Jaroslava Halper - Academia.edu (original) (raw)
Papers by Jaroslava Halper
Analytical Biochemistry, Jul 15, 2002
The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hamp... more The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hampered by the lack of sensitive methods. In this paper we describe the modification and development of three sensitive assays capable of detecting nanogram quantities of GAGs in biological samples. The first assay detects total GAGs. It is a modified Alcian blue dye precipitation assay in which the dye binds to the negatively charged GAGs in CsCl-fractionated extracts from chicken tendons. This assay compares favorably with the widely used uronic acid assay in terms of its sensitivity and ability to detect all classes of GAGs, including keratan sulfate (KS). Two other assays, dot-blotting and immunoblotting, detect KS in complex mixtures and can be easily adapted for the detection of other GAGs. Both take advantage of binding of carboxyl and sulfate groups of GAGs to trivalent neodymium. In dot-blotting, samples were directly blotted onto nitrocellulose membrane soaked in Nd 2 (SO 4 ) 3 buffer, and KS was detected with the monoclonal anti-KS 5-D-4 antibody and an avidin-biotin complex detection system. In immunoblotting, the samples were first separated in 28% polyacrylamide gels, transferred onto a Nd 2 (SO 4 ) 3 -soaked nitrocellulose membrane using a phosphate buffer system, and stained and developed using the same protocol as in dot-blotting. Whereas dot-blotting allows the use of very low quantities of samples because of its high sensitivity (lower detection limit was 5 ng), immunoblotting provides more specificity.
Cancer Research, May 1, 1983
SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small c... more SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small colonies when sus pended in soft agar at low cell densities. The number and size of colonies increased dramatically following stimulation with serum-free medium conditioned by SW-13 cells, indicating the possibility of autostimulation in these malignant cells. Evidence is presented suggesting that SW-13 cells form progressively growing soft agar colonies upon stimulation by epithelial tissuederived growth factor-like polypeptides. Both acid-ethanol ex tracts and conditioned media from three human carcinoma cell lines (A431, D562, and A549) caused similar increases in colony number and size of SW-13 cells. Extracts from 26 of 32 freshly excised human carcinomas and five freshly excised nonneoplastic human kidneys and one human lung stimulated soft agar growth of SW-13 cells as well. None of the nine extracts from nonepithelial human solid malignant tumors stimulated SW-13 cells. However, a benign nonepithelial tumor (uterine leiomyoma) caused a low level of soft agar growth of SW-13 cells. Cell extract from A204 human sarcoma cells and both conditioned medium and acid-ethanol cell extract from A375 human mela noma cells lacked SW-13 activity, whereas medium conditioned by A204 cells stimulated soft agar growth of SW-13 cells.
Archives of Biochemistry and Biophysics, Apr 15, 2003
Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown ... more Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown that proteoglycans (PGs) regulate the organization of collagen fibrils, the main structural components of tendons. We hypothesized that moderate exercise alters PG synthesis in the avian gastrocnemius tendon. To test our hypothesis we compared the PG content in gastrocnemius tendons from control 6.5-week-old chickens with that in tendons from 6.5-week-old chickens that underwent exercise. Our results show high levels and a wide variety of glycosaminoglycans (GAGs) in 6.5-week-old tendons. Chondroitin-4-sulfate disaccharide was the major GAG disaccharide in control and exercised 6.5-week-old gastrocnemius tendons. Exercise led to an increase in the size of the tendons, the content of hyaluronic acid, and the level of decorin. High levels of keratan sulfate (KS) were found in the lower halves of gastrocnemius tendons, although the amount of KS decreased with exercise. This corresponded well with lower content of aggrecan in the lower halves of exercised tendons. In conclusion, our data support the hypothesis that exercise alters the content of PGs in chicken tendons.
