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Biomedical subjects

G J Todaro

Publications and source records attributed to G J Todaro.

At least 37 records · Page 2Linked to original sources
PubMed1986

Isolation and characterization of a putative endogenous benzodiazepineoid (endozepine) from bovine and human brain.

A protein termed endozepine (EP) which inhibits the binding of benzodiazepines to synaptosomal membranes (Ki approximately 5 microM) has been purified to electrophoretic homogeneity from bovine and human brain using acidic ethanol/chloroform extraction, Bio-Sil TSK-250 gel permeation chromatography, and reverse-phase high performance liquid chromatographies. Bovine and human EP are single-chain polypeptides and have molecular weights of approximately 10,000. Both proteins are very hydrophilic and contain an abundance of lysine, glutamic, and aspartic residues. Antisera prepared against bovine EP have been used to develop a sensitive radioimmunoassay for the detection of EP in tissue and body fluids. EP immunoreactivity is widely distributed in mammalian tissues, body fluids, and various cell lines. Substantial variation in the concentrations of EP is observed in different regions of the brain.

Animals↗
PubMed1986

Complete amino acid sequences of bovine and human endozepines. Homology with rat diazepam binding inhibitor.

The complete amino acid sequences of bovine and human brain endozepines have been determined. The amino-terminal serine of both endozepines is acylated. Assignment of the first 7 residues was achieved through Edman degradation after acid-induced rearrangement and subsequent acid hydrolysis of the amino-terminal blocking group. Cleavage of endozepine by chemical and enzymatic techniques established all the fragments in an unambiguous sequence. Bovine and human endozepines are single-chain polypeptides of 86 residues, with calculated molecular weights of 9913, displaying 93% homology. A comparison between the sequences of bovine and human endozepines with the partial sequences of the functionally related diazepam binding inhibitor from rat brain reveals significant sequence homology. The reported results suggest that bovine and human endozepines as well as rat diazepam binding inhibitor belong to a new family of polypeptides which presumably take part in the modulation of gamma-aminobutyric acid-ergic transmission.

Amino Acid Sequence↗
PubMed1986

Oncostatin M: a growth regulator produced by differentiated histiocytic lymphoma cells.

A polypeptide termed oncostatin M, which inhibits the replication of A375 melanoma and other human tumor cells, but not normal human fibroblasts, has been isolated from serum-free supernatants of U-937 histiocytic lymphoma cells that have been induced to differentiate into macrophage-like cells following treatment with the phorbol ester phorbol 12-myristate 13-acetate. No such growth inhibitory activity is detected in the supernatant of untreated U-937 cells, indicating that the protein is induced or increased in expression in the phorbol ester-induced differentiated cells. Oncostatin M is stable between pH 2 and 11 and after heating for 1 hr at 56 degrees C but is not stable at 90 degrees C. Purification of oncostatin M has been achieved by gel chromatography and reversed-phase HPLC, using sequentially acetonitrile and n-propanol in the presence of aqueous trifluoroacetic acid. The apparent molecular weight of oncostatin M is approximately 18,000, as determined by gel chromatography, and 28,000, as determined by polyacrylamide gel electrophoresis. The amino-terminal amino acid sequence of the purified polypeptide has been determined. No substantial sequence homology between oncostatin M and other proteins was found, including other tumor cell inhibitory proteins produced by mononuclear cells. Oncostatin M, therefore, appears to represent a distinct cell growth regulator.

Amino Acid Sequence↗
PubMed1985

A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties.

A fragment of rat transforming growth factor alpha (TGF alpha) comprising the third disulfide loop (residues 34-43) was selected as a potential antigenic and receptor binding region. Immunization of rabbits with a peptide conjugate resulted in antibodies which were specific for both the peptide and rat TGF alpha, but not for the homologous epidermal growth factor (EGF). The synthetic decapeptide exhibited low affinity for EGF receptors on human cells. Affinity was increased 100x to 0.2% of EGF or TGF alpha binding by blocking the peptide ends. The blocked decapeptide had no mitogenic activity but prevented the mitogenic effect of EGF and TGF alpha on fibroblasts. This decapeptide is an antagonist and contains an important receptor binding region of TGF alpha.

Amino Acid Sequence↗
PubMed1985

Stimulation of bone resorption in vitro by synthetic transforming growth factor-alpha.

Experiments were conducted to test the hypothesis that tumor-derived transforming growth factor-alpha (TGF-alpha) is responsible for the increased bone resorption and hypercalcemia seen in some malignant diseases. Homogeneous synthetic TGF-alpha prepared by the solid-phase synthesis method stimulated bone resorption directly in vitro in a concentration-dependent manner. Incubation times of 72 hours or more were required to stimulate resorption, which is similar to the time course of bone resorption by epidermal growth factor.

Animals↗
PubMed1985

Vaccinia virus-infected cells release a novel polypeptide functionally related to transforming and epidermal growth factors.

