Biomedical subjects

S Seetharam

Publications and source records attributed to S Seetharam.

At least 19 recordsLinked to original sources

PubMed2003

Transcobalamin II receptor interacts with megalin in the renal apical brush border membrane.

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.

Animals↗

PubMed2001

Identification and characterization of two distinct ligand binding regions of cubilin.

Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.

Albumins↗

PubMed2001

Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy.

Cubilin is a 460-kDa multipurpose, multidomain receptor that contains an NH(2)-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B(12)) complex and promotes the ileal transport of cbl. The 460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a 230-kDa form of cubilin is also noted in the intestinal membranes. In thyroidectomized (TDX) rats, levels of intestinal brush-border IF-[(57)Co]-labeled cbl binding, 460-kDa cubilin protein levels and tissue (kidney) accumulation of cbl were reduced by approximately 70%. Immunoblot analysis using cubilin antiserum of intestinal total membranes from TDX rats revealed cubilin fragments with molecular masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the 460-kDa form, failed to react with antiserum to EGF. Mucosal membrane cubilin associated with megalin was reduced from approximately 12% in control to approximately 4% in TDX rats, and this decreased association was not due to altered megalin levels. Thyroxine treatment of TDX rats resulted in reversal of all of these effects, including an increase to nearly 24% of cubilin associated with megalin. In vitro, megalin binding to cubilin occurred with the NH(2)-terminal region that contained the EGF-like repeats and CUB domains 1 and 2 but not with a downstream region that contained CUB domains 2-10. These studies indicate that thyroxine deficiency in rats results in decreased uptake and tissue accumulation of cbl caused mainly by destabilization and deficit of cubilin in the intestinal brush border.

Animals↗

PubMed2001

Treatment of head and neck and esophageal xenografts employing Alimta and concurrent ionizing radiation.

We examined the interaction between Alimta and ionizing radiation (IR) as a potential strategy to enhance the therapeutic ratio of combined-modality cancer treatment. Mice bearing human esophageal adenocarcinoma xenografts (Seg-1) or squamous cell carcinoma xenografts (SQ-20B) were treated with Alimta and IR employing a fractionated treatment schedule. Treatment with Alimta alone slowed the growth of Seg-1 but not SQ-20B tumors compared with control tumors. In Seg-1 xenografts combined treatment with Alimta and IR produced significant tumor growth inhibition compared with Alimta alone or IR alone. In SQ-20B xenografts, treatment with Alimta did not enhance IR-mediated tumor growth inhibition suggesting that sensitivity to Alimta is necessary for an interactive cytotoxic effect with IR. The present data suggest the potential clinical efficacy of combining Alimta administration with radiotherapy for Alimta-sensitive cells and indicate that further testing needs to be conducted to optimize the dosing schedule to enhance the interaction between the therapeutic agents.

Adenocarcinoma↗

PubMed2000

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Adenocarcinoma↗

PubMed1999

Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Adenocarcinoma↗

PubMed1999

Enhanced eradication of local and distant tumors by genetically produced interleukin-12 and radiation.

Ionizing radiation (IR) is frequently unsuccessful in the treatment of cancer because of local failure or distant metastases. The efficacy of systemically administered cytokines for cancer therapy is often limited by toxicity. We report that intratumoral injection of an adenoviral vector with interleukin-12 (IL-12) enhances local anti-tumor effects of irradiation (IR). We demonstrate that microscopic tumor growth at a distant site is suppressed following treatment of the primary tumor with adeno-murine IL-12 (Adm.IL-12). The results support a model in which the anti-angiogenic effects of IL-12 contribute to the local anti-tumor effects of radiation, while IL-12 induced immunity suppresses growth of microscopic tumors distant from the primary irradiated site. These data suggest that combining radiotherapy with IL-12 improves both local and distant tumor control compared to either treatment alone. Immunoradiotherapy may be employed in addition to or in place of current conventional therapies to increase local control and decrease distant tumor growth.

Animals↗

PubMed1998

Modulation of apoptotic response of a radiation-resistant human carcinoma by Pseudomonas exotoxin-chimeric protein.

