In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine 3'5'-monophosphate.

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.

Specific nuclear binding of adenosine 3',5'-monophosphate-binding protein complex with subsequent poly(A) RNA synthesis in embryonic chick cartilage.

We used embryonic chick pelvic cartilage as a model to study the mechanism by which cyclic AMP increases RNA synthesis. Isolated nuclei were incubated with [32P]-8-azidoadenosine 3,5'-monophosphate ([32P]N3cAMP) with no resultant specific nuclear binding. However, in the presence of cytosol proteins, nuclear binding of [32P]N3cAMP was demonstrable that was specific, time dependent, and dependent on a heat-labile cytosol factor. The possible biological significance of the nuclear binding of the cyclic AMP-protein complex was identified by incubating isolating nuclei with either cyclic AMP or cytosol cyclic AMP-binding proteins prepared by batch elution DEAE cellulose chromatography (DEAE peak cytosol protein), or both, in the presence of cold nucleotides and [3H]uridine 5'-triphosphate. Poly(A) RNA production occurred only in nuclei incubated with cyclic AMP and the DEAE peak cytosol protein preparation. Actinomycin D inhibited the incorporation of [3H]uridine 5'-monophosphate into poly(A) RNA. The newly synthesized poly(A) RNA had a sedimentation constant of 23S. Characterization of the cytosol cyclic AMP binding proteins using [32P]N3-cAMP with photoaffinity labeling three major cAMP-binding complexes (41,000, 51,000, and 55,000 daltons). The 51,000 and 55,000 dalton cyclic AMP binding proteins were further purified by DNA-cellulose chromatography. In the presence of cyclic AMP they stimulated poly(A) RNA synthesis in isolated nuclei. The 51,000-dalton cyclic AMP-binding protein was the predominant one that bound to the nuclei. While cyclic AMP-dependent protein kinsae activity was present in the cytosol and DEAE peak cytosol proteins, it was not present in the DNA-cellulose-bound, cyclic AMP-binding proteins. We conclude that one possible mechanism by which cyclic AMP increases RNA synthesis is by complexing to a 51,000-dalton cytosol cyclic AMP-binding protein and being subsequently translocated to the nucleus, where it is specifically bound and associated with induction of poly(A) RNA synthesis.

Triiodothyronine stimulates maturation of porcine growth-plate cartilage in vitro.

We studied the effect of triiodothyronine (T3) on mammalian growth-plate cartilage in vitro. Growth-plate cartilages from fetal pigs scapulae were incubated for 3 to 7 d in serum-free medium alone or medium containing T3. Alkaline phosphatase activity, a marker of hypertrophied chondrocytes, was increased in T3 (10 nM)-treated growth-plate cartilage 152 +/- 36% above that of cartilage incubated in medium alone after 3 d of incubation, and 324 +/- 47% after 7 d of incubation. There was a dose-response increase in alkaline phosphatase activity to T3 over the range of 0.01-10 nM. The rise in alkaline phosphatase activity was specific for T3 since growth-plate cartilage alkaline phosphatase activity was not increased by cortisol, insulin, parathyroid hormone, or 5% fetal calf serum. Histological studies of growth-plate cartilage showed that T3 in a concentration-dependent manner increased the width of the zone of maturation (hypertrophied chondrocytes). Histochemical staining for alkaline phosphatase activity demonstrated that T3 caused the recruitment of new cells into the zone of maturation. T3 also stimulated incorporation of L-[3H]leucine into protein and 35SO4 into proteoglycan in growth-plate cartilage. In contrast, T3 did not increase alkaline phosphatase activity or radiolabeled precursor incorporation into nongrowth-plate scapular cartilage. These studies demonstrate that T3 directly stimulates maturation and, to a lesser degree, growth-related processes in fetal mammalian growth-plate cartilage.

Adenosine 3',5'-monophosphate: a modulator of embryonic chick cartilage growth.

