PubMed Health⌕ Search

Biomedical subjects

K Siminoski

Publications and source records attributed to K Siminoski.

32 records · Page 2Linked to original sources
PubMed1990

Hypogonadotropic hypogonadism in idiopathic hemochromatosis: evidence for combined hypothalamic and pituitary involvement.

Hypogonadism is a common finding in idiopathic hemochromatosis. Most studies have localized the defect to either the pituitary gland or the testes. We describe a case with evidence that favors the likely concomitant involvement of the hypothalamus as a factor in the observed hypogonadism. A clinically hypogonadal male with hemochromatosis had a low testosterone concentration with inappropriately normal serum LH levels. Leydig cell function was intact, as demonstrated by a normal increase in serum testosterone following HCG administration. However, although the pituitary secretion of LH was normal in response to GnRH stimulation, clomiphene administration did not produce an increase in LH and FSH, suggesting that there was a defect in the hypothalamic GnRH response. Since the FSH and prolactin responses to stimulatory testing were inadequate, coexisting pituitary dysfunction was likely also present. We conclude that this man had hypogonadism with laboratory evidence for a combined defect in hypothalamic and pituitary function.

Chorionic Gonadotropin↗
PubMed1989

Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands.

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.

Amino Acid Sequence↗
PubMed1987

Detection of molecules with nerve growth factor binding activity in medium conditioned by L-929 fibroblasts.

L-929 fibroblasts (L cells) secrete a high molecular weight form of nerve growth factor (NGF) that is non-covalently bound and contains as part of its structure a molecule similar, if not identical, to beta-NGF in mouse submandibular glands. The other components of the NGF complex have not been characterized. In this study we used radiolabeled beta-NGF as a probe to detect molecules with NGF binding activity in L cell conditioned medium. The L cell NGF complex was dissociated at low pH, or with denaturants or detergents, and allowed to reassociate in the presence of 125I-beta-NGF. Radioactivity became associated with a complex that eluted in a high molecular weight volume on columns of Sephadex G-200 and Sephacryl S-500. Incorporation was saturable and did not occur under non-dissociating conditions. The complex was affinity cross-linked and studied by SDS gel electrophoresis. Radiolabeled molecules were observed with molecular weights of 151,000, 56,000 and 53,000. Labeling did not occur in the presence of excess unlabeled NGF or when cross-linking was done with fetal bovine serum, indicating that binding is specific and that binding activity is not derived from serum added to tissue culture medium. Solutions containing 7S NGF from mouse salivary glands were cross-linked by similar procedures but different banding patterns were observed. The data show that NGF binding molecules dissimilar from those in salivary glands are present in L cell conditioned medium.

Animals↗
PubMed1987

Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum.

Epidermal growth factor (EGF), an acid-stable peptide present in rodent and human milk, is absorbed and promotes intestinal growth when fed to suckling rats. To determine whether absorptive cells of suckling rat ileum conduct selective transepithelial transport of EGF, we followed uptake of 125I-EGF from ileal loops by autoradiography and biochemical methods. Specific binding sites for 125I-EGF were localized by electron microscope autoradiography on apical membranes of ileal epithelial sheets in vitro. During uptake in vivo, radiolabeled molecules were concentrated in apical endosomal compartments and were also associated with lysosomal vacuoles, basolateral cell surfaces, and lamina propria. Excess cold EGF reduced basolateral label by 44% and TCA precipitable serum label by 38%. After 30 and 60 min of continuous uptake, radiolabeled molecules in epithelium, denuded mucosa, blood, and liver were analyzed under reducing conditions by reversed-phase high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although considerable degradation of 125I-EGF occurred after uptake from the lumen, a portion of radiolabel in epithelium and mucosa represented 125I-EGF which eluted somewhat more rapidly from C18 HPLC columns and showed a slight decrease in apparent molecular weight by SDS-PAGE. All radiolabel in blood and liver represented breakdown products. Thus, EGF is selectively transported across the ileal epithelium in suckling rats but is modified during transport. Milk EGF may accumulate in the lamina propria where it could influence growth and maturation of the suckling intestine.

