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J C Jennings

Publications and source records attributed to J C Jennings.

33 records · Page 2Linked to original sources
PubMed1990

Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin.

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors. Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulin-like growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-beta) by bioassay. Gu-EDTA extracts stimulated bone cell proliferation. At 10 micrograms/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/micrograms Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-beta, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.

Calcium↗
PubMed1990

Heterogeneity of latent transforming growth factor-beta isolated from bone matrix proteins.

Transforming growth factor-beta (TGF beta) is a family of 25-kDa peptides that appear to be important regulators of cell proliferation, differentiation, and differentiated function in many tissues. TGF beta is present in platelets and serum and is released by cultured cells as several distinct large mol wt complexes of TGF beta with other proteins. These complexes are biologically inactive and are generically called latent TGF beta (L-TGF beta). Large quantities of TGF beta are present in bone matrix. This study was undertaken to determine whether the TGF beta in bone matrix was present as a free 25-kDa peptide or a large mol wt L-TGF beta complex. TGF beta activity was determined by inhibition of [3H]methylthymidine incorporation in mink lung epithelial cells. The specificity of inhibition was determined by treating fractions with a polyclonal rabbit antiporcine TGF beta-blocking antibody before assay. Latency was examined by assaying untreated and acid-treated fractions for TGF beta activity. Acid treatment of EDTA extracts of the bovine bone matrix proteins increased TGF beta activity from a mean of 0.8 pg/microgram protein to 56 pg/micrograms. Under native conditions L-TGF beta eluted from S400 between the 600-400 kDa mol wt standards. No activity eluted in the fractions with authentic 25-kDa TGF beta. Eighty-five percent of the L-TGF beta bound to lentil lectin, and this separated into four discrete L-TGF beta peaks (I-IV) at 0.22, 0.25, 0.35, and 0.42 M NaCl with Mono-Q anion exchange chromatography. Mono-Q pools II and III were reseparated by molecular size on Superose-12 under native and dissociative conditions. Under native conditions TGF beta activity was latent and eluted in the large mol wt fractions. No 25-kDa TGF beta was present. With dissociating conditions (4 M GuHCl) all TGF beta activity eluted in the small mol wt fractions identical to the elution position of authentic 25-kDa TGF beta. The active fractions from the dissociative separation of Mono-Q pool III were separated by C4 reverse phase HPLC. There were three discrete peaks of TGF beta activity corresponding to TGF beta 1, TGF beta 2, and an unidentified form of TGF. Maximum activation of L-TGF beta in each Mono-Q peak occurred at pH 3-3.5. There was partial activation at pH 4.5, but no additional activation at pH 1.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗
PubMed1989

Copurification of osteolytic and transforming growth factor beta activities produced by human lung tumor cells associated with humoral hypercalcemia of malignancy.

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.

Animals↗
PubMed1989

Beta 2-microglobulin is not a bone cell mitogen.

During the purification of skeletal growth factor/insulin-like growth factor-II and transforming growth factor-beta (TGF beta) from EDTA extracts of bovine bone matrix significant mitogenic activity for cultured osteoblast (Ob)-like cells eluted in fractions that contained a nearly homogeneous peptide with a mol wt of about 14,000. This peptide has been purified to apparent homogeneity and identified as bovine beta 2-microglobulin (beta 2M) by amino-terminal amino acid sequencing. During the final purification of beta 2M by CN reverse phase HPLC the mitogenic activity for bone cells separated from the beta 2M protein peak. In spite of this the apparently homogeneous beta 2M preparation retained some mitogenic activity. The ED50 of the bone-derived beta 2M (4,890 +/- 462 ng/ml) was several orders of magnitude (2 x 10(3) to 1.4 x 10(5) times) greater than the ED50 of simultaneously assayed purified growth factors and was no different from the ED50 of the crude EDTA matrix extract (3,350 +/- 890 ng/ml). The beta 2M accounted for less than 0.002% of the total mitogenic activity for Ob-like cells present in the extracted matrix proteins. The following lines of evidence suggest that the mitogenic activity of bone matrix-derived beta 2M (BMD-beta 2M) is due to contamination of the BMD-beta 2M with TGF beta rather than an intrinsic property of beta 2M: 1) the coelution of TGF beta through four successive purification procedures and purification of TGF beta from adjoining fractions from C4 reverse phase HPLC; 2) the abolition of biological activity of BMD-beta 2M and TGF beta with reducing agents; 3) the lack of additive stimulation of [3H] methylthymidine incorporation into bone cells when beta 2M was added to maximally active concentrations of purified TGF beta; 4) the reduction of mitogenic activity when the BMD-beta 2M was incubated with anti-TGF beta; and 5) the inhibition of mink lung epithelial proliferation by the beta 2M preparation. Based on these findings we conclude that although beta 2M is present in bovine bone matrix extracts, it is not a mitogen for Ob-like cells.

