detection of the virus genome, is controversial. To investigate the seroprevalence, we measured antibodies to a recombinant capsid-related (lytic cycle) KSHV antigen and a latent antigen complex. METHODS: We selected potentially immunoreactive capsid-related proteins of KSHV by expressing them as recombinant proteins and testing them in western blot assays. We used a truncated recombinant protein encoded by KSHV open reading frame 65 (orf 65) to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) and tested sera from HIV-infected individuals with KS, HIV-uninfected patients with "classic" KS, other HIV risk groups, and blood donors. We also compared the antibody response to this capsid-related protein to the response to latent antigen(s) in an immunofluorescence assay. FINDINGS: 77/92 (84%) sera from KS patients reacted with the KSHV orf 65 protein and 84/103 (81.5%) reacted with KSHV latent antigen(s). The dominant immunogenic region of orf 65 is within the carboxyterminal 80 aminoacids, a region with little sequence similarity to the related Epstein-Barr virus, suggesting that orf 65 is a KSHV specific antigen. Only three sera from patients with haemophilia (1/84) or from intravenous drug users (2/63) had KSHV specific antibodies in the orf 65 assay whereas none of these sera reacted with latent antigen. Antibodies to KSHV were also infrequently found in UK and US blood donors by either assay (UK, 3/174 with orf 65 and 4/150 with latent antigen; US, 6/117 with orf 65 and 0/117 with latent antigen). They were more common among HIV-infected gay men without KS (5/16 by orf 65 ELISA, 10/33 by IFA), HIV-uninfected STD clinic attenders (14/166 by IFA), and Ugandan HIV-uninfected controls (6/17 by orf 65 ELISA, 9/17 by IFA). Antibody reactivity to the orf 65 protein (ELISA) and to latent antigen(s) (IFA) was concordant in 89% of 462 sera tested but reactive blood donor sera were discordant in both assays. Four AIDS-KS sera were unreactive in both assays. INTERPRETATION: The distribution of antibodies to both a capsid-related recombinant protein and latent antigen(s) of KSHV strongly supports the view that infection with this virus is largely confined to individuals with, or at increased risk for, KS. However, infection with KSHV does occur, rarely, in the general UK and US population and is more common in Uganda. Antibodies to latent antigen(s) or to orf 65 encoded capsid protein will not detect all cases of KSHV infection, and a combination of several antigens will probably be required for accurate screening and confirmatory assays.

Adult↗
PubMed1996

Pharmacokinetic studies of amiloride and its analogs using reversed-phase high-performance liquid chromatography.

We have studied the pharmacokinetics of amiloride and its analogs. A high-performance liquid chromatographic method has been adapted for the measurement of amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N, N-hexamethylene)amiloride (HMA) in mouse plasma, kidney, liver and tumor tissues. The method uses a C8 preparative solid-phase column, followed by separation using a reversed-phase C18 column (250 x 4 mm I.D., 5 microns particle size) with detection by ultraviolet absorption at 365 nm. Reversed-phase separations were performed at ambient temperature using a non-linear gradient method with two different mobile phases: mobile phase A was 100% acetonitrile while mobile phase B was 0.15 M perchloric acid at pH 2.20 (flow-rate was 1.2 ml/min). The retention times for amiloride, benzamil (used as an internal standard), EIPA and HMA are 13.4, 19.5, 21.8 and 23.5 min, respectively. The calibration curves are linear over the range of 0.1-50 microM in plasma and in tissues. The half-lives of amiloride, EIPA and HMA (and their confidence intervals) in plasma after intraperitoneal injection of drugs into mice were 68.8 +/- 0.2, 31.2 +/- 2.5 and 39.3 +/- 7.9 min, respectively. Amiloride was detected as a metabolite of EIPA but not of HMA. When EIPA was injected at a dose of 10 micrograms/g body weight, it was cleared rapidly from liver, but concentrations > 1 microM were sustained for at least 2 h in murine kidney and in a transplantable tumor.

Amiloride↗
PubMed1996

The inter-locus recombinant HLA-B*4601 has high selectivity in peptide binding and functions characteristic of HLA-C.

The vast majority of new human HLA class I alleles are formed by conversions between existing alleles of the same locus. A notable exception to this rule is HLA-B*4601 formed by replacement of residues 66-76 of the alpha 1 helix of B*1501 by the homologous segment of Cw*0102. This inter-locus recombination, which brings together characteristic elements of HLA-B and HLA-C structure, is shown here to influence function dramatically. Naturally processed peptides bound by B*4601 are distinct from those of its parental allotypes B*1501 and Cw*0102 and dominated by three high abundance peptides. Such increased peptide selectivity by B*4601 is unique among HLA-A,B,C allotypes. For other aspects of function, presence of the small segment of HLA-C-derived sequence in an otherwise HLA-B framework converts B*4601 to an HLA-C-like molecule. Alloreactive cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and cellular glycosidases all recognize B*4601 as though it were an HLA-C allotype. These unusual properties are those of an allotype which has frequencies as high as 20% in south east Asian populations and is associated with predisposition to autoimmune diseases and nasopharyngeal carcinoma.

Amino Acid Sequence↗
PubMed1996

Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands.

Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.

Amino Acid Sequence↗
PubMed1996

Resetting the Drosophila clock by photic regulation of PER and a PER-TIM complex.