We tested the hypothesis that cyclic AMP plays a significant role in modulating the growth of embryonic chick cartilage by determining whether cyclic AMP levels change in growing embryonic cartilage and whether cyclic AMP could stimulate embryonic cartilage growth in a long term in vitro organ culture. Cyclic AMP levels were low (0.1 pmol/mg wet wt) in 8-d chick embryo pelvic cartilage, and increased progressively through the 11th d of embryonic development at which time they reached a maximum (1.8 pmol/mg wet weight) and thereafter remained constant. We developed an in vitro organ culture system to determine whether cyclic AMP, a factor known to stimulate radiolabeled precursor incorporation into macromolecules in short-term studies does, in fact, stimulate growth of cartilage. Individual pelvic cartilages were isolated from 9-d chick embryos, placed in serum-free medium (BGJb-FJ modification) and incubated for 3 to 5 d during which time they increased in size (39 and 60% in length, respectively), wet weight (90 and 141%, respectively), and content of total soluble protein (30 and 48%, respectively). N6-monobutyryl cyclic AMP (BtcAMP) added to the medium caused a dose-dependent (0.05 to 1.0 mM) stimulation of growth. After 3 d of incubation, 1.0 mM BtcAMP increased wet weight (125%), [14C]leucine incorporation into protein (75%), and [3H]thymidine incorporation into DNA (48%) compared with control cartilages incubated in medium alone. 1-methyl-3-isobutyl xanthine, a phosphodiesterase inhibitor, also increased cartilage growth above control while sodium butyrate, AMP, and ATP had no effect. Histological examination of cartilage grown in medium was similar to that of cartilage developing in ovo, whereas, cartilage grown in medium containing BtcAMP showed marked hypercellularity with many immature chondrocytes. Our observations are compatible with the hypothesis that cyclic AMP can significantly modulate the growth of embryonic cartilage.

Altered activity of the nucleotide regulatory site in the parathyroid hormone-sensitive adenylate cyclase from the renal cortex of a patient with pseudohypoparathyroidism.

A series of clinical studies suggest that the primary defect underlying pseudohypoparathyroidism is an abnormality of the parathyroid hormone-receptor-adenylate cyclase complex of the renal cortical cell plasma membrane. In the present study we compared parathyroid hormone-stimulated adenylate cyclase activity in membrane preparations from the renal cortex of three controls and a patient with pseudohypoparathyroidism. In the pseudohypoparathyroid preparation the Km for ATP was significantly greater and parathyroid hormone elicited markedly diminished adenylate cyclase activity at a subsaturating concentration of ATP. In contrast, the dose-response effect of enzyme activity to parathyroid hormone was the same in the control preparations, and that of the pseudohypoparathyroidism kidney, at a saturating concentration of ATP. The apparent alteration in enzyme kinetics, however, was normalized upon addition of guanosine 5'-triphosphate to the reaction mixtures. These results indicate that the defect in the parathyroid hormone-receptor-adenylate cyclase complex of the renal cell membranes, in our patient with pseudohypoparathyroidism, is an abnormal nucleotide receptor site of decreased activity. Such a defect may result in partial uncoupling of the parathyroid hormone receptor and adenylate cyclase, rendering the organ refractory to hormonal stimulation.

Deficient adenylate cyclase regulatory protein in renal membranes from a patient with pseudohypoparathyroidism.

Recent studies have established that some patients with pseudohypoparathyroidism have a deficiency of the adenylate cyclase regulatory protein (the G unit) in plasma membranes from erythrocytes, platelets, and fibroblasts. We have directly measured the activity of the G unit in renal membranes from a patient with pseudohypoparathyroidism who, in addition to parathyroid hormone resistance, has resistance to thyrotropin and gonadotropins. Erythrocyte membrane G unit activity was 57% that of control erythrocyte membranes. Lubrol PX extracts of renal membranes had only 30% of the G unit activity of control renal membrane extracts, whether assayed with sodium fluoride or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S). In cholate extracts, the G unit activity was 37 and 48% of control with fluoride or GTP-gamma-S, respectively. Cholera toxin-dependent incorporation of [32P]ADP-ribose into the 42,000-Mr subunit of the G unit was decreased in renal membranes from the patient compared with control renal membranes. The data demonstrate that the membrane G unit deficiency in pseudohypoparathyroidism extends to the cells of a clinically relevant parathyroid hormone target tissue.

