Appropriate frontal barrier tests for belted occupants.

Vehicle damage from frontal impacts was classified and investigated together with injuries sustained by belted front seat occupants. The sample consisted of 1872 frontal crashes from the Midlands of England. Analysis focused on impacts with broad objects that might conceivably be simulated by a barrier test. Two asymmetrical front-end damage patterns were commonly identified, and these gave the greatest rates of non-minor (Abbreviated Injury Scale (AIS) > or = 2) injuries in a range of Estimated Test Speeds from 35 to 52km/h which is the regime of current legislative crash tests. The most injurious type involved oblique damage caused by a substantial overlap of the struck object. The other type was from a small overlap. Objects struck and passenger compartment intrusions were compared. Appropriate asymmetrical and deformable barrier concepts were discussed. Other findings were connected with the future role of full face barriers as used in current tests such as Federal Motor Vehicle Safety Standard 208. Fuller overlaps (> 50%) tended to give more torso injuries rated > or = AIS 2 caused by seat belt loads and, at high speeds (53-79km/h), caused the most fatalities. Full overlaps (100%) rarely resulted in symmetrical intrusion into the passenger compartment.

Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells.

Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.

Increased secretion of insulin-like growth factor-binding proteins and decreased secretion of insulin-like growth factor-II by muscle from growth-retarded neonatal rats.

Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) may be important factors in the control of neonatal growth. We have examined the production, in vitro, of IGFBPs and IGFs by hind-limb skeletal muscle from normal and small-for-gestational age (SGA) neonatal rats. Conditioned medium was collected from muscle strips after incubation at 37 degrees C for 2 h in Ham's F-12 medium. The conditioned medium was subjected to acid-gel permeation chromatography to separate IGFBPs from IGFs. The binding of 125I-labelled IGF-I to IGFBPs from both control and SGA muscle was displaced equipotently by IGF-I and IGF-II and not at all by insulin. IGFBPs from control and SGA muscles bound IGF-I with comparable affinities (Kd = 0.071 and 0.069 nmol/l respectively). When IGF-II was used as tracer, neither IGF-I nor insulin competed for binding. Western ligand blots of IGFBPs in conditioned media from both control and SGA muscles showed three bands of radioactivity at molecular masses equivalent to 24, 30 and 40 kDa. When the release of IGFBPs by muscle tissue in vitro was quantified by measuring the number of IGF-I binding sites in acid-fractionated medium it was apparent that the muscles from SGA pups secreted significantly more IGFBPs (39.3 +/- 7.5 fmol/mg muscle protein per 2 h) than the muscles from control pups (17.8 +/- 2.7 fmol/mg protein per 2 h; P less than 0.05). In contrast to the IGFBPs, more IGF activity was secreted by the muscles from the control pups (61.1 +/- 15.6 fmol/mg muscle protein per 2 h) than the muscles from the SGA pups (12.6 +/- 5.8 fmol/mg muscle protein per 2 h; P less than 0.05). Analysis of the IGF activity with assays specific for IGF-I and IGF-II showed that both SGA and control muscles secreted predominantly IGF-II with approximately 10% of the total IGF activity measurable as IGF-I. This differential secretion of IGFBPs and IGFs may be associated with the reduced growth potential of the SGA neonate.

Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats.

To investigate the response of the growth retarded neonatal rat to insulin-like growth factor-I (IGF-I) we have measured the effect of IGF-I on in vitro muscle protein synthesis and degradation rates in growth retarded and control neonatal rat pups. The growth retarded pups were growth retarded in utero by ligation of the uterine blood supply at day 17 of gestation. Basal levels of muscle protein synthesis in vitro were significantly lower in growth retarded pups compared with controls. Protein degradation rate were not different in muscles taken from the two groups. IGF-I stimulated protein synthesis in muscle from control pups by 12% and 15% at 20 ng/ml and 200ng/ml respectively. Net protein degradation was inhibited by 20% in the presence of 20ng/ml IGF-I. IGF-I had no effect on net protein synthesis or degradation in muscle from growth retarded pups. Neither Multiplication Stimulating Activity (at 20ng/ml or 200ng/ml) nor insulin (at 40ng/ml or 800ng/ml) was able to increase synthesis or decrease degradation of protein. Specific receptors for IGF-I are present on muscle membranes from both groups. Unlabelled IGF-I was more effective than MSA or insulin in competing with 125I-IGF-I for binding to the receptor. The relative affinities are consistent with type I IGF receptors. The affinity of these receptors for IGF-I was similar (Kd approximately 5nM) in both groups and the receptor concentration in both cases was approximately 250 fmol/mg protein. The refractility of tissue from growth retarded pups to IGF-I may be partially responsible for the lack of catch up growth in growth retarded neonates.

Effect on the fetus of infusing a commercial amino acid preparation into a pregnant sheep.