The recent discovery, that a vaccinia virus (VV) gene encodes a polypeptide with structural homology to transforming growth factor (TGF-alpha) and epidermal growth factor (EGF), led us to look for a virus-induced protein with the predicted biological activity. The supernatants of VV-infected cell cultures were found to contain an acid stable Mr 25,000 polypeptide that competes with EGF for binding to EGF membrane receptors. This VV-induced growth factor (VGF) like EGF and TGF-alpha is mitogenic and stimulates anchorage-independent cell growth in the presence of TGF-beta. However, VGF did not cross-react in a radioimmunoassay specific for small and large forms of TGF-alpha and exhibited minimal cross-reactivity with antisera to EGF. VGF was detectable in the culture medium within 2 hr, and maximal amounts were present 12 hr after infection. The level of VGF was proportional to the multiplicity of VV used. Inhibition of viral DNA synthesis enhanced VGF production, consistent with the hypothesis that VGF is an early gene product encoded by VV. The demonstration of a novel growth factor, released from cells infected with VV, may have important implications regarding the nature of virus-host interactions.

Animals↗
PubMed1985

Detection of larger polypeptides structurally and functionally related to type I transforming growth factor.

Peptides corresponding to various regions of type I rat transforming growth factor (rTGF) have been chemically synthesized, conjugated to carrier protein, and used to immunized rabbits. An antiserum raised against one of these peptides, corresponding to the carboxyl-terminal 17 amino acids of rTGF, has been used to develop a competitive RIA for the immunizing peptide. In this assay, the antiserum reacts only with a restricted region of the immunizing peptide corresponding to the 11 carboxyl-terminal amino acids of the rTGF molecule. Intact rTGF competes as well as the immunizing peptide on a molar basis, indicating that the cognate sequence in rTGF is recognized by this antiserum. Mouse epidermal growth factor (mEGF) was ineffective as a competitor, even at a 10,000-fold greater concentration, showing that the RIA was capable of distinguishing TGF from functionally related proteins. Several immunoreactive species were detected by gel filtration of conditioned medium from cultured retrovirus-transformed rat cells; both a low molecular weight species that corresponds to rTGF, and a higher molecular weight specie(s) copurify with biologic activity in the EGF radioreceptor assay. Immunoblotting analysis of the higher molecular weight peak fractions revealed three polypeptides (MrS, 24,000, 40,000, and 42,000) that reacted specifically with the antiserum. These findings suggest the existence of a family of larger polypeptides containing both structural and functional determinants of rTGF.

Animals↗
PubMed1985

Developmental expression of rat transforming growth factor-alpha mRNA.

Expression of the gene encoding transforming growth factor-alpha (TGF alpha) was examined in developing rat embryos by using a cloned TGF alpha cDNA as a hybridization probe. Northern blot analysis of RNA isolated from whole fetuses revealed that TGF alpha mRNA was present at relatively high levels in 8- through 10-day-old embryos and then declined to the low or undetectable level, which is characteristic of adult tissues before birth. The level of TGF alpha mRNA present during early gestation was similar to that present in retrovirus-transformed cells in culture, suggesting that TGF alpha expression is not highly localized in the embryo. These observations are consistent with the hypothesis that TGF alpha plays a role in development, possibly as a fetal growth factor.

Animals↗
PubMed1985

Two distinct tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line.

Tumor cell growth-inhibiting factors (TIFs) have been shown to inhibit the growth of tumor cell lines in culture. TIF-1, the first TIF to be described, is a low-molecular-weight, acid- and heat-stable polypeptide with no antiviral activity. A second class of TIFs (TIF-2) has now been isolated from the conditioned media of a human rhabdomyosarcoma cell line and partially purified by polyacrylamide gel filtration, cation exchange, and reverse-phase high-pressure liquid chromatography. Partially purified preparations of TIF-2 inhibit the growth of a variety of human tumor cells in soft agar and monolayer cultures and are mitogenic for normal human and mouse cells. TIF-2 has no antiviral activity. The growth-inhibitory effects of TIF-2 are reversible when the affected cells are no longer exposed to the factor. Although both TIF-1 and TIF-2 are obtained from the same source, they can be distinguished by their molecular weight, heat lability, elution pattern from reverse-phase high-pressure liquid chromatography, and their effect on the growth of mink lung epithelial cells. The growth of a human tumor cell variant, selected for resistance to growth inhibition by TIF-1, is inhibited by TIF-2. TIFs may therefore be a family of related polypeptides which selectively inhibit the growth of tumor cells.

Cell Line↗
PubMed1985

Isolation of tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line.