Cushing's disease. A review.

Cushing's syndrome continues to tax the most discerning clinician. I review pituitary-dependent adrenal hyperplasia (Cushing's disease), including recent experiences with Cushing's disease at Duke University, Durham, NC, and relate these observations to the current ideas as to pathophysiology, etiology, and management of Cushing's disease. Transsphenoidal microsurgery (TPS) performed by an experienced neurosurgeon offers selective removal of corticotropin (ACTH)-secreting adenoma, immediately cures the hypercortisolism, preserves pituitary function, and is associated with minimal morbidity. Postoperative hypoadrenalism appears to be the best marker of surgical cure. Transsphenoidal surgery has revolutionized our thoughts as to etiology and treatment of Cushing's disease, yet failures with TPS and uncertainty of recurrences leave room for radiotherapy, adrenalectomy, and adjunctive drug therapy in the management of this entity.

1,25-Dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro.

We addressed the question of whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) could directly stimulate cartilage growth in vitro. Pelvic leaflets from chick embryos and scapular growth plates from fetal pigs were organ cultured in serum-free medium in the presence and absence of 1,25-(OH)2D. After 3 days of incubation, 1,25-(OH)2D had increased the pelvic cartilage wet weight 42% and the dry weight 32% above the weight of cartilages incubated in medium alone. 1,25-(OH)2D (10(-9) M-10(-12) M) caused a dose-dependent increase in weight, with maximal increases at 10(-9) M. Furthermore, two deuterized derivatives of 1,25-(OH)2D, 26,27-D6-1,25-(OH)2D3 and 24,26,27-D8-1,25-(OH)2D3, stimulated pelvic cartilage growth in vitro. 26,27-D6-1,25-(OH)2D stimulated increases in growth plate weight above growth plates incubated in medium alone. 26,27-D6-1,25-(OH)2D3 appeared to be potent at lower concentrations than 1,25-(OH)2D on growth plate cartilage. Thus, 1,25-(OH)2D stimulated in vitro growth in two growing cartilage models, the avian pelvic cartilage and the mammalian scapular growth plate cartilage.

Calcitonin stimulates growth and maturation of embryonic chick pelvic cartilage in vitro.

To determine whether calcitonin (CT) affects the growth of avian embryonic skeletal tissue, pelvic cartilages from 9-day-old chick embryos were incubated in a serum-free medium containing CT for 3 days. Porcine CT (PCT), salmon CT (SCT), and human CT (HCT) stimulated increases in cartilage wet weight that were dependent upon the concentration of CT within the medium. Maximal growth was seen with PCT (1.0 U/ml), which increased cartilage wet weight 107% and dry weight 53% above those of cartilage incubated in medium alone. SCT (1.0 U/ml) and HCT (1.0 U/ml) stimulated a 55% increase in cartilage wet weight and a 20% increase in cartilage dry weight over those of cartilage incubated in medium alone. The reason for PCT's apparent potency was due to trace contamination of thyroid hormone in the PCT preparation, since synthetic PCT caused an increase in cartilage wet weight equivalent to those produced by SCT and HCT. Although each calcitonin increased wet and dry cartilage weights, the DNA content was not changed. Alkaline phosphatase activity, a marker of cartilage maturation, was found to be stimulated by CT. SCT, HCT, PCT, and synthetic PCT increased alkaline phosphatase activity over 2-fold above that in cartilage incubated in medium alone. Histological sections of CT-treated cartilage showed large round nuclei, vacuolated cytoplasm with lacuna formation, and an increased amount of cartilage matrix compared to those of cartilage incubated in medium alone. Thus, CT stimulates cartilage growth primarily through cellular hypertrophy and matrix formation. This study demonstrates that CT is a growth and maturation factor for avian embryonic cartilage in vitro.

Triiodothyronine stimulation of in vitro growth and maturation of embryonic chick cartilage.

