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Biomedical subjects

G Lee

Publications and source records attributed to G Lee.

At least 181 records · Page 10Linked to original sources
PubMed1996

Mutational effects on inclusion body formation in the periplasmic expression of the immunoglobulin VL domain REI.

BACKGROUND: Inclusion body (IB) formation in bacteria is an important example of protein misassembly, a phenomenon which also includes folding-dependent aggregation in vitro and amyloid deposition in human disease. Previous studies of mutational effects in other systems implicate the stability of a folding intermediate-rather than the native state-as playing a key role in IB formation. To contribute to an understanding of the comparative biophysics of VL misassembly in different biological settings, we have studied mutation-dependent periplasmic IB formation by the VL domain REI in Escherichia coli. RESULTS: A series of mutants were produced in periplasmic IBs, where, in all cases, the signal peptide was removed. In addition, the intradomain disulfide was clearly formed before deposition into IBs. IB formation in these mutants does not correlate with monomer/dimer equilibrium constants, but does correlate with the thermodynamic stability of the native state. CONCLUSIONS: The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date. As equilibrium unfolding intermediates have also been implicated in light chain amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases.

Amino Acid Sequence↗
PubMed1996

Non steady-state descriptions of drug permeation through stratum corneum. I. The biphasic brick-and-mortar model.

PURPOSE: The diffusion equation should be solved for the non-steady-state problem of drug diffusion within a two-dimensional, biphasic stratum corneum membrane having homogeneous lipid and corneocyte phases. METHODS: A numerical method was developed for a brick-and-mortar SC-geometry, enabling an explicit solution for time-dependent drug concentration within both phases. The lag time and permeability were calculated. RESULTS: It is shown how the barrier property of this model membrane depends on relative phase permeability, corneocyte alignment, and corneocyte-lipid partition coefficient. Additionally, the time-dependent drug concentration profiles within the membrane can be observed during the lag and steady-state phases. CONCLUSIONS: The model SC-membrane predicts, from purely morphological principles, lag times and permeabilities that are in good agreement with experimental values. The long lag times and very small permeabilities reported for human SC can only be predicted for a highly-staggered corneocyte geometry and corneocytes that are 1000 times less permeable than the lipid phase. Although the former conclusion is reasonable, the latter is questionable. The elongated, flattened corneocyte shape renders lag time and permeability insensitive to large changes in their alignment within the SC. Corneocyte/lipid partitioning is found to be fundamentally different to SC/donor partitioning, since increasing drug lipophilicity always reduces both lag time and permeability.

Administration, Cutaneous↗
PubMed1996

Phosphorylation of native and truncated isoforms of protein tau by the double-stranded DNA-dependent protein kinase (DNA-PK) shows that the primary phosphorylation sites are localized between amino acid residues 212-231 of the longest tau.

Alzheimer's disease (AD) is pathologically characterized by the appearance of neurofibrillary tangles (NFT), senile plaques, and loss of subpopulation of neuronal cells. The NFT is composed of paired helical filaments (PHT) with extensively modified protein T as its primary constituent. Previously we had reported on the hyperphosphorylation of T by a double-stranded-DNA-stimulated protein kinase (DNA-PK). In this communication, we have compared the DNA-PK mediated phosphorylation of native and truncated TS with that catalyzed by the cAMP-dependent protein kinase (PKA). In addition, we have attempted to map the primary site(s) of phosphorylation of T by DNA-PK. Our results suggest that DNA-PK phosphorylates T at sites substantially different from those targeted by PKA. Furthermore, we show that the primary phosphorylation sites lie between amino acid residues 212-231 (using numbering system for the longest T, which is the isoform with four "repeats" and a 58 amino acid "insert" at the carboxyl- and amino-termini, respectively, of the protein molecule).

Alzheimer Disease↗
PubMed1996

Adenovirus vector technology: an efficient method for constructing recombinant adenovirus and on/off switching of gene expression.

An efficient method of constructing recombinant adenoviruses (Ad) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus. This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors. Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ad showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy.

Adenoviridae↗
PubMed1996

An apparent functional correlation between variations in amino acid residues in HLA-DR4.1 and 4.2 serological subtypes and oligonucleotide characterization.

HLA-DR4 can be subdivided serologically into two specificities, DR4.1 and DR4.2, using well-defined monospecific alloantisera used in the 11th International Histocompatibility Workshop. In this study, a total of 1095 random DR4-positive individuals from several ethnic groups were tested first for serotype DR4.1/4.2 and then for DRB1*04 alleles using polymerase chain reaction (PCR) followed by sequence-specific oligonucleotide probe hybridization (SSOPH). An almost 100% correlation between samples testing positive for DR4.1 and the presence of alanine at position 74 was observed, while samples testing positive for DR4.2 correlated with the presence of glutamic acid at position 74. DRB1*04 alleles 0401, 0402, 0404, 0405, 0408, 0409 and 0410 are aligned in functional groups which coincide with the serological subtype of DR4.1. DRB1*04 alleles 0403, 0406, 0407 and 0411 coincide with subtype DR4.2. Amino acid substitutions at positions 57, 71 and 86 indicate other significant variations between alleles within the serological subgroup of DR4.1 and define five minor subgroups. The serologic and oligonucleotide allelic subgroups are in turn correlated with recognized cellular Dw antigens. While sequence data provide evidence of structural differences, data on cellular antigens support a functional association between these designated groups and their significance in transplantation and GVHD. Testing results are categorized by ethnic group in order to establish frequency data for donor selection criteria.