Animals↗
PubMed1987

Cortisone, testosterone, and aldosterone reduce levels of nerve growth factor messenger ribonucleic acid in L-929 fibroblasts.

Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (NGF) in medium conditioned by L-929 fibroblasts (L cells). In this study, we extend those observations and have measured in L cells the effects of hormone treatment on mRNA encoding NGF. L Cells were grown for 3 days in the presence or absence of hormones. NGF in conditioned medium was measured by NGF RIA; NGF mRNA was measured in cell extracts by Northern blot analysis. Cortisone reduced NGF levels in conditioned medium below the limit of detection of the RIA (less than 10% of control values) with an ED50 of 5 X 10(-9) M; NGF mRNA was reduced to 12% of control levels with an ED50 of 1 X 10(-8) M. Reductions in mRNA were maximal within 3 h and were completely reversed 12 h after removal of the hormone. Levels of NGF in conditioned medium were also undetectable in cultures treated with testosterone, and mRNA levels were reduced by 80%; the ED50 for both effects was 4 X 10(-9) M. Aldosterone (1 X 10(-6) M) reduced NGF to below detectable levels and NGF mRNA by 70%. Progesterone and thyroid hormone had no effect on NGF or NGF mRNA. 17 beta-Estradiol reduced levels of NGF in medium by 50%, but had no detectable effect on levels of NGF mRNA. These results suggest that cortisone, testosterone, and aldosterone decrease NGF levels in L cell-conditioned medium by reducing the cellular content of NGF mRNA.

Aldosterone↗
PubMed1986

Comparison of 7S nerve growth factor and nerve growth factor I from mouse submandibular glands.

7S nerve growth factor (7S NGF) and nerve growth factor I (NGFI) are NGF-containing protein complexes isolated from mouse submandibular glands by different protocols, and reports suggest that the molecules differ chemically. In this study, we compared the molecular properties and subunit compositions of the two proteins. Purified 7S NGF and NGFI electrophoresed to identical positions on polyacrylamide gels in nondissociating buffers, with electrophoretic mobilities indistinguishable from that of unpurified NGF in salivary gland extracts. Ultraviolet absorption curves were identical, and sedimentation coefficients were similar (7.3 +/- 0.25 S for 7S NGF; 7.2 +/- 0.2 S for NGFI) as determined by sedimentation velocity analysis. By sedimentation equilibrium analysis, molecular weights of 135 000-140 000 were obtained for both complexes at protein concentrations in the centrifuge cell greater than 85 micrograms/mL; when protein concentrations within the centrifuge cell ranged from approximately 30 to 100 micrograms/mL at equilibrium, both complexes dissociated. Molecular weight values determined by gel filtration on Bio-Gel P300 and Sephadex G200 resins were similar for both proteins, and the values determined on Sephadex agreed with those obtained by ultracentrifugation. The subunit compositions of the complexes were also similar as determined by nonequilibrium isoelectric focusing, NGFI being composed of proteins that migrated to positions identical with those of the alpha, beta, and gamma subunits of 7S NGF. Furthermore, the stoichiometry of the subunits was similar in the two complexes as determined by radioimmunoassays to each of the subunits and by densitometric analysis of electrophoretic gels. Both methods showed that the complexes contain approximately 2 mol of the alpha and gamma subunits per mole of beta-NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies.

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.

Animals↗
PubMed1986

Uptake and transepithelial transport of nerve growth factor in suckling rat ileum.

Nerve growth factor (NGF) is necessary for the development of sympathetic and some sensory neurons. Milk may be a source of NGF for suckling young, but sites of intestinal absorption of the protein have not been identified. To determine whether NGF is transported across the absorptive epithelium of suckling rat ileum, we assessed binding, uptake, and transport of 125I-NGF by light microscopy and EM autoradiography. Blood and tissue extracts were analyzed by biochemical and immunological methods to determine whether NGF was taken up structurally intact. NGF binding sites were identified on microvilli and apical invaginations of ileal absorptive cells in vitro. Injected into ileal loops in vivo, NGF radioactivity retained by fixation was evident after 20 min in apical regions of absorptive cells, in endocytic tubules (which mediate the uptake of membrane-bound ligands), in vesicles (which mediate nonspecific endocytosis), and in the supranuclear lysosomal vacuole. At 1 and 2 h, radiolabel in these compartments increased and silver grains were evident at the basal cell surface, and in cells, matrix, and vessels of the lamina propria. In blood and liver, radiolabeled molecules that were immunologically and electrophoretically indistinguishable from NGF and that co-eluted with NGF on gel filtration columns were detected, confirming that some NGF was transported across the epithelium structurally intact. Thus, absorptive cells of suckling rat ileum can take up NGF by both receptor-mediated and nonspecific endocytosis, and direct NGF either to the lysosome for degradation, or into a transepithelial transport pathway.