Archives of Biochemistry and Biophysics, Sep 15, 2010
Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the ... more Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the galactosyl transferase I gene is the underlying defect of a progeroid form of Ehlers-Danlos syndrome. We have previously described pathological changes in equine systemic proteoglycan accumulation (ESPA, formerly degenerative suspensory ligament desmitis) as consisting of excessive presence of decorin and other proteoglycans in organs and structures with a high content of connective tissue. Using liquid chromatography/mass spectrometry, and one-and two-dimensional immunoblotting we have determined that decorin from ESPA-tendons had a higher molecular weight than decorin from non-affected control tendons. Glycosaminoglycan structure and monosaccharide composition were determined with HPLC analysis of chondroitinase ABC-digested glycosaminoglycans and gas chromatography/mass spectrometry. This analysis revealed an increase in the total content of sulfated disaccharides, particularly due to enhanced sulfation at 6-position of N-acetyl galactosamine (GalNAc) with a subsequent decrease in the ratio of 4-sulfation to 6-sulfation disaccharides in the ESPA decorin. The ESPA-affected decorin also exhibited altered biological activity resulting in (1) diminished binding of TGFb1 (and of anti-decorin antibody) to ESPA decorin, and (2) increased expression of TGFb1 in ESPA tissues.
Biochemical and Biophysical Research Communications, Apr 1, 2003
Transforming growth factor beta 4 (TGF-beta 4) is unique to avian species, though its roles in vi... more Transforming growth factor beta 4 (TGF-beta 4) is unique to avian species, though its roles in vivo have not yet been well established. In this paper we describe the expression and partial characterization of recombinant chicken TGF-beta 4. By using a GC-rich PCR system in a modified 5'RACE methodology we generated the 5'-end of cDNA sequence encoding the TGF-beta 4 precursor, which was in-frame cloned into pcDNA3.1/V5-His-TOPO and transfected into the Chinese hamster ovary cell line (CHO-K1). A cell line stably expressing TGF-beta 4 precursor protein was established from CHO-K1 cells. Acid-activated mature TGF-beta 4 inhibited the growth of mink lung epithelial (Mv1Lu) cell line. TGF-beta 4 also stimulated the expression of type I procollagen and enhanced heat shock protein 47 (Hsp47) expression in chicken tendon fibroblasts. Hsp47 expression by TGF beta 4 is likely regulated through activation of heat shock transcription factor 1 (HSF1). Because the presence of TGF-beta 1 has not been documented in avian cells and our data show that TGF-beta 4 elicits biological activities in chicken tendon cells, which closely parallel that of TGF-beta 1, we propose that TGF-beta 4 plays roles in avian species similar to what TGF-beta 1 plays in mammalian species.
Biochem Biophys Res Commun, 2003
Transforming growth factor β4 (TGF-β4) is unique to avian species, though its roles in vivo have ... more Transforming growth factor β4 (TGF-β4) is unique to avian species, though its roles in vivo have not yet been well established. In this paper we describe the expression and partial characterization of recombinant chicken TGF-β4. By using a GC-rich PCR system in a modified 5′RACE methodology we generated the 5′-end of cDNA sequence encoding the TGF-β4 precursor, which was in-frame cloned into pcDNA3.1/V5-His-TOPO and transfected into the Chinese hamster ovary cell line (CHO-K1). A cell line stably expressing TGF-β4 precursor protein was established from CHO-K1 cells. Acid-activated mature TGF-β4 inhibited the growth of mink lung epithelial (Mv1Lu) cell line. TGF-β4 also stimulated the expression of type I procollagen and enhanced heat shock protein 47 (Hsp47) expression in chicken tendon fibroblasts. Hsp47 expression by TGFβ4 is likely regulated through activation of heat shock transcription factor 1 (HSF1). Because the presence of TGF-β1 has not been documented in avian cells and our data show that TGF-β4 elicits biological activities in chicken tendon cells, which closely parallel that of TGF-β1, we propose that TGF-β4 plays roles in avian species similar to what TGF-β1 plays in mammalian species.