Amino Acid Sequence↗
PubMed1996

Role of stool screening tests in diagnosis of inflammatory bacterial enteritis and in selection of specimens likely to yield invasive enteric pathogens.

The Leuko-Test yielded a negative predictive value of 98.4% when it was used to screen 325 patients for inflammatory bacterial enteritis and a negative predictive value of 99.4% when it was used to screen 416 stool specimens for those from which enteric pathogens would likely be recovered when cultured. Neither microscopy for fecal leukocytes nor an assay for fecal occult blood, alone or in combination, allowed for the reliable detection of invasive bacterial enteritis or the reliable selection of specimens for culture. When positive in the Leuko-Test, specimens collected from patients after the third day of hospitalization did not yield enteric pathogens when the specimens were cultured, and specimens collected from inpatients within the first 3 days of hospitalization or from outpatients did not contain Clostridium difficile toxin A. As a screening test, the Leuko-Test has the ability to generate rapidly a result which can support the presumptive diagnosis of inflammatory bacterial enteritis or which can be used to determine the suitability of stool specimens for bacteriologic culture.

Bacterial Infections↗
PubMed1996

Insoluble collagen matrices for prolonged delivery of proteins.

The purpose of this investigation was to evaluate the application of high molecular weight, insoluble collagen as a carrier material for proteins. Matrices were formulated and their behavior in buffer solution was investigated with focus on swelling and inner structure. Cross-linking with glutaraldehyde was introduced prior to the formation of the devices and its influence characterized. In addition, the enzymatic degradation process was studied and release experiments with systems loaded with fluorescent-labeled bovine serum albumin were carried out. Insoluble collagen matrices were characterized by intensive swelling in buffer resulting in development of a coarse porous character. Cross-linking strongly reduced the water penetration, leading to denser structures of the swollen devices. The continuous enzymatic degradation of the disk-shaped matrices by collagenase followed the kinetics of an heterogeneous enzymatic process with hindrance of proteolysis by the addition of glutaraldehyde. Release studies demonstrated that large amounts of model protein were held in the matrices with increased cross-linking degree. In presence of collagenase a prolonged release of the trapped protein over several days by matrix cleavage could be achieved. Insoluble collagen can be effective as a carrier material for proteins with an in vitro release characteristic by both diffusion-controlled and enzymatic degradation mechanisms. Cross-linking at the stage of preparing the aqueous dispersion offers an alternative to subsequent cross-linking processes.

Animals↗
PubMed1995

Cyclic 3'-5'-adenosine monophosphate binds to annexin I and regulates calcium-dependent membrane aggregation and ion channel activity.

The annexin (Anx) gene family comprises a set of calcium-dependent membrane binding proteins, which have been implicated in a wide variety of cellular processes including membrane fusion and calcium channel activity. We report here that cAMP activates Ca(2+)-dependent aggregation of both phosphatidylserine (PS) liposomes and bovine chromaffin granules driven by [des 1-12]annexin I (lipocortin I, Anx1). The mechanism of cAMP action involves an increase in AnxI-dependent cooperativity on the rate of such a reaction without affecting the corresponding k1/2 values. Cyclic AMP causes the values of the Hill coefficient (nH) for AnxI to change from 3 to 6 in both PS liposomes and chromaffin granules. By contrast, ATP inhibits the rate of aggregation activity without affecting the cooperativity or the extent of aggregation process. We were also able to photolabel Anx1 specifically with an 8-azido analogue of cAMP by a calcium-independent process. Such a process is saturable, yielding a Kd = 0.8 microM by Scatchard analysis. Specific displacement occurs in the presence of cAMP and ATP. Finally, we found that cAMP alters the conductance of calcium channels formed by AnxI in planar lipid bilayers. We interpret these data to indicate that AnxI binds both calcium and cAMP independently, and that both actions have functional consequences. This is the first report of a nucleotide binding function for a member of the annexin gene family.

Adenosine Triphosphate↗
PubMed1995

Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.

Adenoviridae↗
PubMed1995

Bone marrow B lymphocyte development in c-abl-deficient mice.

Mice homozygous for a mutation in the c-abl tyrosine kinase gene have multiple defects including high postnatal mortality, runting, morphological abnormalities, susceptibility to infections, and reductions in lymphocytes and their precursors. FACS analysis of bone marrow from mutant mice demonstrates variable reductions in pro-B and pre-B cells. While the numbers of cells in these populations are profoundly reduced in some mutants (16 and 1.2% of control pro-B and pre-B cells, respectively), normal levels are found in other individuals. In the affected mutants, some reductions are observed in many stages of B cell development. The response of B cell precursors to the cytokine interleukin-7 is variably affected while that of several other cytokines (stem cell factor, interleukin-3, GM-CSF, G-CSF, and erythropoietin) is normal in c-abl mutants. The population defects caused by the c-abl mutation can be recreated in normal mice by the transfer of adult bone marrow but, surprisingly, not fetal liver. These studies demonstrate that c-Abl signaling pathways may play a role in the earliest stages of B cell development in a developmental stage-specific manner. In spite of these variable abnormalities, however, the hemopoietic system of c-abl mutant animals is surprisingly intact.