Animals↗
PubMed1986

Steroids and triiodothyronine reduce nerve growth factor concentrations in medium conditioned by L-929 fibroblasts.

Thyroid hormone, testosterone, and glucocorticoids stimulate the production of nerve growth factor (NGF) in mouse submandibular glands, but little is known about the influences of these hormones on other sources of NGF. In this study we tested the effects of thyroid hormone and steroids on NGF levels in medium conditioned by L-929 fibroblasts (L-cells), a mouse cell line that secretes NGF in culture. NGF concentrations in conditioned medium were measured by RIA and were normalized for cell number. In the absence of hormones, NGF levels in medium averaged 5.57 ng/ml. T3, cortisone, and aldosterone decreased NGF levels below the sensitivity of the RIA (less than 0.37 ng/ml), with respective ID50 values of 1 X 10(-5), 1 X 10(-8), and 5 X 10(-8) M. Testosterone decreased NGF levels by 66%, with an ID50 of 9 X 10(-9) M, and 17 beta-estradiol decreased it by up to 50%, although the hormone's effects had not plateaued at 10(-6) M. NGF concentrations were unaffected by progesterone at doses as high as 10(-6) M. Cell viability was unaffected by hormone treatment, and the total amount of secreted protein in medium decreased only in cultures treated with high doses of cortisone and aldosterone. Qualitatively similar results were obtained by bioassay; steroids markedly reduced, but did not eliminate, the nerve growth-promoting effects of L-cells on embryonic chick dorsal root ganglia in cocultures. These data indicate that hormones that stimulate NGF production in salivary glands inhibit NGF production by L-cells in culture.

Aldosterone↗
PubMed1985

Cellular and subcellular colocalization of nerve growth factor and epidermal growth factor in mouse submandibular glands.

Immunocytochemical methods have been used to compare the cellular and subcellular distribution of nerve growth factor (NGF) and epidermal growth factor (EGF) in mouse submandibular glands. Rabbit antisera raised against purified proteins were characterized by immunoblot methods and were used to stain sections of salivary glands embedded in plastic. For light microscopy, antibodies were visualized by indirect immunofluorescence. For electron microscopy, thin sections were treated simultaneously with IgG against NGF and EGF coupled to colloidal gold particles of different size. Data indicate that NGF and EGF are present in all granular convoluted tubule cells and in no other cell type within the salivary gland. Ultrastructural analyses indicate that NGF and EGF are evenly distributed together within mature secretory granules, although a population of small granules was identified that is not immunoreactive for either protein. Taken together, the data suggest that granular convoluted tubule cells are homogeneous in the production and storage of NGF and EGF.

Animals↗
PubMedDate not supplied

Differential movement during swallowing as an aid in the detection of thyroid pseudonodules.

The clinical examination of neck masses is clearly imperfect and false-positive detection of thyroid nodules, termed "pseudonodules," remains a common problem. Movement on swallowing has been emphasized as a highly specific feature of thyroid masses, but lesions in the vicinity of the thyroid can be displaced by underlying structures during deglutition, causing them to mimic thyroid nodules. Two cases are presented to illustrate how additional features of neck mass movement can help determine whether a lesion is located within the thyroid or whether it is extrathyroidal. These include the range of motion of the mass, the presence or absence of a stationary phase prior to descent, and the timing of the movements of the mass. Assessment of these parameters during the physical examination of anterior neck masses may help reduce the incidence of thyroid pseudonodules.

Adult↗