The common clinical practice of intravenous feeding of the pregnant woman poses the question of the effect on the fetus of such infusions. We have used the sheep as a model to study the change in fetal amino acid levels after a maternal infusion of Synthamin 13. The maternal plasma aminogram largely reflects the amino acid pattern in the infusate. However, in the fetal circulation only the branched chain amino acids (leucine, isoleucine and valine), phenylalanine and alanine rose significantly after infusion. Only leucine and isoleucine were observed to spill into the fetal urine. The results suggest that the ovine placenta selectively modifies the amino acid profile presented to the fetus when the maternal plasma aminogram is distorted. However, the fetus is not totally protected from changes in phenylalanine, which in high concentrations, is detrimental to normal development.

Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D3 in human cultured cell lines.

Several human cancer cells possess receptors for 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]. In these cells 1,25-(OH)2D3 has a biphasic concentration-dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24-carbon and of 1,24R,25-trihydroxyvitamin D3[1,24R,25-(OH)3D3]. The difluorinated analogue 24,24-difluoro-1,25-(OH)2D3[24,24-F2-1,25-(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25-(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25-(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25-(OH)2D3 at 0.05 nM for 6 h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6.7-fold. None of the other compounds had a similar effect at this concentration. At 10 nM all 1-hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17-fold); this effect is still specific since 25-OH D3 had no such effect even at 10 nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in plasma amino acids in the developing chick.

We have sampled arterial blood from chicken embryos during development and measured the changes in plasma amino acids from mid-gestation to hatching. During gestation, several amino acids rise to a peak concentration at 16 days and fall prior to hatching. After hatching, most amino acids fall, although the plasma concentrations of aspartate, glutamate and taurine rise significantly.

Regulation of the 1,25-dihydroxyvitamin D3 receptor by 1,25-dihydroxyvitamin D3 in intact human cancer cells.

Human breast cancer cells have been shown to be targets of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]action with specific dose-dependent effects on growth in vitro. These cells possess specific, high affinity receptors for 1,25-(OH)2D3. We have studied the interaction of the vitamin D hormone with its receptor in these intact human target cells. Specific binding of tritium-labeled 1,25-(OH)2D3 by these cells reaches a maximum with 3-9 h depending on hormone concentration; subsequently there is a rapid loss of hormone-binding activity to virtually undetectable levels by 12-24 h. Although the hormone induces its own metabolism in these cells, binding cannot be restored by the addition of fresh hormone. This loss of hormone-binding activity of the receptor, is analogous to the processing of the estrogen receptor in estrogen receptor-positive cells. Two inhibitors of DNA transcription, actinomycin D and cordycepin, delay the onset and slow the processing of receptor if added with the hormone. They are ineffective if added after hormone binding is maximal. Inhibitors of protein synthesis have no protective effect at all and, in fact, these agents inhibit both the attainment of maximum specific binding and the recovery of receptor over the subsequent 24-72 h. These data demonstrate for the first time that the 1,25-(OH)2D3 receptor undergoes regulation in the intact human target cell. This event, which resembles processing of the estrogen receptor and down-regulation of peptide-hormone receptors, likely represents a nuclear mechanism for the control of cellular responsiveness to hormone in the intact cell.

1 alpha, 25-Dihydroxyvitamin D3 specifically induces its own metabolism in a human cancer cell line.

Human breast cancer and malignant melanoma cells possess specific, high affinity receptors for 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of these cells is affected by 1,25-(OH)2D3 with a biphasic dose response, i.e. stimulation at low concentration and inhibition at high concentration. Several 1,25-(OH)2D3 metabolites hydroxylated at the 23,24 and 26 carbons have reduced affinity for the receptors but are still able to inhibit cell replication at high concentrations; none of these metabolites are able to stimulate cell growth. In this context, it is of considerable interest that T47-D breast cancer cells in culture actively metabolize 1,25-(OH)2D3 to chloroform- and aqueous-soluble compounds. Based on multiple solvent high pressure liquid chromatography, double label studies, and periodate sensitivity it is apparent that metabolism occurs about the 23,24 and 26,27 carbon atoms. Three metabolites have been tentatively identified as 1,24,25-(OH)3D3 and the 24-oxo derivatives of 1,25-(OH)2D3 and 1,23,25-trihydroxyvitamin D3. Although calcitroic acid may be produced, other aqueous-soluble metabolites with partially intact side chains are major metabolic products. The metabolic activity is low in the 1,25-(OH)2D3-depleted cell and is induced in 3-4 h by a process requiring new protein synthesis. The induction by 1,25-(OH)2D3 is highly specific since 25-OH D3 is quite ineffective. The studies reported here carried out in intact cells in culture demonstrate unequivocally that 1,25-(OH)2D3 metabolism, through chloroform-soluble to aqueous-soluble metabolites, occurs entirely within a proven target cell of 1,25-(OH)2D3 action. Since other 1,25-(OH)2D3 metabolites hydroxylated at the 23,24 and 26 carbons lack the ability to stimulate cell replication, it is hypothesized that the hormone-inducible metabolic activity represents a sensitive mechanism for the control of cellular responsiveness to 1,25-(OH)2D3.