Circadian clocks can be reset by light stimulation. To investigate the mechanism of this phase shifting, the effects of light pulses on the protein and messenger RNA products of the Drosophila clock gene period (per) were measured. Photic stimuli perturbed the timing of the PER protein and messenger RNA cycles in a manner consistent with the direction and magnitude of the phase shift. In addition, the recently identified clock protein TIM (for timeless) interacted with PER in vivo, and this association was rapidly decreased by light. This disruption of the PER-TIM complex in the cytoplasm was accompanied by a delay in PER phosphorylation and nuclear entry and disruption in the nucleus by an advance in PER phosphorylation and disappearance. These results suggest a mechanism for how a unidirectional environmental signal elicits a bidirectional clock response.

Animals↗
PubMed1996

Constitutive expression of class 3 aldehyde dehydrogenase in cultured rat corneal epithelium.

Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5'3' RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation-derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner.

Aldehyde Dehydrogenase↗
PubMed1996

Modulation of sensitivity to transforming growth factor-beta 1 (TGF-beta 1) and the level of type II TGF-beta receptor in LNCaP cells by dihydrotestosterone.

Transforming growth factor-beta 1 (TGF-beta 1) and androgen are potential physiological regulators of prostate cancer cells. In the present study, we have used LNCaP cells as a model of androgen-responsive prostate cancer to investigate the effects of dihydrotestosterone (DHT) on the sensitivity to TGF-beta 1. The ability of LNCaP cells to respond to TGF-beta has been controversial. In some studies, LNCaP cells were insensitive to TGF-beta 1 while, in others, they were sensitive to the growth inhibitory effect of TGF-beta 1. The present study was carried out to establish androgenic conditions that rendered LNCaP cells sensitive to TGF-beta 1. Cells were cultured in phenol-red-free RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum. DHT was added at the following concentrations: 0, 10(-12), 10(-10), and 10(-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth-arrest dose, respectively. The effects of TGF-beta 1 observed on LNCaP cells included inhibition of cell proliferation, decrease in cell viability, alteration in cell morphology, and enhancement of gene transcriptional activity through activation of a TGF-beta responsive promoter. Of the various DHT concentrations investigated in this study, these effects of TGF-beta 1 on LNCaP cells were consistently demonstrated only at 10(-10) M. At other concentrations, the effects of TGF-beta 1 were either minimal or undetectable. Accompanying these effects of TGF-beta 1, a low but statistically significant level of TGF-beta 1-specific binding and an increased protein level of TGF-beta receptor type II were detected by a competitive binding assay and Western blot analysis respectively. These results indicate that LNCaP cells can be induced by DHT to respond to TGF-beta 1 and that DHT modulates the sensitivity to TGF-beta 1 and the level of TGF-beta receptor type II in these cells.

Binding, Competitive↗
PubMed1996

Genetic change in transforming growth factor beta (TGF-beta) receptor type I gene correlates with insensitivity to TGF-beta 1 in human prostate cancer cells.

Transforming growth factor beta 1 (TGF-beta 1), a potential regulator of growth of prostate cancer cells, exerts its effects through interaction with membrane receptors. In the present study, an attempt was made to establish a correlation between TGF-beta 1 sensitivity and TGF-beta receptor expression in three prostate cancer cell lines (PC3, DU145, and LNCaP). In a dose-dependent manner, TGF-beta 1 inhibited the proliferation of PC3 and DU145 cells but not LNCaP cells. Since TGF-beta signals through a heteromeric complex composed of TGF-beta receptors type II and type I, the expression of these receptors was investigated by Western blot analysis and reverse transcriptase-PCR. These studies demonstrated that all three prostate cancer cell lines express type II receptor. In contrast, type I receptor was detected only in the TGF-beta 1-sensitive PC3 and DU145 cells but not in the TGF-beta 1-insensitive LNCaP cells. To investigate the possibility that the undetectable expression of type I receptor in LNCaP cells is due to a change in the respective gene, Southern blot analysis was performed. The result demonstrated that there was a genetic change in type I receptor gene in these cells. Subsequently, when LNCaP cells were transiently transfected with T beta R-I cDNA, sensitivity to TGF-beta 1 was restored. These observations indicate that LNCaP cells contain a defective T beta R-I gene which rendered these cells insensitive to the action of TGF-beta 1.

Base Sequence↗
PubMed1996

Anterior paralimbic mediation of procaine-induced emotional and psychosensory experiences.

BACKGROUND: Procaine activates limbic structures in animals. In humans, acute intravenous administration of procaine yields emotional and psychosensory experiences and temporal lobe fast activity. We studied procaine's acute effects on cerebral blood flow (CBF) in relationship to clinical responses. METHODS: Cerebral blood flow was assessed by positron emission tomography with oxygen-15-labeled water in 32 healthy volunteers. Data were analyzed with statistical parametric mapping and magnetic resonance imaging-directed regions of interest. RESULTS: Procaine increased global CBF and, to a greater extent, anterior paralimbic CBF. Subjects with intense procaine-induced fear compared with those with euphoria had greater increases in left amygdalar CBF. Absolute and normalized left amygdalar CBF changes tended to correlate positively with fear and negatively with euphoria intensity. Procaine-induced visual hallucinations appeared associated with greater global and occipital CBF increases. Absolute occipital CBF increases appeared to correlate positively with visual hallucination intensity. CONCLUSIONS: Procaine increased anterior paralimbic CBF, and different clinical responses appeared to be associated with different patterns of CBF changes.

Adolescent↗