Cancer Research
Surgically removed solid human benign and malignant neo plasms and nonneoplastic tissues were exa... more Surgically removed solid human benign and malignant neo plasms and nonneoplastic tissues were examined for the pres ence of transforming growth factors (TGFs). TGFs are polypeptide growth factor-like substances which cause the appearance of a reversible neoplastic phenotype in nontransformed, anchor age-dependent cells in culture, including the induction of the ability to grow while suspended in semisolid medium. Acidethanol extracts from adenocarcinomas of the breast, colon, kidney, and ovary; fibrosarcoma and leiomyosarcoma; Hodgkin's lymphoma; fibroadenoma of the breast; uterine leiomyoma; and nonneoplastic kidney and lung were found to cause growth in soft agar of both nontransformed mouse AKR-2B and rat NRK cells. This colony-stimulating activity, where tested, was heat and acid stable but was destroyed by trypsin and dithiothreitol treatment, indicating that the activity is due to a polypeptide with disulfide bonds. Extracts from several of the tumors provided sufficient material for purification by molecular sieve chromatography. Peaks of colony-stimulating activity from a Bio-Gel P-60 column eluted with 1 M acetic acid were detected in the M, 3,000 to 25,000 range with the apparent molecular weight varying depending on the type of tumor being studied and the indicator cells used. The data suggest that at least three TGFs are present in human-tumors. Evidence is presented differentiating these TGFs into TGFa, which has selective activity for stimulating AKR-2B cells, and TGFn, which has selective activity for stimulating NRK cells. The TGFn activity was further subdivided into a TGFns fraction and TGFnl fraction, denoting small (less than 6,000) and large (12,000 to 20,000) apparent molecular weights, respec tively. The TGFa and TGFnl activities were present in malignant and nonneoplastic (kidney and lung) tissue, whereas the TGFns activity predominated in benign neoplasms. These TGFs exhib ited no competition with epidermal growth factor for binding to the epidermal growth factor receptor, and the TGFnl activity was potentiated by epidermal growth factor.
Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed... more Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angio- genic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-a (TNF-a), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized
Archives of pathology & laboratory medicine
Three patients with classic Takayasu's arteritis affecting the aorta and its major branch... more Three patients with classic Takayasu's arteritis affecting the aorta and its major branches were found to have a unique small pulmonary arteriopathy characterized by deficiency of the outer media, with capillary ingrowth both here and in the thickened fibrosed intima. In one patient there was also evidence of a mononuclear cellular arteritis of the affected arteries and widespread focal angiomatoid like lesions. Another patient had similar, but less well-defined dilatation lesions. This patient's major coronary arteries showed an active arteritis with ingrowth of reparative granulation tissue capillaries into both media and intima, yielding an appearance not dissimilar to that seen in the small pulmonary arteries. The outer pulmonary artery medial defects observed in our patients bear some similarity to experimental acute immune-complex arteritis and may thus provide tenuous evidence for an autoimmune basis for Takayasu's disease.
Journal of Neuropathology and Experimental Neurology
ABSTRACT
Cancer Research
SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small c... more SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small colonies when sus pended in soft agar at low cell densities. The number and size of colonies increased dramatically following stimulation with serum-free medium conditioned by SW-13 cells, indicating the possibility of autostimulation in these malignant cells. Evidence is presented suggesting that SW-13 cells form progressively growing soft agar colonies upon stimulation by epithelial tissuederived growth factor-like polypeptides. Both acid-ethanol ex tracts and conditioned media from three human carcinoma cell lines (A431, D562, and A549) caused similar increases in colony number and size of SW-13 cells. Extracts from 26 of 32 freshly excised human carcinomas and five freshly excised nonneoplastic human kidneys and one human lung stimulated soft agar growth of SW-13 cells as well. None of the nine extracts from nonepithelial human solid malignant tumors stimulated SW-13 cells. However, a benign nonepithelial tumor (uterine leiomyoma) caused a low level of soft agar growth of SW-13 cells. Cell extract from A204 human sarcoma cells and both conditioned medium and acid-ethanol cell extract from A375 human mela noma cells lacked SW-13 activity, whereas medium conditioned by A204 cells stimulated soft agar growth of SW-13 cells.
