Biomedical subjects

C A Carter

Publications and source records attributed to C A Carter.

At least 37 records · Page 2Linked to original sources

PubMed1998

Cytoskeletal reorganization induced by retinoic acid treatment of human endometrial adenocarcinoma (RL95-2) cells is correlated with alterations in protein kinase C-alpha.

We have shown previously that treatment of human endometrial adenocarcinoma (RL95-2) cells with either 13-cis or all-trans retinoic acid results in reorganization of actin filaments, indicating reversion to a stationary phenotype. In the present study, we investigated the role of protein kinase C (PKC) in this process. Treatment of cells with PKC inhibitors (staurosporine, bisindolylmaleimide, or G¿6976) resulted in morphological alterations and reorganization of actin filaments similar to retinoic-acid-treated cells. For example, RL95-2 cells treated with staurosporine flattened, exhibited cell surface extensions and some actin filaments. Bisindolylmaleimide-treated cells flattened, and actin filaments reorganized similar to retinoic-acid-treated cells. RL95-2 cells treated with G¿6976, which inhibits only PKC, alpha, beta and gamma, exhibited many cell surface extensions and some actin filament reorganization. We then investigated whether retinoic acid affected the subcellular localization of PKC-alpha. In control cells, PKC-alpha was mainly evident as diffuse cytoplasmic immunostaining, with a small percentage of total PKC-alpha also evident in the plasma membrane. Retinoic acid treatment dramatically altered PKC-alpha localization, since a more distinct cytoplasmic and perinuclear staining pattern was apparent. Western blot analysis confirmed these results, since the amount of cytosolic PKC-alpha increased following retinoic acid treatment. Thus, retinoic-acid-induced endometrial differentiation may be associated with alterations in PKC-alpha localization and signaling.

Adenocarcinoma↗

PubMed1997

2-Chloroethyl-3-sarcosinamide-1-nitrosourea, a novel chloroethylnitrosourea analogue with enhanced antitumor activity against human glioma xenografts.

Nitrosoureas are among the most widely used agents used in the treatment of malignant gliomas. Here, the activity of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) was compared with that of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), in vivo against s.c. implanted SF-295 and U-251 central nervous system (CNS) tumor xenografts. When given i.v., q4d for 3 doses, to athymic mice bearing s.c. SF-295 tumors, SarCNU, at an optimum of 167 mg/kg/dose, produced 9 tumor-free animals of 10 total animals, 1 regression, and no evidence of overt toxicity (> or =20% body weight loss). With a similar dosing schedule, BCNU produced no tumor-free animals, six regressions, and one drug-related death at its optimum of 30 mg/kg/dose. Furthermore, SarCNU retained high antitumor activity at two lower dose levels, 66 and 45% of the optimal dose, whereas BCNU demonstrated a progressive loss of antitumor activity at lower doses. Following p.o. administration, SarCNU similarly demonstrated antitumor activity that was superior to that of BCNU. In the U-251 CNS tumor model, SarCNU yielded six of six tumor-free animals at 80 mg/kg/dose with i.p. administration q.d. for 5 days, starting on day 14, whereas BCNU, at 9 mg/kg/dose, yielded three of six tumor-free mice and one drug-related death. Again, SarCNU resulted in tumor-free animals at 66 and 45% of its optimal dose and was relatively nontoxic, in contrast to BCNU. Results of testing to date indicate that SarCNU is clearly more effective than BCNU against the human CNS tumors SF-295 and U-251 in vivo. These results encourage the initiation of clinical trials for SarCNU, in an effort to improve therapeutic approaches to glioma, but clinical trials must determine whether superiority of SarCNU in preclinical models can be extrapolated to patients.

Administration, Oral↗

PubMed1997

Multiple cellular proteins are recognized by the adeno-associated virus Rep78 major regulatory protein and the amino-half of Rep78 is required for many of these interactions.