We studied the direct effect of T3 on cartilage growth and maturation in vitro. Pelvic cartilages from 9-day-old chick embryos were incubated in a serum-free organ culture system which supported cartilage growth over a 5-day interval. The addition of T3 (15 nM) to the medium increased cartilage wet weight (approximately 100%), dry weight (77%), length (35%), and total soluble protein (67%) over 3 days compared to cartilage incubated in medium alone. The DNA content was only slightly increased (2%) by T3 over the interval. T3 stimulated the same parameters of growth similarly after 5 days of incubation. In addition, T3 increased the incorporation of [14C]leucine into protein (82%) and 35SO4 into proteoglycan (53%). A dose-dependent increase in cartilage wet weight was seen with T3 (0.0015-15 nM) over 3 days of incubation. Cartilage incubated with T3 demonstrated microscopic changes in maturation, with development of large numbers of hypertrophied chondrocytes, and biochemical evidence of maturation, with increased alkaline phosphatase activity (130%). The dose-response range for T3 stimulation of alkaline phosphatase activity (0.015-0.15 nM) was considerably more restricted than that for stimulation of growth (0.0015-15 nM). These studies demonstrate that T3 in physiological concentrations directly affects cartilage growth and maturation, primarily through stimulating chondrocyte hypertrophy.

Calcitonin stimulates maturation of mammalian growth plate cartilage.

To determine whether calcitonin (CT) might effect maturation of mammalian growth plate cartilage, we administered salmon CT (sCT) to young rats and used the growth plate from the distal metatarsal as our in vivo growth plate model. Growth plate alkaline phosphatase activity and histological examination were assessed after 3 days of sCT treatment. Alkaline phosphatase activity, a marker of hypertrophied chondrocytes, increased 85%, and the zone of maturation enlarged in rats receiving sCT. In addition, sCT treatment was associated with an increased growth plate 35SO4 incorporation 84% above animals receiving buffer alone. We tested whether this might be a direct effect of CT by using an in vitro model, the growth plate from the fetal pig scapula. Organ culture of these cartilages in serum-free medium with and without sCT (1 U/ml) was performed for 3 days. sCT stimulated alkaline phosphatase activity 49% above growth plates incubated in medium alone. Furthermore, sCT treatment increased the number of hypertrophied chondrocytes leading to widening of the zone of maturation. These studies suggest that CT might have a role growing mammals by promoting maturation of growth plate cartilage.

Embryonic chick cartilage produces its own somatomedin-like peptide to stimulate cartilage growth in vitro.

Embryonic chick pelvic cartilages increase in size and weight when incubated in a chemically defined medium in the absence of serum. We addressed the question of whether endogenous production of growth factors by the cartilage was responsible for this growth. We found that conditioned medium, in which pelvic cartilages from 9-day-old chick embryos had been incubated for 3 days, increased cartilage dry weight 32% over weights of cartilages incubated in fresh medium. Increasing concentrations of conditioned medium stimulated cartilage weight and proline incorporation in a dose-dependent manner. To determine the molecular size(s) of potential growth-stimulating factors, conditioned medium was dialyzed at acid pH, lyophilized, and fractioned over HPLC-TSK Spherogel 3000. The collections were pooled into five fractions (greater than 100K, 30-100K, 20-30K, 12-20K, 1-12K, and less than 1K). Each fraction was readded to organ culture, and growth was assessed 3 days later. Only the 1-12K fraction stimulated growth above that of control cartilage. We assayed cartilage and conditioned medium for somatomedin-C (Sm-C) by RIA to determine if Sm-like peptides were present. Although Sm-C was not detectable within the cartilage, it was readily measurable in concentrated medium (248 +/- 35 pg/ml). Since Sm-like peptides might play a functional role in the growth process, we used a monoclonal antibody to Sm-C to determine whether immunoneutralization of the Sm-like peptides would inhibit cartilage growth in vitro. Addition of anti-Sm-C to organ culture of chick cartilage prevented increases in cartilage wet and dry weights (only 16% and 0%, respectively, above preincubation weights). The inhibitory effect of anti-Sm-C could be reversed by the addition of high doses of insulin to the medium. These studies suggest that endogenously produced Sm-like peptides have a functional role in promoting cartilage growth and support the hypothesis that growth factors may regulate growth through autocrine mechanisms.