Two types of growth-modulating factors were derived from the serum-free conditioned media of a human rhabdomyosarcoma cell line, A673. One type, Mr 18,000 to 22,000, competes for binding to epidermal growth factor receptors and enhances the growth of normal and tumor cells in soft agar. It has all of the biological properties ascribed to transforming growth factor type alpha (TGF alpha). A673 cells also produce factors which inhibit the growth of human tumor cells in soft agar and in monolayer cultures. These tumor cell growth-inhibiting factors (TIFs) are acid- and heat-stable peptides. The major TIF activities have molecular weights in the ranges of greater than 28,000, 18,000 to 22,000, 10,000 to 16,000, and 5,000 to 10,000 and do not possess the antiviral activity associated with interferon. Partially purified preparations of TIF-1 (Mr 10,000 to 16,000) inhibit the growth of all human tumor cell lines tested and stimulate the growth of normal human fibroblasts and epithelial cells in monolayer cultures. The growth of human lung carcinoma A549 cells in soft agar, which was enhanced by treatment with TGF alpha from A673-conditioned media, was inhibited by treatment with TIF-1 derived from the same media. The ratio of the two types of tumor cell-derived, growth-modulating factors (TIFs and TGF alpha), which are antagonistic in their biological effects, may determine the capacity of tumor cells for anchorage-independent growth.

Cell Line↗
PubMed1985

Comparison of growth factors functionally related to epidermal growth factor in the urine of normal and human tumor-bearing athymic mice.

Urine from nude mice contains epidermal growth factor (EGF) and a minor acid-stable component with an apparent molecular weight of 20,000, which competes with EGF for binding to EGF membrane receptors and which promotes colony formation by normal rat kidney cells in soft agar. The levels of this Mr 20,000 urine-derived growth factor are increased approximately 4- to 10-fold in nude mice bearing tumors following s.c. injection of cultured human tumor cells. Following removal of the primary tumor, the concentration of this factor is reduced to basal levels, and thus, elevated levels of this growth factor appear to be dependent on tumor burden. The Mr 20,000 urinary component is separable into four EGF competing activities by high-performance liquid chromatography; the major species is immunologically related to mouse submaxillary gland EGF and therefore appears to be of host origin. However, in addition to elevated levels of host growth factor, urine from tumor-bearing mice also contains transforming growth factor activity in amounts comparable to that released by the tumor cells in culture. The tumor-derived urinary transforming growth factor activity is immunologically unrelated to EGF but is immunoreactive with an antiserum to transforming growth factor-alpha. We propose that the nude mouse may be a useful model to examine the role of both host- and tumor-derived growth factors in tumorigenesis and the usefulness of these factors as biological markers of response to therapy and tumor progression.

Animals↗
PubMed1984

Rat transforming growth factor type 1: structure and relation to epidermal growth factor.

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.

Amino Acid Sequence↗
PubMed1984

Neuroblastoma cells produce transforming growth factors during exponential growth in a defined hormone-free medium.

Mouse neuroblastoma Neuro-2A cells have been cultured in a chemically defined serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 30 nM selenite and 10 micrograms of transferrin per ml. In this medium, which does not contain any externally added polypeptide growth factor, cells proliferate rapidly with a doubling time of approximately equal to 10 hr. During exponential growth in this serum-free medium, Neuro-2A cells secrete a 15- to 20-kDa transforming growth factor with strong mitogenic action and the ability to induce anchorage-independent growth on nontransformed cells. This neuroblastoma-derived transforming growth factor (ND-TGF) is acid and heat stable but is sensitive to treatment with trypsin or dithiothreitol. However, it does not compete with epidermal growth factor (EGF) for receptor binding and does not require EGF receptors for its mitogenic activity. Experiments on the effects of EGF on ND-TGF-induced soft agar growth of normal rat kidney cells indicate that Neuro-2A cells secrete an EGF-potentiated TGF in addition to ND-TGF. It is suggested that Neuro-2A cells can proliferate in the absence of externally added growth factors as a result of autocrine production of polypeptide growth factors.

Animals↗
PubMed1983

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus.

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Synder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (less than 10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.

Animals↗
PubMed1983

Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor.

Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.

Amino Acid Sequence↗
PubMed1983

High-molecular-weight transforming growth factor activity in the urine of patients with disseminated cancer.

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.

Binding, Competitive↗
PubMed1982

Transforming growth factor and epidermal growth factor stimulate the phosphorylation of a synthetic, tyrosine-containing peptide in a similar manner.

A partially purified preparation of a transforming growth factor (TGF) obtained from serum-free growth medium conditioned by a human melanoma tumor line was found to stimulate the phosphorylation of a synthetic tyrosine-containing peptide. The sequence of the peptide is related to that of the known site of tyrosine phosphorylation in the Rous sarcoma virus-encoded transforming protein, pp60src. In A431 membranes, the characteristics of TGF- and epidermal growth factor (EGF)-stimulated peptide phosphorylation are nearly identical. The effects of the two growth factors are not additive, suggesting that TGF and EGF stimulate peptide phosphorylation through the same EGF receptor system. This conclusion is supported by the finding that both TGF and EGF stimulate peptide phosphorylation in wild type Swiss 3T3 cell membranes, but neither factor is effective in stimulating peptide phosphorylation in membranes prepared from EGF receptor-deficient NR6 3T3 cells.

Animals↗