Strategies to sensitize human tumors that are resistant to apoptosis have been clinically unsuccessful. We demonstrate that a structurally modified chimeric Pseudomonas exotoxin, PEdelta53L/TGF-alpha/KDEL, with binding specificity for the epidermal growth factor receptor, markedly enhances sensitivity of human xenografts to radiation killing. Exposure to PEdelta53L/TGF-alpha/KDEL decreases the apoptotic threshold through protein synthesis inhibition and simultaneous production of ceramide in tumor cells that lack functional p53 protein. In contrast, no increase in local or systemic toxicity was observed with the chimeric toxin and radiation. We conclude that biochemical targeting of the chimeric toxin and physical targeting of ionizing radiation may increase the therapeutic ratio in the treatment of human cancers with alterations of p53 expression. This strategy offers a high therapeutic potential for Pseudomonas exotoxin A chimeric proteins and irradiation.

ADP Ribose Transferases↗

PubMed1998

Characterization of the human transcobalamin II promoter. A proximal GC/GT box is a dominant negative element.

Deletion and mutagenesis of the 5'-flanking region of the human transcobalamin II (TC II) transfected in human intestinal epithelial Caco-2 cells have revealed that TC II promoter activity is: (a) very weak; (b) restricted to a core region (-29 to -163) that contained multiple transcription initiation sites; (c) not dependent on other potential elements, such as a distally localized CCAAT box, a CF1, a HIP1 binding motif and a MED-1 element; (d) modulated weakly by a positive-acting GC box (-568-GAGGCGGTGC) and strongly by a proximal GC/GT overlapping box (-179 CCCCCGCCCCACCCC). Gel shift and immunosupershift analyses demonstrated that both the positive-acting GC box and the negative-acting GC/GT box were recognized by Sp1 and Sp3. Co-transfection studies using Sp1 and/or Sp3 expression plasmids revealed that while Sp1 stimulated, Sp3 repressed Sp1-mediated transactivation of TC II transcription. The proximal GC/GT box also acted as a negative element in human chronic myelogenous leukemia K-562 and HeLa cells. These results suggest that tissue/cell specific expression of the TC II gene may be controlled by the relative ratios of Sp1 and Sp3 that bind to the GC/GT box and the weak promoter activity of TC II is due to the transcriptional repression caused by the binding of Sp3 to the proximal GC/GT box.

Base Sequence↗

PubMed1998

Brefeldin A (BFA) inhibits basolateral membrane (BLM) delivery and dimerization of transcobalamin II receptor in human intestinal epithelial Caco-2 cells. BFA effects on BLM cholesterol content.

Brefeldin A (BFA) treatment of Caco-2 cells (5 microg/ml for 12 h) reduced by 90% the cholesterol, but not the phospholipid (PL), levels of the basolateral membrane (BLM), thus altering its PL/cholesterol molar ratio from 2.6 to 22.0, and decreasing its steady state fluorescent anisotropy (rs) from 0.27 to 0.15. BFA treatment for 12 h also resulted in complete loss of transcobalamin II receptor (TC II-R) activity/protein levels in the BLM and the disappearance of trans-Golgi network (TGN) morphology as revealed by confocal immunofluorescence microscopy using antibody to TGN 38. However, BFA treatment had no effect on either total cellular cholesterol, TC II-R activity, or PL levels. When cells treated with BFA for 12 h were exposed to BFA-free medium for 0-24 h, all of the effects were reversed, including reappearance of normal TGN morphology. TC II-R delivered to the BLM during this period was progressively sialylated and changed its physical state from a monomer (8 h) to a dimer (12 h), coinciding with increased delivery (11-53 pmol) of cholesterol to the BLM and an increase in the BLM rs from 0.15 to 0.21. These results indicate that cholesterol, but not PL, delivery to the BLM of Caco-2 cells is BFA-sensitive, and cholesterol, by influencing the higher order of the BLM, is essential for TC II-R dimerization.

Anti-Bacterial Agents↗

PubMed1997

Bipolar functional expression of transcobalamin II receptor in human intestinal epithelial Caco-2 cells.