Adeno-associated virus (AAV) encoded Rep78 is a multi-functional protein which is required for AAV DNA replication, is able to regulate both AAV and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of AAV DNA into human chromosome 19. By comparison with the analogous replication protein of the polyomaviruses, large T antigen, it seemed likely that Rep78 would interact with cellular proteins to carry out at least some its functions. This study demonstrates that Rep78 is able to interact with multiple cellular proteins, from cellular extracts as measured by West(far)-western, coimmunoprecipitation, and Rep78-affinity chromatography analysis. Eight cellular proteins of approximately 180, 140, 120, 95, 75, 55, 45, and 35 kDa (+/- 10%), were observed to bind Rep78 in all three assay systems. Two others, of 30 and 24 kDa, were observed in two of three assay systems. Furthermore, using truncated Rep78 proteins, it is demonstrated that the amino-terminus is required for most Rep78-cellular protein interactions. However, the extreme carboxy-terminus of Rep78 was found to be all that is required for binding to the 55 kDa cellular protein.

Binding Sites↗

PubMed1997

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.

Amino Acid Isomerases↗

PubMed1997

Tamoxifen alters the localization of F-actin and alpha 5/beta 1-integrin fibronectin receptors in human endometrial stromal cells and carcinoma cells.

We have investigated F-actin and the integrin fibronectin receptor as possible targets of tamoxifen (TAM) signaling in a cell-based model of the endometrium. Normal human endometrial stromal cells and RL95-2 human endometrial adenocarcinoma cells were treated for 1 h with TAM, a known antagonist of protein kinase C (PKC), or with staurosporine or HA1004, two broad-spectrum protein kinase antagonists capable of inhibiting PKC and PKA, respectively. We utilized fluorescein-phalloidin and confocal microscopy to visualize the cellular distribution of F-actin. Normal stromal cells and RL95-2 cells differed in the arrangement of F-actin in control cells and in their response to TAM. In control stromal cells, actin stress fibers were well organized throughout the cell, but in RL95-2 cells, they were disorganized and present mainly at the cell periphery. F-actin in RL95-2 cells treated with TAM (0.1 and 1.0 microM) or with staurosporine (0.7 and 7.0 nM) exhibited a reorganization into stress fibers consistent with a more stationary phenotype. In contrast, TAM- or staurosporine-treated normal stromal cells exhibited an increase in the amount of organized F-actin. Interestingly, in normal stromal cells treated with staurosporine but not TAM or HA1004, these F-actin fibers appeared to terminate in dense plaques proximal to the plasma membrane. The alpha 5/beta 1 integrin fibronectin receptor mediates between the extracellular matrix and the actin cytoskeleton. TAM induced clustering of the fibronectin receptor at the plasma membrane in normal stromal cells, but not in carcinoma cells. This study supports the importance of plasma membrane-cytoskeletal protein interactions in the response of normal and carcinoma cells to TAM.

Actins↗

PubMed1996

The major homology region of the HIV-1 gag precursor influences membrane affinity.

Assembly of retroviruses, including HIV-1, involves movement of newly synthesized viral proteins and RNA to the plasma membranes of host cells. The major homology region (MHR, aa 285-304), a highly conserved sequence in the capsid domain of the HIV-1 Gag precursor polyprotein, plays a critical, but unknown, role in infectious particle assembly. Mutations of invariant residues in the sequence have pleiotropic effects: Mutation of Gln287 blocks viral assembly while mutation of Arg299 permits assembly, but blocks formation of infectious particles. In this report, we demonstrate that Gag proteins lacking the entire MHR accumulated in the cytoplasm of transfected COS-1 cells, as did the wild-type protein, but were processed in a defective manner at the cellular membrane resulting in impaired particle assembly. To further examine the role of the MHR in membrane association, membrane binding of unmyristylated recombinant Gag proteins with alterations in the MHR was investigated in vitro. The wild-type Gag precursor bound to acidic phospholipid vesicles highly efficiently, as determined by fluorescence spectroscopy or velocity sedimentation. In contrast, deletion of the entire MHR reduced membrane affinity an average of approximately 3-fold or greater. Mutation of the invariant Gln287 residue disrupted membrane affinity approximately 6-fold relative to the wild-type, which was similar to the level of inhibition obtained by deletion of a membrane-binding signal previously identified in the matrix domain of the Gag precursor. Mutation of the invariant Arg299 residue reduced the affinity to a lesser extent. The results indicate that correct membrane binding is determined not only by signals in the MA domain of the precursor but also by sequences in the CA domain of Gag. We speculate that defects in the highly conserved MHR affect a Gag conformation that is required for productive interactions at the membrane assembly site.

Amino Acid Sequence↗

PubMed1996

Balancing the clinical outcomes and economics associated with thromboembolic prophylaxis.