Characterization of the insulinotropic potency of polyunsaturated fatty acids.

In this study we have assessed the individual abilities of the essential fatty acids, linoleic and linolenic acids, to release insulin and compared their insulinotropic potencies with those of the more established nutrient insulin secretagogues, glucose and arginine. In each experiment, a total of six islets microdissected from three mice were preperifused at the rate of 1 ml/min with Krebs-Ringer bicarbonate buffer, pH 7.4, containing 2% bovine albumin and 5.5 mM glucose (basal) with a continuous supply of 95% O2-5% CO2 at 37 C for 1 h. After collecting basal samples, the effects of 27.7 mM glucose, 20 mM arginine, 10 mM linoleic acid (18:2, omega 6), and 5 mM linolenic acid (18:3, omega 3) were tested using a sandwich protocol that entails 20-min alternating periods of stimulation with a secretagogue and a washout with basal perifusion. These nutrient concentrations were selected from initial experiments performed to characterize their dose-response effects on insulin secretion. Effluent samples were collected throughout each experiment for measurement of insulin by RIA. In one series of experiments, islets were challenged three times with 27.7 mM glucose, 10 mM linoleic acid, and 5 mM linolenic acid. In another set of experiments, islets were perifused with 20 mM arginine, 27.7 mM glucose, and 10 mM linoleic acid. All of these nutrients stimulated insulin release in a dose-dependent manner. In comparing the insulinotropic potencies of these secretagogues, we assessed insulin secretion as the integrated areas under the curve during 20 min of perifusion with a given nutrient. Thus, the mean integrated area under the curve per 20 min above basal in the presence of 27.7 mM glucose was 6,516 +/- 1,435 pg, which was not significantly different from the value of 4,772 +/- 866 pg obtained during arginine perifusion. However, the area under the curve during 20 min above basal obtained in the presence of linoleate and linolenic acid (8,712 +/- 1,949 and 10,506 +/- 1,490 pg, respectively) were significantly different (P less than 0.05) from those calculated during arginine and glucose perifusions. There was no statistically significant difference between the effects of these two fatty acids at the concentrations tested. In conclusion, our data suggest that linoleic acid and linolenic acid may be, at least in this murine islet preparation, as effective in stimulating insulin release as glucose and arginine, hitherto used to assess the abilities of nutrients to stimulate insulin secretion. However, it remains to be seen whether the efficacy of these polyunsaturated fatty acids in insulin release by murine islets will be obtained in experiments performed on human islets.

Hypoparathyroidism after I-131 therapy with subsequent return of parathyroid function.

A 22-year-old woman developed hypoparathyroidism in 1970, 10 months after treatment of hyperthyroidism with I-131. The hypocalcemia was corrected with Vitamin D2 and oral calcium and she remained normocalcemic for 8 yr. In 1979 hypercalcemia was found and Vitamin D2/calcium was discontinued. Because she remained normocalcemic without therapy for 3 yr, we measured the levels of immunoreactive and bioactive PTH in plasma stored since 1970 and in plasma obtained in 1982 to determine whether there had been restoration of parathyroid function. Indeed, PTH levels in 1970 while the patient was hypocalcemic were low. The bioactive PTH was 0.26 pg/ml (normal 1.5-30), whereas--COOH terminal immunoreactive PTH was 620 pg/ml (normal 600-1500) and midmolecule immunoreactive PTH was 433 pg/ml (normal 300-900). In 1982 while normocalcemic the bioactive PTH and immunoreactive PTH were normal (5.18 pg/ml;--COOH, 970 pg/ml; midmolecule, 789 pg/ml, respectively). Thus, an unusual case of hypoparathyroidism after I-131 therapy with return of parathyroid function is documented by measurements of both immunoreactive and bioactive PTH.

The 25-kilodalton insulin-like growth factor (IGF)-binding protein inhibits both basal and IGF-I-mediated growth of chick embryo pelvic cartilage in vitro.

