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Biomedical subjects

T J Lobl

Publications and source records attributed to T J Lobl.

At least 19 recordsLinked to original sources
PubMed2001

N-Succinyl-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin: an extracellularly tumor-activated prodrug devoid of intravenous acute toxicity.

Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.

Animals↗
PubMed2001

Extracellularly tumor-activated prodrugs for the selective chemotherapy of cancer: application to doxorubicin and preliminary in vitro and in vivo studies.

Oligopeptidic derivatives of anthracyclines unable to penetrate cells were prepared and screened for their stability in human blood and their reactivation by peptidases secreted by cancer cells. N-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin was selected as a new candidate prodrug. The NH2-terminal beta-alanine allows a very good blood stability. A two-step activation by peptidases found in conditioned media of cancer cells ultimately yields N-L-leucyl-doxorubicin. In vitro, when MCF-7/6 cancer cells are exposed to the prodrug, they accumulate about 14 times more doxorubicin than MRC-5 normal fibroblasts, whereas when exposed to doxorubicin the uptake is slightly higher in fibroblasts than in MCF-7/6 cells. This increased specificity of the prodrug over doxorubicin was confirmed in cytotoxicity assays using the same cell types. In vivo, the prodrug proved about nine times less toxic than doxorubicin in the normal mouse and also much more efficient in two different experimental chemotherapy models of human breast tumors.

Animals↗
PubMed1998

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Amino Acid Sequence↗
PubMed1994

Cyclic RGD peptide inhibits alpha 4 beta 1 interaction with connecting segment 1 and vascular cell adhesion molecule.

The integrin supergene family includes receptors for a variety of extracellular matrix as well as cell surface proteins. Integrin alpha 4 has been shown to play an important role in leukocyte adhesion and extravasation during immune and inflammatory reactions. One recognition sequence known to interact with alpha 4 is the Leu-Asp-Val (LDV) site contained in the connecting segment 1 region of fibronectin. Here we present evidence that shows that a conformationally restricted RGD-containing peptide is a potent inhibitor of cell adhesion mediated by alpha 4 beta 1, a receptor not convincingly documented to interact with RGD peptides. This peptide, 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8), blocks Jurkat cell adhesion to connecting segment 1-containing peptides as well as cell adhesion to cytokine-activated endothelial cells. Adhesion of Jurkat cells to either vascular cell adhesion molecule-expressing cells or recombinant vascular cell adhesion molecule-coated plates was likewise inhibited by this peptide. Furthermore, alpha 4 beta 1 can bind directly to a cyclic RGD peptide immobilized to Sepharose. Integrins, alpha 5 beta 1, alpha v beta 3, alpha IIb/beta IIIa, alpha 2 beta 1, alpha v beta 1, alpha v beta 5, alpha v beta 6, and alpha 3 beta 1, have been shown to recognize the Arg-Gly-Asp (RGD) sequence present in a variety of extracellular matrix proteins, and our data support the addition of alpha 4 beta 1 to this group. Further studies using molecular modeling of such cyclic RGD peptides could help in the design of more potent peptides or nonpeptide mimetics that could effectively block alpha 4-mediated activity and have potential application in a number of inflammatory diseases.

Amino Acid Sequence↗
PubMed1993

A novel cyclic pentapeptide inhibits alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated cell adhesion.

Lymphocytes and monocytes initiate and modulate inflammatory and immune responses for host defense. This process is dependent upon extravasation of leukocytes from the circulation to sites of antigenic challenge and is controlled, in part, by various integrins, including alpha 4 beta 1 and alpha 5 beta 1. A small cyclic pentapeptide that inhibits, in vitro, both alpha 4 beta 1 and alpha 5 beta 1 activity is described. This peptide, Arg-Cys-Asp-Thioproline-Cys (RC*D[ThioP]C*), is cyclized by a disulfide bond through the cysteine residues (the asterisks denote cyclizing residues). RC*D(ThioP)C* inhibits alpha 5 beta 1-mediated leukocyte adhesion to the 120-kDa Arg-Gly-Asp (RGD)-containing binding site of fibronectin. Two different adhesion activities of alpha 4 beta 1 are also inhibited: alpha 4 beta 1-mediated cell adhesion to the alternatively spliced CS-1 site of fibronectin and the alpha 4 beta 1-dependent binding of leukocytes to cytokine-activated endothelial cells. Both alpha 4 beta 1 and alpha 5 beta 1 can be purified by affinity chromatography using the immobilized pentapeptide. The peptide does not inhibit adhesion to other extracellular matrix proteins including laminin and vitronectin. The specificity of the RC*D(ThioP)C* peptide for alpha 4 beta 1 and alpha 5 beta 1 suggests potential therapeutic utility for inhibiting inflammatory disease.