The approach to the treatment of patients is being influenced by the movement towards integrating health care services. This trend presents several challenges to health care professionals as patients move through the cycles of wellness, prevention, acute treatment, and after-care. Consequently, the development of treatment guidelines based upon clinical and economic parameters is critical. Preventing thromboembolic complications is receiving a great deal of attention due to the tremendous medical and economic consequences of deep venous thrombosis and pulmonary embolism. The early discharge of patients from acute surgical and medical facilities is increasing the need to consider outpatient prophylaxis. Pharmacoeconomic modeling demonstrates that enoxaparin may be more cost-effective than warfarin for outpatient prophylaxis when used for 3 weeks or less. The requirement of a subcutaneous injection of enoxaparin is not a detraction to this recommendation. Patient self-medication is a common practice and is growing in prevalence. Patients are eager to learn about their illnesses and treatment options as a result of their attitudes regarding empowerment and responsibility for their own health care.

Cost of Illness↗

PubMed1996

Preclinical antitumor activity of bizelesin in mice.

Bizelesin (U-77779, NSC 615291), a synthetic analogue of the cytotoxic antibiotic CC-1065, is a bifunctional alkylating agent that produces DNA interstrand cross-links. Bizelesin was evaluated for antitumor activity against a broad spectrum of syngeneic murine tumors and human tumor xenografts in mice. Systemic drug administration produced >6.7 log10 cell kill against i.p. implanted P388 and L1210 leukemias and 80% tumor-free survivors against s.c. implanted L1210. Against i.p. implanted B16 melanoma, i.p. drug administration produced a 158%; increase in life span with 25% tumor-free survivors, whereas i.v. drug administration produced only a 67% increase in life span with no tumor-free survivors. More than 1.0 log10 cell kill was observed at low microgram/kg doses in several human tumor models representing diverse histiotypes (CAKI-1 renal, LX-1 lung, HT-29 colon, LOX IMVI and UACC-62 melanomas, and MX-1 mammary). Less than 1.0 log10 cell kill was exhibited in other tumor models (Lewis lung, colon 38, pancreatic 02, MCF7 mammary, and SK-MEL-3 melanoma). Bizelesin was optimally active when administered i.v. Although antitumor activity was independent of the schedule of administration, greater total doses were tolerated on the more prolonged schedules in any given experiment. Therapeutic doses of bizelesin did not produce delayed deaths, which had previously been observed for the parent compound CC-1065. However, recovery of lost weight was not attained until 16-30 days posttherapy. Bizelesin was as active against murine leukemia sublines resistant to cisplatin, melphalan, and 1,3-bis-(2-chloroethyl)-1-nitrosourea as against the parental line but was totally inactive against a doxorubicin-resistant subline. The complete cross-resistance of the doxorubicin-resistant subline to bizelesin suggests that bizelesin may be a substrate for the efflux pump that causes multidrug resistance. Due to its breadth of antitumor activity, potency, unique mechanism of action, and lack of cross-resistance with other alkylating agents, bizelesin was selected for development in clinical trials by the National Cancer Institute and the Upjohn Company. Toxicological studies and pharmaceutical development have been completed, and clinical trials are planned to start in the summer of 1996.

Animals↗

PubMed1995

Phenotype and differentiation potential of a novel rat tracheal epithelial cell line.

In this report we described the establishment and characterization of a continuous rat tracheal epithelial (RTE) cell line spontaneously derived from secondary RTE cell cultures. Designated SPOC1, this cell line is nontumorigenic and maintains a diploid karyotype with specific, nonrandom chromosomal alterations involving chromosomes 1, 3, and 6. SPOC1 cells demonstrate decreased requirements for peptide growth factors, compared with primary RTE cells. Upon inoculation into denuded rat tracheas, which are then implanted into syngeneic hosts, SPOC1 cells initially form a stratified squamous epithelium, which becomes less stratified with time and forms glandlike invaginations into the surrounding lamina propria. No evidence of ciliated cell differentiation is detected. The epithelium formed by SPOC1 cells in tracheal grafts reacts with antibodies specific for keratin 14, 13, and 19 (but not keratin 18) at both early and late time points, although the localization of antibody staining changes as the epithelium becomes less stratified with time. The suprabasal epithelial cells become positive for alcian blue-periodic acid-Schiff staining at later time points. The near-normal karyotype and differentiation potential of SPOC1 cells make this cell line a unique window into early changes occurring during immortalization of airway epithelial cells and will allow studies of relationships between differentiation state and neoplastic transformation.