Insulin-like growth factor (IGF)-I stimulates the growth of many tissues, including growth plate cartilage. However, the role of IGF-binding proteins in the growth process is controversial. We purified a 25-kDa IGF-binding protein (BP-25) from amniotic fluid. We tested the effect of this BP-25 preparation on both basal and IGF-I-stimulated growth of chick embryo pelvic cartilages maintained in serum-free organ culture. Cartilage wet weight was 4.1 +/- 0.3 mg/cartilage initially; after 3 days, BP-25 inhibited both basal and IGF-I-stimulated growth. Control cartilages weighed 7.4 +/- 0.7 mg/cartilage, while those incubated with 100 nM BP-25 weighed 5.8 +/- 0.5 mg/cartilage (P less than 0.001 vs. control); BP-25 concentrations as low as 0.2 nM significantly inhibited basal cartilage growth. Cartilages incubated with 1.25 nM IGF-I weighed 10.4 +/- 0.8 mg/cartilage (P less than 0.001 vs. control), while those incubated with both 100 nM BP-25 and 1.25 nM IGF-I weighed 8.1 +/- 0.5 mg/cartilage (P less than 0.001 vs. cartilage incubated with IGF-I alone); BP-25 concentrations as low as 0.4 nM significantly inhibited IGF-I-stimulated cartilage growth. BP-25 also inhibited basal and IGF-I-stimulated increases in cartilage dry weight, [3H]thymidine incorporation into DNA, and 35SO4 incorporation into proteoglycan. A second BP-25 preparation, which in the presence of 1% platelet-poor plasma acts synergistically with IGF-I to stimulate DNA synthesis and cell replication of fibroblasts and smooth muscle cells in tissue culture, inhibited IGF-I-stimulated cartilage growth to the same degree as did our BP-25 preparation. In separate experiments, proteins present in serum-free medium conditioned for 3 days by chick cartilages were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with [125I]IGF-I. This medium was found to contain two IGF-binding proteins; one appeared to be the chick equivalent of BP-25, while the other had a molecular mass similar to that of a poorly characterized human 34-kDa IGF-binding protein. We conclude that purified BP-25 inhibits the growth of chick embryo pelvic cartilage in our serum-free organ culture system. Since conditioned medium from these cartilages contains both IGF-I-like peptides and IGF-binding proteins such as BP-25, we suggest that the IGF-binding proteins present may act to down-regulate the growth-promoting effects of the local IGF peptides.

Homologous and heterologous growth hormones fail to stimulate avian cartilage growth in vitro.

Recent reports that GH has a direct effect on growing cartilage have raised questions as to the role of somatomedins (Sm) in cartilage growth. To test the hypothesis that GH directly stimulates cartilage growth, we added homologous and heterologous GHs to organ cultures of embryonic chick pelvic cartilage. Pelvic rudiments from 9-day-old chick embryos were incubated in serum-free medium for 3 days in medium alone or medium containing chicken GH, turkey GH, bovine GH, human GH, and bovine GH produced by recombinant DNA methodology. None of the GH preparations studied stimulated avian cartilage growth in vitro. However, cartilage wet weight increased in response to sera from normal and growth hormone-treated hypophysectomized rats. In addition, 20 ng/ml purified Sm-C caused a 78% increase in cartilage weight above that of cartilage incubated in medium alone. Insulin also caused an increase in cartilage weight, but in concentrations 50,000-100,000 times that of Sm-C. Our studies demonstrate that homologous and heterologous GH have no effect on growing avian cartilage and support the hypothesis that Sm directly mediate cartilage growth.

Ia+ T cells in new onset Graves' disease.

The Ia (immune-associated, DR) antigen is a cell surface glycoprotein which is absent on normal circulating T lymphocytes but present on activated T lymphocytes. We studied the expression of this antigen on circulating T lymphocytes from patients with untreated hyperthyroid Graves' disease. All patients (n = 33) with recent onset hyperthyroid Graves' disease studied had an increased percentage and number of circulating Ia+ T cells. Patients with non-Graves's hyperthyroidism or Graves' disease patients more than 1 yr after thyroid ablation had normal values for Ia+ T cells. Other cell surface activation antigens, recognized by monoclonal antibodies 4F2 and 5E9, were not present on circulating T cells in patients with Graves' disease. The 100% positivity for increased numbers of Ia antigen-bearing T cells in hyperthyroid Graves' disease contrasts with our finding of 70% positivity in another HLA-DR-associated disease, recent onset type I diabetes mellitus. The pathogenic significance of these cells is not known, but they seem to represent selective activation of the immune system in patients with untreated hyperthyroid Graves' disease.