Amino Acid Sequence↗
PubMed1990

SV40 large T-antigen nuclear signal analogues: successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates.

The signal sequence of a nuclear-directed protein encodes the necessary information for targeting the attached proteins to the cell nucleus. The sequence/structural requirements for a functional transport signal were explored with a series of peptides derived from the simian virus 40 large T-antigen nuclear signal 126-134 (CPKKKRKVED-NH2, wild type) conjugated to bovine serum albumin (BSA) through an N-terminal Cys (1) with m-maleimidobenzoyl-N-hydroxysuccinimide ester. Nuclear accumulation was virtually complete 15 min after microinjection into green monkey kidney cells (TC-7). Peptides with Asn, Orn, and Gln substituted for Lys128, the reverse wild-type peptide (DEVKRKKPC-NH2) and the long 34-residue wild-type analogue (CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS-NH2), were synthesized and conjugated similarly to BSA. The Orn peptide and the 34-residue wild-type analogue conjugated to BSA also transported to the nucleus but at a slower rate than 1. The reverse wild-type, Asn- and Gln-BSA conjugates of these signal analogues did not show transport to the nucleus after 6 h of incubation. In an effort to learn if such signal sequences would also target a small molecule such as a fluorescent tag to the nucleus, 1 fluorescently tagged with monobromobimane was prepared and microinjected into TC-7 cells. The peptide was distributed throughout the cell. These results support the notion that a positively charged residue at position 128 is needed for rapid nuclear transport and that the intracellular transport machinery has spatial recognition. The results with fluorophore-peptide conjugates suggest nuclear localization of these low molecular weight peptides will be difficult to attain even if attached to a functional nuclear localization sequence.

Amino Acid Sequence↗
PubMed1989

Use of the Membrane Invasion Culture System (MICS) as a screen for anti-invasive agents.

The Membrane Invasion Culture System (MICS) assay was adapted for relatively rapid screening of compounds and used to identify anti-invasive drugs that inhibit human and murine tumor cell migration through a reconstituted basement membrane in vitro. Cell lines demonstrating low and high invasive and metastatic potentials were tested with all compounds for tumoricidal effects prior to evaluation in MICS at non-cytotoxic doses. The effect on invasive potential in the MICS assay was determined in 3 categories: (1) 48 hr drug pre-treatment prior to seeding in the MICS (exceptions: 90 min pre-treatment with pertussis toxin and, for some studies, continuous exposure for 2-7 days); (2) peptide or prostaglandins 2 hr after seeding and attachment to the membranes in MICS followed by continuous exposure; and (3) cells receiving neither drug nor peptide treatment and serving as controls in each MICS chamber. Since invasion involves cellular motility and deformability, some cytoskeleton disrupting agents were selected. Of these, vincristine, colcemid and colchicine inhibited invasion but taxol did not. Pre-treatment with cAMP agonists produced conflicting results: dibutyryl cAMP and 8-(4-chloro-phenylthio) cAMP resulted in 50% and 38% reduction in invasion, respectively, whereas 8-bromo cAMP stimulated invasive potential by 30%. Forskolin and cholera toxin both significantly reduced invasiveness. Pre-treatment with 5-azacytidine and araC, to consider the role of methylation and proliferations decreased invasive ability. Anti-metastatic drugs such as gamma-interferon and razoxane inhibited invasive potential but to varying degrees. Treatment of cells with prostaglandins E2, F2 alpha, A2, and D2 were ineffectual; however, indomethacin mildly inhibits invasion (less than 30%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

Amino Acid Sequence↗
PubMed1989

Characterization of monoclonal antibodies against human prorenin profragment and identification of active prorenins in plasma.