Cancer Research
Previous studies have indicated that an autostimulatory transforming growth factor was required f... more Previous studies have indicated that an autostimulatory transforming growth factor was required for the optimal growth of SW-13 adrenal carcinoma cells in soft agar. The production of SW-13 colony-stimulating activity by other human malignant cell lines of both epithelial and mesenchymal origin has been demonstrated. Evidence was presented indicating that the stimulating activity detected in crude acid-ethanol extracts was an acid-and heat-stable polypeptide requiring disulfide bonds for full activity. This activity was detected more frequently in tumors and human cancer cells in culture of epithelial origin than of mesenchymal origin and in a variety of nonneoplastic tissues. In the present study, this activity, termed epithelial transforming growth factor (TGFe) because of its ability to stimulate soft agar growth of certain epithelial cells, was partially purified from bovine kidney. Fourfold purification of the kidney acid-ethanol extract with 50% maximal growthstimulatory activity of 10 «ig was achieved using molecular sieve chromatography where TGFe eluted with an apparent molecular weight of 20,000-25,000. The next purification step, molecular sieve high perform ance liquid chromatography, yielded a 50% maximal growth-stimulatory activity of 50 ng and an 800-fold purification from the initial acid-ethanol extract. TGFe eluted in the M, 11,000 range. Reversed phase high performance liquid chromatography with a Ci« column was then used, yielding a single or double peak of SW-13 colony-stimulating activity at 30-35% acetonitrile. The degree of purification was 11,000-fold with a 50% maximal growth-stimulatory activity of 3.5 ng. Analysis of the peak on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major and sometimes single band with a molecular weight of 23,000-25,000. Extraction of protein from the polyacrylamide gel dem onstrated that only the M, 23,000-25,000 band stimulated soft agar growth of SW-13 cells. The biological activity of the partially purified TGFe was found to differ from other known growth factors with regard to its ability to stimulate soft agar growth of SW-13 cells with the exception of basic fibroblast growth factor (FGF). The acid lability of FGF, the different molecular weights of these two growth factors, the lack of stimulation of soft agar growth of A431 cells, and the lack of binding of TGFe to FGF receptors indicated that TGFe was not related to basic FGF. Partially purified TGFe was also found to stimulate soft agar growth of two squamous cell carcinoma lines, A431 and 1)562. and the mouse embryo-derived AKR-2B cells. These data suggest that TGFe is a unique factor distinct from those previously described and it is speculated that TGFe may play a role in normal and neoplastic growth of epithelial cells.
BioTechniques
The purification and recovery of biologically active epithelial-type transforming growth factor (... more The purification and recovery of biologically active epithelial-type transforming growth factor (TGFe) is described. In the final phase of purification, micropreparative electrophoretic chromatography was employed using a Tris-glycine-sodium dodecyl sulfate buffer system in an automated instrument. Briefly, partially purified protein preparations were separated in 2.5 x 50 mm, 10% polyacrylamide gel in electrophoresis tubes installed in the apparatus, electrophoresed under constant current of 1.5 mA for 400 min and recovered by automated fractionation and collection of the eluant from the tube gel. Aliquots of the eluted fractions were assayed in a biological system using SW-13 cell growth stimulation as an indicator of the presence of biologically active TGFe. Using the above procedure, TGFe was purified to within 95% homogeneity as assessed by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Laboratory Investigation
Recent reports indicate that fibroblast growth factors known to be present in the pituitary in hi... more Recent reports indicate that fibroblast growth factors known to be present in the pituitary in high levels regulate the action of growth hormone and prolactin. New data also suggest a regulatory role in the pituitary for other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Since in most systems cooperation of several growth factors is required for their optimal function, we sought to demonstrate the presence of certain growth factors in the pituitary. Acid ethanol extracts from approximately 50 autopsy-derived human pituitaries were subjected to molecular sieve chromatography and were tested for growth factors. Low molecular weight protein (10 micrograms) eluted from the molecular sieve column contained 10-20 ng material binding to the EGF/TGF-alpha receptor as determined by the EGF/TGF alpha radioreceptor binding assay which represents 11 ng EGF/TGF alpha per pituitary. By Western blotting we found EGF but could not document the presence of TGF-alpha in this material. Radioimmunoassay for insulin-like growth factor I detected 0.4-0.8 ng insulin-like growth factor-I/100 micrograms extract. TGF-beta eluted between 14,000 and 20,000 M(r) at levels of 3-4 ng/pituitary. Its ability to inhibit growth of CC164 mink lung cells was abolished by antibody to TGF-beta 1 but not by antibody raised against TGF-beta 2. The detection of platelet derived growth factor was equivocal and not fully reproducible. We have partially purified TGFe from the pituitary; it stimulated soft agar growth of carcinoma SW-13 cells, and it followed an elution pattern identical to bovine kidney TGFe on molecular sieve column and high pressure liquid and high performance electrophoretic chromatography. Our data show that in addition to fibroblast growth factors, the human pituitary contains other growth factors, such as EGF/TGF-alpha, TGF-beta, insulin-like growth factor I, and TGFe.