Amino Acid Sequence↗
PubMed1988

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II.

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.

Amino Acid Sequence↗
PubMed1988

Comparison of the biological actions of TGF beta-1 and TGF beta-2: differential activity in endothelial cells.

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.

Animals↗
PubMed1988

Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro.

1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues.

Angiotensin II↗
PubMed1986

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones.

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.

Bone and Bones↗
PubMed1986

Characterization of mitogenic activities extracted from bovine bone matrix.

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 micrograms protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained beta transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that BBE contains more than one growth factor, including a beta TGF and a factor that is not specific for bone cells, and all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE. Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.

Animals↗
PubMed1979

Primary dysmenorrhea treated with indomethacin.

Primary dysmenorrhea is a difficult entity to treat, and therapy is usually directed at relieving symptoms. There is some indication that this disorder is caused by an increase in prostaglandin F2alpha. Therefore, logically the treatment may include antiprostaglandin agents. We have studied 32 women with the diagnosis of primary dysmenorrhea in a randomized double-blind fashion using a placebo and indomethacin. Both agents were taken three times a day over four cycles, and therapy was begun two days before the usual onset of pelvic pain. Only two of 16 patients in the placebo group were significantly improved in the four-month treatment cycles while all 16 in the treatment group showed some improvement, 11 having cessation of pain. In the six months following the study period, all patients were given indomethacin. The original treatment group did not change significantly. However, all in the placebo group when switched to indomethacin had some relief, 12 of the 16 showing complete cessation of pain. Gastric irritation was the main side effect and was present in 18% of the treatment group and 12% in the placebo group. Indomethacin appears to effectively relieve primary dysmenorrhea and does not appear to be associated with a high incidence of side effects.

Adolescent↗
PubMed1978

SQ 20,881: effect on eclamptic--pre-eclamptic women with postpartum hypertension.

The renin-angiotensin system has been implicated in the genesis of pre-eclampsia. To avoid fetal toxicity, five women were studied who developed hypertension, proteinuria, and edema in the last trimester of pregnancy and whose BP elevation persisted immediately postpartum. At about 6 hours after delivery the CE enzyme inhibitor (SQ 20,881) was given in incremental doses ranging from 0.25 to 3.0 mg. per kilogram intravenously, before and after diuresis with furosemide, 40 mg. intravenously. BP was measure every 2 minutes and PRA and angiotensin II concentration before treatment, 30 minutes after 0.25 to 0.30 mg. per kilogram, and 30 minutes after 2.0 to 3.0 mg. per kilogram. Echocardiographic assessment of CI and PVR was performed before treatment and after a maximum dose in three patients. Before diuresis, CE blockade had no effect on heart rate, BP, CI, PVR, or PRA, regardless of whether the patient was in positive or negative fluid balance or was sodium loaded or restricted over the preceding 24 hours. Angiotensin II fell by 77 and 10 per cent, respectively, after 0.25 mg. per kilogram was given to two patients, but rose slightly in the other three patients, then fell an average of 46 per cent after 1.0 to 3.0 mg. per kilogram were given. After diuresis, 1.0 mg. per kilogram resulted in a 24 per cent fall in BP which persisted for 3 hours in two patients and a 14 per cent fall which lasted for 30 minutes after 1.0 or 3.0 mg. per kilogram in a third patient. It is concluded that the BP elevation which persists after delivery in certain patients with pre-eclampsia is not angiotensin II dependent.

Adolescent↗
PubMed1977

Hemoglobinopathies in pregnancy.

Sickle cell trait is associated with an increased incidence of asymptomatic bacteriuria, low birth weight infants and premature rupture of membranes. Hemoglobin SS and hemoglobin SC in pregnancy result in significantly increased maternal morbidity and mortality and reproductive wastage. Heterozygous thalassemias in pregnancy carry increased risks but to a lesser degree than hemoglobins SS and SC. Partial exchange transfusion can provide effective prophylaxis for many of the maternal and fetal problems of sickle cell disease.

Anemia, Sickle Cell↗