Transcobalamin II (TC II) receptor is expressed in the apical and basolateral membranes of human intestinal mucosa and in post-confluent human intestinal epithelial Caco-2 cells with a 6-7-fold enrichment in basolateral membranes. Caco-2 cells grown on culture inserts bound (at 5 degrees C) 30 and 180 fmol of the ligand, TC II-[57Co]cobalamin (Cbl), to the apical and the basolateral surfaces, respectively. Within 5 h at 37 degrees C, all apically bound Cbl was internalized and subsequently transcytosed as TC II-Cbl. In contrast, all basolateral surface-bound Cbl was internalized and retained by the cells, but transferred from TC II to other cellular proteins. Chloroquine or leupeptin had no effect on the apical to basolateral transcytosis of either [57Co]Cbl or 125I-TC II. In contrast, following basolateral internalization of the ligand, both chloroquine and leupeptin inhibited the intracellular degradation of 125I-TC II, which resulted in secretion of 60-65% of TC II-Cbl complex into the basolateral medium. When 125I-TC II-Cbl was orally administered to rats, intact labeled TC II was detected in the portal blood 4 and 8 h later. These studies suggest that TC II-Cbl is processed when presented to the (a) apical/luminal side by a hitherto unrecognized non-lysosomal pathway in which both TC II and Cbl are transcytosed and (b) basolateral side by the lysosomal pathway in which TC II is degraded and the released Cbl is utilized.

Animals↗

PubMed1996

Dimerization of transcobalamin II receptor. Requirement of a structurally ordered lipid bilayer.

Transcobalamin II receptor (TC II-R) exists as a monomer and a dimer of molecular masses of 62 and 124 kDa in the microsomal and plasma membranes, respectively, and in vitro, pure TC II-R monomer dimerizes upon insertion into egg PC/cholesterol (molar ratio, 4:1) liposomes (Bose, S., Seetharam, S., and Seetharam, B. (1995) J. Biol Chem. 270, 8152-8157 and Bose, S., Seetharam, S., Hammond, T., and Seetharam, B. (1995) Biochem. J. 310, 923-929). The current studies were carried out to define the mechanism of TC II-R dimerization. Both the mature TC II-R (62 kDa) and the enzymatically deglycosylated TC II-R (45-47 kDa) demonstrated optimal association and formed dimers of molecular masses of 95 and 124 kDa, respectively, at 22 degrees C when bound to egg PC vesicles containing at least 10 mol % of cholesterol. Mature TC II-R dimerized upon insertion into synthetic phosphatidylcholine vesicles of different fatty acyl chain length (dimyristoyl, dipalmitoyl, and disteroyl phosphatidylcholine) in the absence or the presence of cholesterol at temperatures below or above their transition temperatures, respectively. Dimerization of TC II-R also occurred with vesicles prepared using lipid extract from the plasma but not microsomal membranes. Cholesterol depletion of native intestinal plasma membranes or its enrichment in the microsomal membranes resulted in the in situ conversion of the 124-kDa dimer to the 62-kDa monomer or of the monomer into the dimer form, respectively. Treatment of plasma membranes with phospholipase A2 resulted in the conversion of the dimer form of the receptor to the monomer form and spin label studies using 1-palmitoyl, 12 doxylsteroyl phosphatidylcholine revealed that interactions of TC II-R with PC vesicles increased order around the probe. Based on these results we suggest that dimerization of TC II-R is mediated by its interactions with a rigid more ordered lipid bilayer membrane, is regulated in plasma membranes by cholesterol levels, and is independent of glycosylation-mediated folding.

Animals↗

PubMed1996

In vitro and in vivo inactivation of transcobalamin II receptor by its antiserum.

Rabbits injected with pure human placental transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, transcobalamin II, in their plasma. Exogenously added transcobalamin II-[57Co]cyanocobalamin bound very poorly (2-5%) to the affected rabbit liver, kidney, and intestinal total or intestinal basolateral membrane extracts relative to the binding by membrane extracts from normal rabbit tissues. The activity was restored to normal values following a wash of affected rabbit tissue membranes with pH 3 buffer containing 200 mM potassium thiocyanate. Immunoblot analysis of normal and affected rabbit kidney and liver total membranes revealed similar amounts of 124-kDa TC II-R dimer protein. The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II-[57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl-dependent enzymes. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes. Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and thus cause severe Cbl deficiency and the noted failure to thrive.

Animals↗

PubMed1995

Regulation of expression of transcobalamin II receptor in the rat.