Animals↗
PubMed1995

A prospective follow-up study of brain morphology and cognition in first-episode schizophrenic patients: preliminary findings.

Brain morphological abnormalities have been reported in several independent investigations of chronic schizophrenic patients. The present study is a prospective 4-year follow-up of first-episode schizophrenic patients to determine whether some of these abnormalities may be a consequence of regional brain structural change over time after the onset of a first psychotic episode. Whole hemisphere, temporal lobes, superior temporal gyrus, hippocampus, caudate, corpus callosum, and lateral ventricles were measured in a series of MRI scans taken over a 4-year period in 20 patients and five controls. Total volume reduction was noted in both hemispheres to a greater degree in patients than controls. When adjusted for total brain size, left ventricular enlargement occurred in patients, but not controls, over time. These preliminary data suggest that subtle cortical atrophy may be occurring over time after the onset of illness.

Adult↗
PubMed1995

The antiinflammatory activity of topically applied novel calcium-channel antagonists.

The antiinflammatory activities of two novel calcium-channel antagonists, AGN 190742 and AGN 190744, were evaluated in murine models of cutaneous inflammation. These 2(5H)-furanone ring compounds block both depolarization-dependent Ca2+ entry and receptor-mediated responses in GH3 cells. Topical application of AGN 190742 or AGN 190744 inhibits neutrophil infiltration and epidermal hyperplasia induced by repeated treatment of mouse skin with phorbol ester. AGN 190744 also is active in an arachidonic acid model of acute inflammation. These data suggest that topical application of calcium-channel antagonists can inhibit cutaneous inflammatory responses and that AGN 190742 and/or AGN 190744 may serve as useful pharmacological probes for examining these responses in vivo.

Administration, Cutaneous↗
PubMed1995

Micrometastases in bone marrow of patients undergoing "curative" surgery for gastrointestinal cancer.

BACKGROUND & AIMS: Immunohistochemical detection of bone marrow micrometastases has been reported as a prognostic marker in colorectal cancer. The aims of this study were to evaluate the potential advantage of flow cytometry as an objective method of identifying and quantifying micrometastatic deposits within bone marrow and to determine the prevalence and quantity of micrometastases in patients undergoing surgery for gastrointestinal cancers. METHODS: Flow cytometry was first validated by a controlled "spike" experiment in which varying numbers of neoplastic epithelial cells were added to bone marrow, and cytometry was performed in a blinded fashion. Three neoplastic cell lines (colonic and esophageal) with varying degrees of expression of cytokeratin-18 were used. Epithelial cells were detected by dual staining with fluorescence-labeled, monoclonal anti-cytokeratin, and propidium iodide. RESULTS: Cytometry reproducibly detected the presence of > or = 10 neoplastic cells per 10(5) marrow cells. Micrometastases were found in 20%-30% of patients undergoing potentially curative resection of colorectal and gastroesophageal adenocarcinomas. There was a trend toward increasing positivity for marrow deposits with advanced Dukes' staging of colorectal cancer. CONCLUSIONS: Flow cytometric assessment of bone marrow is a reliable, objective, and quantitative method of detecting micrometastatic deposits found in a substantial subset of patients undergoing surgery for gastrointestinal adenocarcinomas.

Adenocarcinoma↗
PubMed1995

Clinical evaluations of a new ovarian cancer marker, COX-1.

A monoclonal antibody was generated against an ovarian cancer cell line, OC-3-VGH and was shown to recognize a unique tumor-associated antigen, COX-1 found in ovarian or cervical tumors. COX-1 can be detected in the cultured shed medium of several ovarian/cervical cancer cell lines, and in the sera of ovarian or cervical cancer patients when assayed by a sandwich immunoassay kit employing this monoclonal antibody. Multi-medical centers clinical trials have been conducted since 1989 to evaluate the efficacy of using this immunoassay procedure for the cancer monitoring. In the case of ovarian cancer, COX-1 immunoassay kit has the sensitivity of 68% as compared to 71% for the corresponding CA 125 kit. When both COX-1 and CA 125 kits were combined for the simultaneous monitoring of cancer patients, the sensitivity could be increased to as high as 87%. Further analysis revealed that serum COX-1 levels in cancer patients are correlated with stages of tumor progression. Following the surgical removal of tumors, greater than 50% of the cancer patients showed dramatic and significant decrease of serum levels of COX-1 antigen. The results of these multi-center clinical studies clearly suggested that COX-1 is a suitable marker for the monitoring and diagnosis of ovarian cancer patients, when used alone or in combination with CA 125.

Antigens, Neoplasm↗