Experimental Biology and Medicine
Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, incl... more Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity. We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis. Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor. Some inflammatory cells were scattered among the blood vessels. The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithelialization. Subcutaneous injection of Matrigel impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel. LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells. LS also induced production of TNF-␣ by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells. LS did not appear to have an effect on collagen production. In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-␣ production, and of angiogenesis. Exp Biol
Analytical Biochemistry, Jul 15, 2002
The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hamp... more The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hampered by the lack of sensitive methods. In this paper we describe the modification and development of three sensitive assays capable of detecting nanogram quantities of GAGs in biological samples. The first assay detects total GAGs. It is a modified Alcian blue dye precipitation assay in which the dye binds to the negatively charged GAGs in CsCl-fractionated extracts from chicken tendons. This assay compares favorably with the widely used uronic acid assay in terms of its sensitivity and ability to detect all classes of GAGs, including keratan sulfate (KS). Two other assays, dot-blotting and immunoblotting, detect KS in complex mixtures and can be easily adapted for the detection of other GAGs. Both take advantage of binding of carboxyl and sulfate groups of GAGs to trivalent neodymium. In dot-blotting, samples were directly blotted onto nitrocellulose membrane soaked in Nd 2 (SO 4 ) 3 buffer, and KS was detected with the monoclonal anti-KS 5-D-4 antibody and an avidin-biotin complex detection system. In immunoblotting, the samples were first separated in 28% polyacrylamide gels, transferred onto a Nd 2 (SO 4 ) 3 -soaked nitrocellulose membrane using a phosphate buffer system, and stained and developed using the same protocol as in dot-blotting. Whereas dot-blotting allows the use of very low quantities of samples because of its high sensitivity (lower detection limit was 5 ng), immunoblotting provides more specificity.
Cancer Research, May 1, 1983
SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small c... more SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small colonies when sus pended in soft agar at low cell densities. The number and size of colonies increased dramatically following stimulation with serum-free medium conditioned by SW-13 cells, indicating the possibility of autostimulation in these malignant cells. Evidence is presented suggesting that SW-13 cells form progressively growing soft agar colonies upon stimulation by epithelial tissuederived growth factor-like polypeptides. Both acid-ethanol ex tracts and conditioned media from three human carcinoma cell lines (A431, D562, and A549) caused similar increases in colony number and size of SW-13 cells. Extracts from 26 of 32 freshly excised human carcinomas and five freshly excised nonneoplastic human kidneys and one human lung stimulated soft agar growth of SW-13 cells as well. None of the nine extracts from nonepithelial human solid malignant tumors stimulated SW-13 cells. However, a benign nonepithelial tumor (uterine leiomyoma) caused a low level of soft agar growth of SW-13 cells. Cell extract from A204 human sarcoma cells and both conditioned medium and acid-ethanol cell extract from A375 human mela noma cells lacked SW-13 activity, whereas medium conditioned by A204 cells stimulated soft agar growth of SW-13 cells.
Archives of Biochemistry and Biophysics, Apr 15, 2003
Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown ... more Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown that proteoglycans (PGs) regulate the organization of collagen fibrils, the main structural components of tendons. We hypothesized that moderate exercise alters PG synthesis in the avian gastrocnemius tendon. To test our hypothesis we compared the PG content in gastrocnemius tendons from control 6.5-week-old chickens with that in tendons from 6.5-week-old chickens that underwent exercise. Our results show high levels and a wide variety of glycosaminoglycans (GAGs) in 6.5-week-old tendons. Chondroitin-4-sulfate disaccharide was the major GAG disaccharide in control and exercised 6.5-week-old gastrocnemius tendons. Exercise led to an increase in the size of the tendons, the content of hyaluronic acid, and the level of decorin. High levels of keratan sulfate (KS) were found in the lower halves of gastrocnemius tendons, although the amount of KS decreased with exercise. This corresponded well with lower content of aggrecan in the lower halves of exercised tendons. In conclusion, our data support the hypothesis that exercise alters the content of PGs in chicken tendons.
Archives of Biochemistry and Biophysics, Sep 15, 2010
Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the ... more Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the galactosyl transferase I gene is the underlying defect of a progeroid form of Ehlers-Danlos syndrome. We have previously described pathological changes in equine systemic proteoglycan accumulation (ESPA, formerly degenerative suspensory ligament desmitis) as consisting of excessive presence of decorin and other proteoglycans in organs and structures with a high content of connective tissue. Using liquid chromatography/mass spectrometry, and one-and two-dimensional immunoblotting we have determined that decorin from ESPA-tendons had a higher molecular weight than decorin from non-affected control tendons. Glycosaminoglycan structure and monosaccharide composition were determined with HPLC analysis of chondroitinase ABC-digested glycosaminoglycans and gas chromatography/mass spectrometry. This analysis revealed an increase in the total content of sulfated disaccharides, particularly due to enhanced sulfation at 6-position of N-acetyl galactosamine (GalNAc) with a subsequent decrease in the ratio of 4-sulfation to 6-sulfation disaccharides in the ESPA decorin. The ESPA-affected decorin also exhibited altered biological activity resulting in (1) diminished binding of TGFb1 (and of anti-decorin antibody) to ESPA decorin, and (2) increased expression of TGFb1 in ESPA tissues.