Surface and intracellular membrane distribution and hormonal regulation of transcobalamin II receptor (TC II-R) activity and protein levels have been studied in an effort to understand its regulation of expression in the rat. TC II-R activity and the levels of the 62 kDa monomeric and 124 kDa dimeric forms of TC II-R were highest in the rat kidney and intestine, and in these tissues the receptor expression was not dependent upon the postnatal development of the rat. TC II-R expression was uniform in the various regions of the gut. Surface membrane distribution of TC II-R in the kidney revealed the expression of the 124 kDa dimer form of TC II-R in the apical and basolateral membranes in the ratio of 1:10. Further subcellular distribution of TC II-R in the kidney revealed the expression of the 124 kDa dimer in the intermicrovillar clefts and clathrin-coated vesicles and the 62 kDa monomer in the microsomes. Neither the monomer nor the dimer could be detected in the early endosomes or lysosomes. Membrane TC II-R activity and TC II-R protein levels and cobalamin (Cbl; vitamin B12) transport in vivo were inhibited by about 90% in adrenalectomized rats and all three returned to normal levels by oral treatment of these animals with cortisone acetate. In contrast, thyroidectomy or experimentally induced diabetes had no effect on TC II-R activity or Cbl transport. Based on these observations, we suggest that TC II-R expression is not developmentally or regionally regulated in rat renal and intestinal membranes and its expression in the kidney is asymmetrically distributed between the apical (10%) and basolateral (90%) membranes. In addition, our results also show that the dimerization of TC II-R is a post-microsomal event and that the expression of TC II-R and plasma Cbl transport is regulated by cortisone.

Adrenalectomy↗

PubMed1995

Membrane expression and interactions of human transcobalamin II receptor.

Antiserum raised to purified 62-kDa human placental transcobalamin II receptor (TC II-R) has been used to study its synthesis and membrane expression. The antiserum immunoprecipitated a 45-kDa protein from the cell-free translation using human kidney mRNA and recognized a single 124-kDa band on immunoblotting of placental and other human tissue membranes, and quantitation of the blots revealed high levels of TC II-R expression in the human kidney followed by placenta, intestine, and liver. Triton X-100 extraction of placental membranes resulted in the complete (100%) solubilization of the receptor, and immunoblotting of the Triton X-100-soluble fraction revealed a single band of 62 kDa. Lipid extraction of placental membranes with a mixture of chloroformmethanol (2:1) followed by immunoblotting revealed a single band of molecular mass 62 kDa. The molecular mass of the pure Triton X-100-bound receptor increased on SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its insertion in liposomes prepared using egg phosphatidylcholine and cholesterol. Chemical cross-linking of native membrane-or lipid vesicle-bound TC II-R or detergent-soluble extracts of the membrane with 125I-TC II-cobalamin revealed that both the 124- and 62-kDa forms of the receptor were active in ligand binding. Based on these results we suggest that TC II-R is synthesized as a single polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is expressed in plasma membranes as a noncovalent dimer of 124 kDa. The dimerization of TC II-R in the plasma membranes is due to its interactions with annular lipids.

Biopolymers↗

PubMed1995

Genomic structure of human transcobalamin II: comparison to human intrinsic factor and transcobalamin I.

Human transcobalamin II (TC II) gene was isolated and partially sequenced. The gene is composed of nine exons and eight introns spanning approximately 20 kb. Multiple potential transcription start sites were revealed by primer extension analysis. The 5'-flanking region of the gene contained no TATA-like motif, but a binding motif for HIP1, which is suggested to be important in the transcription of TATA-less housekeeping genes, was identified in a region very close to the initiator methionine codon. In addition, potential binding sites for a variety of transcription factors such as SP1, AP2, CF1, NF-IL6, Ets-1, Myb and E2A were also observed. Comparison of the genomic structure of TC II to other Cbl-binding proteins, human gastric intrinsic factor (IF) and transcobalamin I (TC I) revealed similar intron-exon organizations with respect to the number, position and size of exons. These results suggest that TC II, TC I and IF genes have originated by gene duplications of an ancestral gene and TC II, unlike the other two Cbl-binding proteins, is the product of a "housekeeping" gene.