Animals↗

PubMed1994

Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.

The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precursor expressed in Escherichia coli were compared using circular dichroic (CD) spectral analysis. The spectra obtained for the intact recombinant and viral proteins were indistinguishable, indicating that the backbone configurations directed by the primary amino acid sequences of the proteins were similar or identical. The structure predictions derived from CD were, in general, inconsistent with a model proposing the eight-stranded beta barrel motif found in several RNA viruses. However, aspects of the model were supported by experiments that identified surface-exposed regions. Biochemical analysis indicated that the recombinant CA protein formed nonrandom higher-ordered structures in vitro. Under physiological conditions, the protein assembled into oligomers containing subunits in two packing arrangements. In one arrangement, the central region near Arg100 was exposed and susceptible to tryptic digestion at low enzyme concentrations (enzyme:substrate ratios = 1:5000 to 1:100). Also, in this arrangement, the proteins were susceptible to crosslinking by the bifunctional agent DTSSP. Proteins in the other arrangement were resistant to proteolysis at low enzyme concentrations. The central region of these resistant molecules was inaccessible to monospecific antibodies that recognized antigenic sites between residues 94 and 107 and these proteins were not crosslinked by DTSSP or EGS. Following incubation with trypsin, both the resistant molecules and the fragments derived from the susceptible proteins in the oligomer migrated as smaller complexes, suggesting that the regions digested by trypsin stabilized the oligomer unit. The results indicate that the central region of the HIV-1 CA protein plays a role in formation of higher-ordered structures. Moreover, the relative stability of the N- and C-terminal partial digestion fragments arising from cleavage at Arg100/Gly101 suggests that this exposed central region separates two structural domains of the protein.

Circular Dichroism↗

PubMed1994

Physical mapping of the rat MHC class II genes shows a high level of interspecies conservation.

We report here a pulsed-field gel electrophoresis map of the rat major histocompatibility complex (MHC) class II region. Using probes for the recently discovered Tap-1 and Tap-2 genes and the different MHC class II genes, we found the gene order in the rat MHC (RT1) region to be RT1.H-Tap-1-Tap-2-Bb-Ba-Db-Da. Moreover, the distance between the Tap-1 and the RT1.Da genes is approximately 150 kb. This, together with recent mapping of the RT1 class II region, demonstrates an extensive colinearity in the MHC region of different species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Alterations in the localization of F-actin, fibronectin, and thrombospondin occur prior to neoplastic transformation in rat tracheal epithelial cells.

Structural glycoproteins and cytoskeletal proteins play a major role in the regulation of cellular organization and function. Changes in the structure and function of these proteins are involved in the cascade of events which lead to neoplastic transformation. We evaluated RNA levels, protein localization, and organization of selected proteins in an in vitro model system of respiratory carcinogenesis to examine alterations in cell architecture. Localization of fibronectin (Fn), thrombospondin (Tsp), and F-actin was examined in (1) primary rat tracheal epithelial (RTE) cells; (2) spontaneously immortalized nonneoplastic cells (SPOC-1); and (3) neoplastic cells (EGV5T) derived from tumors arising following transplantation of an N-methyl-N'-nitro-N-nitrosoguanidine-transformed RTE cell line into nude mice. Proteins were stained with fluorescein-labeled antibodies or phalloidin compound and analysis was performed with a confocal laser scanning microscope. Primary RTE cells display organized F-actin stress fibers, perinuclear Fn and Tsp, and pericellular Fn in fibrillar arrays. In larger colonies, Tsp occurs between cells and occasionally in fibrillar arrays. SPOC-1 cells, unlike primary RTE cells and neoplastic EGV5T cells, seldom form junctions and exhibit few cell surface extensions. F-actin stress fibers are reduced in these immortalized cells. F-actin in SPOC-1 cells occurs in the perinuclear region, scattered diffusely throughout the cell and in punctate adhesions. Fn and Tsp are localized to the perinuclear region with Fn staining more intensely. EGV5T neoplastic cells also display a dramatic loss of stress fibers and F-actin is concentrated mainly near the cell periphery. Perinuclear staining of Fn and Tsp occurs in some cells within the colony. Levels of Tsp RNA and Fn RNA and protein are significantly reduced in both cell lines compared to primary RTE cells. We conclude that structural protein disruptions are early events in the transformation of these respiratory epithelial cells.