Biochemical and Biophysical Research Communications, Apr 1, 2003
Transforming growth factor beta 4 (TGF-beta 4) is unique to avian species, though its roles in vi... more Transforming growth factor beta 4 (TGF-beta 4) is unique to avian species, though its roles in vivo have not yet been well established. In this paper we describe the expression and partial characterization of recombinant chicken TGF-beta 4. By using a GC-rich PCR system in a modified 5'RACE methodology we generated the 5'-end of cDNA sequence encoding the TGF-beta 4 precursor, which was in-frame cloned into pcDNA3.1/V5-His-TOPO and transfected into the Chinese hamster ovary cell line (CHO-K1). A cell line stably expressing TGF-beta 4 precursor protein was established from CHO-K1 cells. Acid-activated mature TGF-beta 4 inhibited the growth of mink lung epithelial (Mv1Lu) cell line. TGF-beta 4 also stimulated the expression of type I procollagen and enhanced heat shock protein 47 (Hsp47) expression in chicken tendon fibroblasts. Hsp47 expression by TGF beta 4 is likely regulated through activation of heat shock transcription factor 1 (HSF1). Because the presence of TGF-beta 1 has not been documented in avian cells and our data show that TGF-beta 4 elicits biological activities in chicken tendon cells, which closely parallel that of TGF-beta 1, we propose that TGF-beta 4 plays roles in avian species similar to what TGF-beta 1 plays in mammalian species.
Biochem Biophys Res Commun, 2003
Transforming growth factor β4 (TGF-β4) is unique to avian species, though its roles in vivo have ... more Transforming growth factor β4 (TGF-β4) is unique to avian species, though its roles in vivo have not yet been well established. In this paper we describe the expression and partial characterization of recombinant chicken TGF-β4. By using a GC-rich PCR system in a modified 5′RACE methodology we generated the 5′-end of cDNA sequence encoding the TGF-β4 precursor, which was in-frame cloned into pcDNA3.1/V5-His-TOPO and transfected into the Chinese hamster ovary cell line (CHO-K1). A cell line stably expressing TGF-β4 precursor protein was established from CHO-K1 cells. Acid-activated mature TGF-β4 inhibited the growth of mink lung epithelial (Mv1Lu) cell line. TGF-β4 also stimulated the expression of type I procollagen and enhanced heat shock protein 47 (Hsp47) expression in chicken tendon fibroblasts. Hsp47 expression by TGFβ4 is likely regulated through activation of heat shock transcription factor 1 (HSF1). Because the presence of TGF-β1 has not been documented in avian cells and our data show that TGF-β4 elicits biological activities in chicken tendon cells, which closely parallel that of TGF-β1, we propose that TGF-β4 plays roles in avian species similar to what TGF-β1 plays in mammalian species.