Monoclonal antibodies were raised against a synthetic peptide (43 amino acid residues) that corresponds to the complete profragment of human prorenin. Seven monoclonal antibodies were chosen for further characterization. Two antibodies, 2-X-C1 and 4-X-E1, reacted with the middle region and C-terminus of the profragment and were isotyped IgG1. The affinity constants of these antibodies against the human profragment were 7.6 x 10(8) and 3.0 x 10(7) M-1, respectively. Immunoaffinity columns containing the antibodies 2-X-C1 and 4-X-E1, respectively, were used for the characterization of active prorenin in human plasma. This active prorenin strongly bound to the 4-X-E1 column and eluted as two separate peaks which corresponded to fully and partially active prorenin, respectively. The partially active prorenin had higher activity with a small substrate, tridecapeptide, than with a large one, angiotensinogen, although the fully active prorenin had the same renin activity irrespective of the size of the substrate. These data suggest that new forms of prorenin, active prorenin, exist in human plasma and that their active sites are completely or partially exposed to the substrates. Moreover, the active prorenin in plasma was found not only in human but also in all tested mammalians. Cross-reactivity among the profragments of mammalian plasma prorenins can be explained by conservation of the amino acid sequence (epitope) of the combining site.

Antibodies, Monoclonal↗
PubMed1988

On-resin biotinylation of chemically synthesized proteins for one-step purification.

A general, convenient, one-step purification procedure for chemically synthesized proteins present in low yields using on-resin biotinylation is reported. The protein, terminally deprotected and neutralized on-resin, is stirred in dimethylformamide and then biotinylated with N-hydroxysuccinimidobiotin (2 mg/mg protein on-resin) for 24 h at 45 degrees C. Following low/high hydrogen fluoride cleavage (J. P. Tam, W. F. Heath, and R. B. Merrifield (1983) J. Amer. Chem. Soc. 105, 6442-6455) the crude cleavage product was applied to an avidin agarose column. The column was washed with phosphate-buffered saline until all unbound materials had been eluted off. Then the biotinylated protein was eluted with 0.1 M glycine HCl, pH 2.0. A pilot experiment with two unrelated peptides on-resin established the experimental conditions for biotinylation. We then demonstrated that the chemically synthesized 153 residue [Asp205]-interleukin-1 beta (117-269), present in less than 1% yield in the crude HF cleavage mixture, could be purified to homogeneity in one step. In addition 70 and 114 residue synthetic fragments, (200-269) and (156-269), were also purified in this manner. Biotinylation on-resin appears to be an attractive method of purifying low yield chemically synthesized proteins and for preparing proteins with biotinyl moieties at specific locations such as the amino terminus.

Avidin↗
PubMed1988

Peptides as potential virus inhibitors. Synthesis and bioassay of five respiratory syncytial virus peptide analogs with antimeasles activity.

Five carbobenzoxylated and D-amino acid containing-peptide analogs of the respiratory syncytial virus (RSV) F1 glycoprotein amino terminus were chemically synthesized by solution and FMOC-solid phase peptide synthesis methods. Several of these peptides, ranging from 3 to 6 residues in length, raised the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. None of these peptides were specific inhibitors of RSV or herpes simplex virus infection. Two of the series, CBZ-D-Phe-L-Leu-Gly-D-Phe-D-Leu-D-Leu and CBZ-D-Phe-L-Leu-Gly-D-Phe-D-Leu-D-Leu-Gly, were active in reducing measles virus-induced cytopathic effect at 62 micrograms/mL. Others in the series showed some activity at higher doses or activity simultaneously with some cell toxicity. These results support the view that membrane-stabilizing agents may have non-specific effects on membranes which are responsible for their antimeasles activity.

Amino Acid Sequence↗
PubMed1987

Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide.

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH2 = Z-Gly-Phe-NH2 greater than Z-Ser-Leu-NH2 greater than Z-Gly-Leu-NH2 greater than Z-Gly-Gly-NH2. A linear correlation was found between the respective HPLC retention time parameter k' for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH2, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.

Animals↗
PubMed1984

Affinity labeling of the androgen receptor in rat prostate cytosol with 17 beta-[(bromoacetyl)oxy]-5 alpha-androstan-3-one.