Actins↗

PubMed1994

T-cell recognition of an allogeneic RT1-Dbu class II MHC peptide.

The allo-antibody response of several rat strains to an unconjugated synthetic 20 amino acid peptide derived from the alpha helical region of the RT1-Du beta chain was tested. The LEW (RT1l) and WAG (RT1u) strains produced little or no antibody; the PVG (RT1c) and DA (RT1av1) strains produced moderate amounts of antibody; while the BN (RT1n) strain produced strong primary and secondary antibody responses. This suggested that the BN strain was able to process and present the RT1-Dbu peptide on its class II molecules. In vitro proliferation studies demonstrated that LEW T cells did not respond to the peptide, whereas BN T cells responded strongly, and that the response in the BN strain was found only in the CD4+ T-cell subset. However, immunisation of BN rats with the RT1-Dbu peptide failed to cause any acceleration of rejection of WAG skin or kidney grafts. Moreover, BN rats primed with WAG skin and kidney grafts did not produce T cells reactive to the RT1-Dbu synthetic peptide. This suggests that the T-cell response of the BN strain to the synthetic major histocompatibility complex peptide was not relevant to the indirect T-cell allo-recognition response to naturally processed RT1-Du beta chains.

Amino Acid Sequence↗

PubMed1993

Localization of tenascin in uterine sarcomas and partially transformed endometrial stromal cells.

Normal mesenchymal cells within developing embryonic organs and transformed stromal cells in organs undergoing spontaneous carcinogenesis have the capacity for normal or altered expression of the extracellular matrix glycoprotein tenascin (Tn). Mesenchymal cell constituents of normal adult organs show only a very limited tendency to deposit Tn in their extracellular matrix. In the present study, we investigated whether malignant human mesenchymal cells derived from uterine sarcomas or normal human endometrial stromal cells partially transformed via transfection with selected oncogenes have the capacity to produce and deposit Tn. We reached the following conclusions: (1) compared with normal endometrial tissues, uterine sarcomas show heterogeneous, but increased, immunoreactive staining patterns exclusively within the extracellular compartment, regardless of the histologic subtype of the tumor; (2) in vitro, all normal and transfected stromal cells and cell lines examined secreted Tn into the tissue culture medium; (3) this secretory ability was not translated into morphologic uniformity, since immunoreactivity detected by confocal laser scanning microscopy was observed in only selected cell populations; (4) also, the deposition and the incorporation of Tn depended upon the density of transfected cells, and (5) double-staining experiments revealed that Tn could always be localized in close proximity to fibronectin. In summary, the production of Tn is increased in most cases of human uterine sarcoma. The capacity of stromal cells to deposit Tn in a matrix-like structure in vitro, rather than increase production of Tn, is correlated with the degree of neoplastic progression.

Adult↗

PubMed1993

Enoxaparin: the low-molecular-weight heparin for prevention of postoperative thromboembolic complications.

OBJECTIVE: To introduce readers to a new low-molecular-weight heparin (LMWH) product, enoxaparin. The chemistry, pharmacology, pharmacodynamics, clinical efficacy in thromboembolic prophylaxis following surgery, and adverse effects are reviewed. DATA SOURCES: A MEDLINE search of the English-language literature was used to identify relevant literature. STUDY SELECTION: A focus was placed on human clinical studies with well-accepted measures of antithrombotic efficacy endpoints, i.e., venography and ultrasonography. Emphasis was on pharmacologic and pharmacokinetic studies conducted in humans. DATA EXTRACTION: Most data were extracted from double-blind, controlled clinical studies. Other study designs were accepted if the results were believed to be significant. Pharmacology and pharmacokinetic data were selected from studies with exceptional design conducted in humans. DATA SYNTHESIS: Enoxaparin is a polysaccharide chain produced by the depolymerization of heparin. In comparison with heparin, which has an average molecular weight of 12,000-15,000 daltons, the average molecular weight of enoxaparin is approximately 4500 daltons. Enoxaparin does not form a complex with antithrombin III and thrombin as extensively as does heparin; however, the anti-Xa activity of enoxaparin is similar. The significance of this fact is an enhancement of antithrombotic activity and clinical efficacy. Trials comparing enoxaparin with other thromboembolic prophylaxis techniques are ongoing. CONCLUSIONS: Thromboembolism remains one of the major complications of all surgical procedures. Attempts have been made throughout the last century to develop the most effective means to prevent this complication. Clinical studies performed throughout the world have shown that enoxaparin is superior or equivalent to other antithrombotic agents, including heparin, in preventing the formation of venous thromboembolism. In addition, enoxaparin appears to possess an equivalent or lower incidence of bleeding complications when compared with heparin prophylaxis. Enoxaparin is expected to be joined by other LMWH products in the future. As a result, the methods of providing effective prophylaxis against thromboembolic complications is expected to change in the coming years.