Cancer Research
Surgically removed solid human benign and malignant neo plasms and nonneoplastic tissues were exa... more Surgically removed solid human benign and malignant neo plasms and nonneoplastic tissues were examined for the pres ence of transforming growth factors (TGFs). TGFs are polypeptide growth factor-like substances which cause the appearance of a reversible neoplastic phenotype in nontransformed, anchor age-dependent cells in culture, including the induction of the ability to grow while suspended in semisolid medium. Acidethanol extracts from adenocarcinomas of the breast, colon, kidney, and ovary; fibrosarcoma and leiomyosarcoma; Hodgkin's lymphoma; fibroadenoma of the breast; uterine leiomyoma; and nonneoplastic kidney and lung were found to cause growth in soft agar of both nontransformed mouse AKR-2B and rat NRK cells. This colony-stimulating activity, where tested, was heat and acid stable but was destroyed by trypsin and dithiothreitol treatment, indicating that the activity is due to a polypeptide with disulfide bonds. Extracts from several of the tumors provided sufficient material for purification by molecular sieve chromatography. Peaks of colony-stimulating activity from a Bio-Gel P-60 column eluted with 1 M acetic acid were detected in the M, 3,000 to 25,000 range with the apparent molecular weight varying depending on the type of tumor being studied and the indicator cells used. The data suggest that at least three TGFs are present in human-tumors. Evidence is presented differentiating these TGFs into TGFa, which has selective activity for stimulating AKR-2B cells, and TGFn, which has selective activity for stimulating NRK cells. The TGFn activity was further subdivided into a TGFns fraction and TGFnl fraction, denoting small (less than 6,000) and large (12,000 to 20,000) apparent molecular weights, respec tively. The TGFa and TGFnl activities were present in malignant and nonneoplastic (kidney and lung) tissue, whereas the TGFns activity predominated in benign neoplasms. These TGFs exhib ited no competition with epidermal growth factor for binding to the epidermal growth factor receptor, and the TGFnl activity was potentiated by epidermal growth factor.
Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed... more Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angio- genic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-a (TNF-a), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized
Archives of pathology & laboratory medicine
Three patients with classic Takayasu's arteritis affecting the aorta and its major branch... more Three patients with classic Takayasu's arteritis affecting the aorta and its major branches were found to have a unique small pulmonary arteriopathy characterized by deficiency of the outer media, with capillary ingrowth both here and in the thickened fibrosed intima. In one patient there was also evidence of a mononuclear cellular arteritis of the affected arteries and widespread focal angiomatoid like lesions. Another patient had similar, but less well-defined dilatation lesions. This patient's major coronary arteries showed an active arteritis with ingrowth of reparative granulation tissue capillaries into both media and intima, yielding an appearance not dissimilar to that seen in the small pulmonary arteries. The outer pulmonary artery medial defects observed in our patients bear some similarity to experimental acute immune-complex arteritis and may thus provide tenuous evidence for an autoimmune basis for Takayasu's disease.
Journal of Neuropathology and Experimental Neurology
ABSTRACT
Cancer Research
SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small c... more SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small colonies when sus pended in soft agar at low cell densities. The number and size of colonies increased dramatically following stimulation with serum-free medium conditioned by SW-13 cells, indicating the possibility of autostimulation in these malignant cells. Evidence is presented suggesting that SW-13 cells form progressively growing soft agar colonies upon stimulation by epithelial tissuederived growth factor-like polypeptides. Both acid-ethanol ex tracts and conditioned media from three human carcinoma cell lines (A431, D562, and A549) caused similar increases in colony number and size of SW-13 cells. Extracts from 26 of 32 freshly excised human carcinomas and five freshly excised nonneoplastic human kidneys and one human lung stimulated soft agar growth of SW-13 cells as well. None of the nine extracts from nonepithelial human solid malignant tumors stimulated SW-13 cells. However, a benign nonepithelial tumor (uterine leiomyoma) caused a low level of soft agar growth of SW-13 cells. Cell extract from A204 human sarcoma cells and both conditioned medium and acid-ethanol cell extract from A375 human mela noma cells lacked SW-13 activity, whereas medium conditioned by A204 cells stimulated soft agar growth of SW-13 cells.