An androgen affinity label, 17 beta-[(bromoacetyl)-oxy]-5 alpha-androstan-3-one, has been synthesized in both radioactive and nonradioactive forms. The affinity label (170 Ci/mmol) was characterized and found to have a high degree of purity. Affinity labeling of the androgen receptor from rat ventral prostate was androgen specific and appeared to be directed at the steroid binding site of the protein. Covalent binding was achieved at 0 degrees C; however, heat treatment at 23 degrees C for 30 min enhanced covalent binding by 31%. The covalently bound steroid was resistant to extraction with organic solvents and precipitation with trichloracetate. The Stokes radius (4.2 nm) and sedimentation coefficient (4.5 S) were identical with those found for receptor bound noncovalently to dihydrotestosterone. Gel electrophoresis of the affinity-labeled receptor under denaturing conditions revealed a molecular weight of 86000. The same molecular weight was observed for the receptor from rat seminal vesicle. This affinity label will be useful in future studies on the structure and function of androgen receptors.

Affinity Labels↗
PubMed1984

Molecular properties of the androgen receptor in rat ventral prostate.

Results from these studies demonstrate that we have purified a protein from rat prostate cytosol that is similar to the beta-protein (complex II) but different from the alpha-protein (complex I) reported by Liao et al. The purified receptor was different from androgen binding protein (ABP) in that ABP has a faster dissociation rate (6 min), a lower pI value (4.6), and requires higher concentrations of ammonium sulfate for precipitation (40-50%) than the prostatic androgen receptor. It is not likely that we have purified a serum sex-steroid binding protein since no such protein is found in rat serum. This report presents a rapid and efficient procedure for the purification of androgen receptor from rat ventral prostate. However, the present procedure only allowed us to obtain a limited quantity of purified receptor from each preparation. It is obvious that we need to scale up the purification of the receptor in order to study in detail its physicochemical properties and to produce monospecific antibodies against the protein. This work is in progress. In addition, we have demonstrated that two affinity labels can be used to bind covalently to the androgen receptor. Most importantly, these compounds can be used to characterize androgen receptors under both nondenaturing and denaturing conditions and represent useful tools for future work with androgen receptor proteins and androphilic proteins in general.

Affinity Labels↗
PubMed1983

Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts.

Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.

Binding, Competitive↗
PubMed1983

Contraceptive efficacy of testosterone-estradiol implants in male rhesus monkeys.

Rhesus monkeys (Macaca mulatta) were treated with testosterone (100 micrograms/kg/day) plus estradiol (0.5 micrograms/kg/day) via subcutaneous polydimethylsiloxane (PDS, Silastic) implants. This treatment caused a striking reversible sterility. No pregnancies were observed in females bred to the steroid-treated males. In contrast, there was no difference in pregnancy rate of females bred to control and steroid-treated monkeys for 14 weeks, beginning 17 weeks after removal of the steroid-filled implants.

Animals↗
PubMed1983

Medroxyprogesterone acetate has opposite effects on the androgen binding protein concentrations in serum and epididymis.

In the rat, the effects of progestin and androgen administration on serum, testicular and epididymal androgen binding protein (rABP) concentrations were determined and related to the organ weight and morphology. Adult rats were treated with medroxyprogesterone acetate (MPA; 17 alpha-acetoxy-6 alpha-methylprogesterone), testosterone propionate (TP) and mibolerone (MB; 7 alpha, 17 alpha-dimethyl-19-nortestosterone). MPA reduced testicular and epididymal weights and the concentrations of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. During MPA treatment testicular and epididymal ABP content declined in parallel with organ weights and hormone concentrations, whereas serum ABP concentrations increased. Combinations of MPA and TP reduced testicular and epididymal ABP, but the reductions were less than with MPA alone; this combined treatment also elevated serum AMP. Both MB and TP reduced ABP in the male reproductive tract, but unlike MPA did not increase the concentration of this protein in serum. The results suggest that MPA acts directly on Sertoli cells resulting in increased ABP release into the blood. The comparison was made of steady state polyacrylamide gel electrophoresis (SS-PAGE) and radioimmunoassay (RIA) methods of estimating rABP. The potency ratio of testicular ABP estimated by the two methods (RIA:SS-PAGE) was three times higher than this ratio in the epididymis in normal and hormonally treated animals. Due to differences in end points, these observations imply that these assays do not quantify the molecules in the same way in one or both of these tissues. The results indicate, however, that both assays are suitable for following rABP concentration in animals with altered hormonal states.

Androgen-Binding Protein↗