Clinical Trials as Topic↗

PubMed1992

Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease.

A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-beta-naphthylamide). Fluorescence due to the release of Pro-MeO-beta-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kappa cat for the hydrolysis of N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta- naphthylamide by HIV-1 protease were calculated to be 2.0 +/- 0.2 mM and 75 +/- 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.

Amino Acid Sequence↗

PubMed1992

Differential effects of dioctanoylglycerol on fibronectin localization in normal, partially transformed, and malignant human endometrial stromal cells.

In this study, we describe the effects of direct activation of PKC by dioctanoylglycerol (DiC8) on cellular morphology and the localization of fibronectin (Fn) in normal, oncogene-transfected, and malignant human endometrial stromal cells. We questioned whether DiC8, an endogenous specific activator of PKC, would function as a second oncogene in partially transformed human endometrial stromal cells (HESC). Cells utilized were (1) normal HESC, (2) HESC transfected with a plasmid containing an origin-defective temperature-sensitive SV40 large T antigen alone or (3) in combination with an EJ ras oncogene, and (4) an endometrial sarcoma cell line (S7). Cell cultures were treated for 1 h with sn-dioctanoylglycerol (DiC8) and stained with a monoclonal fluorescein-labeled anti-Fn antibody. In normal HESC, DiC8 induced cell rounding and caused Fn localization to revert from the perinuclear region to the cell periphery. All experiments in this investigation were performed when cells were maintained at the permissive temperature for SV40 large T antigen function. In HESC expressing the SV40 large T antigen alone, Fn was localized to the perinuclear region and also occurred as parallel strands between cells. When these cells were treated with DiC8, Fn localization changed to intense punctate regions at the cell periphery or to matrix-like patterns between cells. Also, in these cells, DiC8 induced greater detachment of cells from the substrate than from other cells, resulting in an apparent piling up of cells. Control and treated SV40/EJ ras cells and uterine sarcoma cells expressed Fn in a matrix-like pattern between cells. The rounded cellular morphology of treated HESC and treated cells expressing SV40 resembled the morphology of control or treated SV40/EJ ras cells and uterine sarcoma cells. Thus, treated cells expressing the SV40 large T antigen resembled the SV40/EJ ras cells and uterine sarcoma cells with respect to Fn localization and cellular morphology. DiC8 did not appear to further transform HESC expressing SV40 and EJ ras. However, with regard to cell shape and Fn localization, our results suggest that DiC8 may function as a second oncogene in the signal transduction pathway, in cells expressing SV40 alone. It appears that, with regard to Fn localization, DiC8 may alter signal transduction analogously to that caused by the activated Ha-ras oncogene in HESC expressing the SV40 large T antigen.

Antibodies, Monoclonal↗

PubMed1992

Stimulation of CD4+ T lymphocytes by allogeneic MHC peptides presented on autologous antigen-presenting cells. Evidence of the indirect pathway of allorecognition in some strain combinations.

A preliminary analysis of the alloantibody response to free, unconjugated class I and class II MHC peptides in several rat and mouse strains was performed, to screen for an effective interaction between the allogeneic MHC peptides and recipient MHC molecules. The PVG rat strain was noted to produce very strong, MHC-restricted, primary and secondary responses to a synthetic peptide derived from the alpha helical region of the alpha 2 domain of an RT1.C/E class I MHC molecule of the DA strain. In vitro proliferation studies demonstrated that CD4+ but not CD8+ T cells of the PVG strain responded in a recipient APC-dependent manner to the peptide, whereas the BN strain (which showed no antibody response to this peptide) gave no T cell proliferation. Immunization of PVG rats with the peptide did not influence the rejection of DA skin allografts. The relevance of these studies to the possible mechanisms of allograft rejection by an indirect pathway are discussed.

Amino Acid Sequence↗