Lung ventilation studies with technetium-99m Pseudogas.

Technetium-99m Pseudogas is an ultrafine near monodisperse aerosol of 0.12-microgram diam particle size. This report describes initial clinical experiences with 27 patients referred for investigation of suspected pulmonary embolism, and in whom Pseudogas ventilation images were compared with a high quality commercial aerosol. An additional group of ten patients with severe COPD was examined in a comparative trial of Pseudogas with 81mKr. Pseudogas was better than a conventional aerosol in reaching a diagnosis of pulmonary embolism using a simple blinded comparison with coded images. In addition, bronchial deposition was minimal unless COPD was severe. Moderately well patients had no difficulty inhaling the necessary activity in one or two breaths, and even severely ill and frail aged persons could accomplish the passive breathing maneuver in less than a minute. Clearance of Pseudogas was directly to the systemic circulation with a half-time of 10 min in normal subjects extending up to 100 min in patients with airways disease.

The physical and chemical nature of technegas.

UNLABELLED: Technegas, the discrete radio-aerosol particle, containing 99mTc has been investigated, and the chemical evolution and physical properties of the particle demonstrated. METHODS: A commercial technegas generator was used to produce aerosols according to standard clinical procedures. The aerosols were collected by electrostatic precipitation and examined with transition electron microscopy (TEM), scanning electron microscopy (SEM) and force microscopy. The chemical evolution was examined by x-ray techniques and thermogravimetric analysis. RESULTS: The active particle was identified as hexagonal platelets of metallic technetium contained within a thin layer of graphitic carbon. This composite structure is discussed in light of the metal particle behaving as a template for the carbon capsule. The average size of the observed hexagonal platelets, 30-60 nm, was only weakly dependent on the concentration of technetium in the crucible. CONCLUSION: The mechanism for the formation of the technegas pancreas has been developed and the particles involved characterized. It appears that the use of other metals also leads to the formation of similar materials.

Physical properties and use of pertechnegas as a ventilation agent.

UNLABELLED: Pertechnegas, a variant of technegas, produces similar ventilation images with a much increased clearance rate. This work aims to determine the properties of pertechnegas and its use as a ventilatory agent. METHODS: Fourteen men and 11 women were scanned for PE, after pertechnegas ventilation. Six were reimaged with technegas within 1 wk. Studies were reported according to PIOPED criteria. Pertechnegas samples were analyzed by transmission electron microscopy (TEM), cascade impaction (CI), aerosol mobility analysis (AMA), Fourier transform mass spectrometry (FTMS), x-ray photoelectron spectroscopy (XPS), paper strip (PC) and gas chromatography (GC). RESULTS: Post-test probabilities were normal in 5, low in 8, high in 5 and indeterminate in 7. There were 15 Grade 1, 6 Grade 2 and 4 Grade 3 studies. All Grade 3 patients had FEV1 < 1.5 liters, 3 with rates < 1.0 liter. Patients with high probability had proven deep venous thrombosis in three by venography and in one by doppler. TEM identified 0.3 micron salt particles. CI demonstrated a 7-min time to half clearance from the chamber for particles in the < 0.1 micron range. AMA indicated all particles were < 0.032 micron when salt was excluded. Pertechnegas behaves in PC as pertechnetate, GC demonstrated CO levels below 516 ppm. CO2 concentrations were 0.146 +/- 0.0009%. FTMS found molecular pertechnetate species including 99TcO3(OH)+, Na99TcO3(OH)3+ and Na99TcO3(OH)3+. XPS confirmed that these Tc species exist in oxidation state +7. CONCLUSION: Comparison with technegas images in the follow-up group proved equivalent in the first five views, but indistinct lung boundaries and a high background activity characterized the final anterior images. The active component of pertechnegas is molecular pertechnetate.