Cancer Research
Previous studies have indicated that an autostimulatory transforming growth factor was required f... more Previous studies have indicated that an autostimulatory transforming growth factor was required for the optimal growth of SW-13 adrenal carcinoma cells in soft agar. The production of SW-13 colony-stimulating activity by other human malignant cell lines of both epithelial and mesenchymal origin has been demonstrated. Evidence was presented indicating that the stimulating activity detected in crude acid-ethanol extracts was an acid-and heat-stable polypeptide requiring disulfide bonds for full activity. This activity was detected more frequently in tumors and human cancer cells in culture of epithelial origin than of mesenchymal origin and in a variety of nonneoplastic tissues. In the present study, this activity, termed epithelial transforming growth factor (TGFe) because of its ability to stimulate soft agar growth of certain epithelial cells, was partially purified from bovine kidney. Fourfold purification of the kidney acid-ethanol extract with 50% maximal growthstimulatory activity of 10 «ig was achieved using molecular sieve chromatography where TGFe eluted with an apparent molecular weight of 20,000-25,000. The next purification step, molecular sieve high perform ance liquid chromatography, yielded a 50% maximal growth-stimulatory activity of 50 ng and an 800-fold purification from the initial acid-ethanol extract. TGFe eluted in the M, 11,000 range. Reversed phase high performance liquid chromatography with a Ci« column was then used, yielding a single or double peak of SW-13 colony-stimulating activity at 30-35% acetonitrile. The degree of purification was 11,000-fold with a 50% maximal growth-stimulatory activity of 3.5 ng. Analysis of the peak on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major and sometimes single band with a molecular weight of 23,000-25,000. Extraction of protein from the polyacrylamide gel dem onstrated that only the M, 23,000-25,000 band stimulated soft agar growth of SW-13 cells. The biological activity of the partially purified TGFe was found to differ from other known growth factors with regard to its ability to stimulate soft agar growth of SW-13 cells with the exception of basic fibroblast growth factor (FGF). The acid lability of FGF, the different molecular weights of these two growth factors, the lack of stimulation of soft agar growth of A431 cells, and the lack of binding of TGFe to FGF receptors indicated that TGFe was not related to basic FGF. Partially purified TGFe was also found to stimulate soft agar growth of two squamous cell carcinoma lines, A431 and 1)562. and the mouse embryo-derived AKR-2B cells. These data suggest that TGFe is a unique factor distinct from those previously described and it is speculated that TGFe may play a role in normal and neoplastic growth of epithelial cells.
BioTechniques
The purification and recovery of biologically active epithelial-type transforming growth factor (... more The purification and recovery of biologically active epithelial-type transforming growth factor (TGFe) is described. In the final phase of purification, micropreparative electrophoretic chromatography was employed using a Tris-glycine-sodium dodecyl sulfate buffer system in an automated instrument. Briefly, partially purified protein preparations were separated in 2.5 x 50 mm, 10% polyacrylamide gel in electrophoresis tubes installed in the apparatus, electrophoresed under constant current of 1.5 mA for 400 min and recovered by automated fractionation and collection of the eluant from the tube gel. Aliquots of the eluted fractions were assayed in a biological system using SW-13 cell growth stimulation as an indicator of the presence of biologically active TGFe. Using the above procedure, TGFe was purified to within 95% homogeneity as assessed by silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Laboratory Investigation
Recent reports indicate that fibroblast growth factors known to be present in the pituitary in hi... more Recent reports indicate that fibroblast growth factors known to be present in the pituitary in high levels regulate the action of growth hormone and prolactin. New data also suggest a regulatory role in the pituitary for other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Since in most systems cooperation of several growth factors is required for their optimal function, we sought to demonstrate the presence of certain growth factors in the pituitary. Acid ethanol extracts from approximately 50 autopsy-derived human pituitaries were subjected to molecular sieve chromatography and were tested for growth factors. Low molecular weight protein (10 micrograms) eluted from the molecular sieve column contained 10-20 ng material binding to the EGF/TGF-alpha receptor as determined by the EGF/TGF alpha radioreceptor binding assay which represents 11 ng EGF/TGF alpha per pituitary. By Western blotting we found EGF but could not document the presence of TGF-alpha in this material. Radioimmunoassay for insulin-like growth factor I detected 0.4-0.8 ng insulin-like growth factor-I/100 micrograms extract. TGF-beta eluted between 14,000 and 20,000 M(r) at levels of 3-4 ng/pituitary. Its ability to inhibit growth of CC164 mink lung cells was abolished by antibody to TGF-beta 1 but not by antibody raised against TGF-beta 2. The detection of platelet derived growth factor was equivocal and not fully reproducible. We have partially purified TGFe from the pituitary; it stimulated soft agar growth of carcinoma SW-13 cells, and it followed an elution pattern identical to bovine kidney TGFe on molecular sieve column and high pressure liquid and high performance electrophoretic chromatography. Our data show that in addition to fibroblast growth factors, the human pituitary contains other growth factors, such as EGF/TGF-alpha, TGF-beta, insulin-like growth factor I, and TGFe.
Experimental Biology and Medicine
Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, incl... more Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity. We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis. Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor. Some inflammatory cells were scattered among the blood vessels. The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithelialization. Subcutaneous injection of Matrigel impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel. LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells. LS also induced production of TNF-␣ by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells. LS did not appear to have an effect on collagen production. In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-␣ production, and of angiogenesis. Exp Biol