Amino Acid Sequence↗

PubMed1995

Herpes simplex type 1:lacZ recombinant viruses. I. Characterization and application to defining the mechanisms of action of known antiherpes agents.

Recombinant viruses with the lacZ gene placed under the control of the HSV-1 ICP4, TK and gD regulatory regions were constructed by recombination into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of beta-galactosidase was overcome by the use of HSV-1 translational initiation sequences of these genes in place of vector-derived sequences. beta-Galactosidase expression displayed the kinetics particular to each viral class. The maximal expression of beta-galactosidase from the recombinant viruses within a 22-h period (m.o.i. 5) (relative to the ICP4 virus) was gD(3) > gC(2) > ICP4(1) > TK(0.5). The ICP4 virus produces easily quantifiable levels of beta-galactosidase activity for multiplicities of infection from 5 x 10(-4) through 5 over 48 h postinfection. At multiplicities of infection between 2 and 5, ICP4-driven activity was measurable within 2 h postinfection from a monolayer of 3 x 10(4) Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals were probed by using the regulated expression of beta-galactosidase from the ICP4 virus as a marker for viral growth. An experimental antiviral (E3925, IC50 1 microgram/ml) and a neutralizing gD MAb (DUP55306, IC50 0.6 microgram/ml) acted prior to immediate early synthesis, consistent with inhibition of viral entry or uncoating. IFN-gamma inhibited expression of immediate-early synthesis, while having no effect on viral entry. IC50 values for E3925 obtained using either the ICP4 or gD viruses at m.o.i. 0.005, were in good agreement with those obtained by standard plaque assays, but were determined in only 1 day, using a microtiter plate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively reproducing the results for IC50 determinations from standard plaque assays within 24 h in a microtiter plate format.

Antibodies, Monoclonal↗

PubMed1995

Herpes simplex type1:lacZ recombinant viruses. II. Microtiter plate-based colorimetric assays for the discovery of new antiherpes agents and the points at which such agents disrupt the viral replication cycle.

A panel of microtiter plate-based colorimetric assays for monitoring HSV-1 growth has been made. The panel consists of 4 different HSV-1 (strain KOS) lacZ recombinant viruses which express beta-galactosidase under the control of different HSV-1 promoters derived from each class of herpes simplex gene expression: immediate-early (ICP4), early (TK), delayed early (gD) and late (gC). Inhibitors of HSV-1 growth were evaluated using differential effects on each of the reporter viruses as a measure of which points in the viral replication cycle an inhibitor was acting. Aphidicolin (DNA synthesis inhibitor) was studied as a model compound. At an m.o.i. of 0.05, at 24 h postinfection (h p.i.), aphidicolin inhibited 80% of viral growth at 1 micrograms/ml, as determined by a reduction in ICP4-driven activity within the second cycle of infection. At m.o.i. 5, within the first infectious cycle, aphidicolin had no effect on the signals from either the ICP4 or TK viruses at 3 micrograms/ml, while largely suppressing gD and fully inhibiting gC-driven signals at 2 micrograms/ml. This profile is consistent with the behavior expected of a DNA synthesis inhibitor. Five inhibitors of unknown mechanism were evaluated. Two compounds inhibited ICP4-driven activity within the first infectious cycle and were classified as potential inhibitors of viral entry, uncoating or IE gene expression (XF884, BT318). One compound inhibited gD and gC-driven activity without inhibiting signal from the ICP4 and TK viruses, and was classified as a potential DNA synthesis inhibitor (DS810). Two compounds (S5193, ER622) had effects on gD- and gC-driven activity which were somewhat different from aphidicolin and DS810, but which could be interpreted as inhibition of viral assembly and/or egress. The potency of XF884 varied with the time postinfection at which it was added to cells (IC50 3.7 to > 10 micrograms/ml) while the effects of BT318 were independent of time of addition (IC50 11.4 micrograms/ml). These results suggest XF884 inhibits viral entry while BT318 is acting after viral entry, possibly as a direct inhibitor of ICP4 gene expression. Together, these results suggest the panel of recombinant herpes viruses has utility in aiding in the identification of the points in the herpes life cycle at which antiherpes drug candidates, of unknown mechanisms, are acting.

Antimetabolites↗