1,25-dihydroxyvitamin D3 and breast cancer.

Studies by this laboratory have demonstrated the presence of specific, high affinity 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptors both in surgical specimens of human breast cancer and in breast cancer cells in culture. We report here that 1,25-(OH)2D3 receptors were found in 54% of 230 human primary breast cancers. Although receptor levels are lower than those of oestrogen receptors, using a modified and more sensitive assay method, the apparent receptor concentration is increased without altering the receptor positivity rate. Also in preliminary studies on lymph node metastases and their primary tumours, the receptor positivity rate is higher in the lymph nodes. These findings suggest that metastatic cells may be selected for the presence of 1,25-(OH)2D3 receptors. These data, taken with the evidence that 1,25-(OH)2D3 and several of its metabolites inhibit the growth of human breast cancer cells in culture, exactly analogously with the effects of oestrogens on cancer cell growth in vitro and in vivo, indicate that 1,25-(OH)2D3 or its metabolites could have a role in the 'hormonal' therapy of metastatic human breast cancer.

Inhibition of human cancer cell growth by 1,25-dihydroxyvitamin D3 metabolites.

Specific high-affinity 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors, which can undergo hormone-dependent activation and nuclear localization, have been demonstrated in a wide variety of established human cancer cell lines and surgically obtained human cancer tissues. 1,25-(OH)2D3 has been reported by some workers to stimulate cancer cell replication at low "physiological" concentrations and by ourselves and others to inhibit at higher concentrations. We report here that 1,25-(OH)2D3 had a biphasic effect on the replication of two distinct human cancer cell lines, i.e., the breast cancer T-47D and the malignant melanoma MM96, an effect analogous to that of estrogens on the breast cancer cell line MCF-7. These inhibitory effects were accompanied by marked morphological changes. Furthermore, two known metabolites of 1,25-(OH)2D3, i.e., 1,24,25-trihydroxyvitamin D3 and 1,25,26-trihydroxyvitamin D3, which compete for binding to the 1,25-(OH)2D3 receptor, did not stimulate but were almost equipotent with 1,25-(OH)2D3 in inhibiting the replication of both cell lines. The stimulatory but not the inhibitory effect of 1,25-(OH)2D3 was abolished by cortisone. These 1,25-dihydroxyvitamin D3 metabolites show promise for the inhibition of cancer growth, analogous to the effect of estrogens and antiestrogens in breast cancer but with potential application in a much wider range of human cancers.

Presence of 1,25-dihydroxyvitamin D3 receptors in established human cancer cell lines in culture.

Receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been described in several human breast cancer cell lines and more recently in human melanoma. The presence of 1,25-(OH)2D3 receptor (1,25-DR) in two cultured breast cancer cell lines was associated with receptors for calcitonin, another hormone thought to have effects on calcium handling. Therefore, it seemed important to examine a range of established human cancer cell lines for the presence of receptors for 1,25-(OH)2D3 and calcitonin. Thirty-three cancer cell lines were examined. 1,25-DR was found to be present in 23 lines, while calcitonin receptors were not detected in any of them. The 1,25-DR from several cell lines sedimented at about 3.5S in sucrose density gradients, had the appropriate specificity for vitamin D metabolites, had Kds of 0.8 to 2.2 x 10(-11) M, and had receptor concentrations of 12 to 99 fmol/mg protein. Ten malignant melanoma and nine colonic carcinoma lines constituted the largest groups of carcinoma cell lines, and seven and eight, respectively, of these were 1,25-DR positive. The high frequency of 1,25-DR positivity in the cultured colonic carcinoma cells is quite different from the low frequency of 1,25-DR in primary colonic carcinomas. It was also interesting that both of two cell lines derived from patients who had had both bone metastases and malignant hypercalcemia were 1,25-DR positive. These various cell lines may provide useful models for the examination of 1,25-(OH)2D3 action in vitro.

Calcitonin and 1,25-dihydroxyvitamin D3 receptors in human breast cancer cell lines.

Five human breast cancer cell lines (MCF 7, T 47D, BT 20, MDA 157, and MDA 231) and a human breast epithelial cell line (HBL 100) have been found to contain specific high-affinity receptors for 1,25-dihydroxyvitamin D3, Kd values ranged from 0.6 to 2.0 X 10(-11) M and receptor concentration from 31 to 150 fmol/mg cytosol protein. Two of the breast cancer lines (MCF 7 and T 47D) contain specific high-affinity receptors for calcitonin and a calcitonin-responsive adenylate cyclase, which have been characterized with the aid of salmon, eel, and human calcitonins and in several substituted analogues of human calcitonin. The 1,25-dihydroxyvitamin D3 receptor may reflect a normal property of the breast cell. Breast cancer cell lines provide a useful source of 1,25-dihydroxyvitamin D3 receptors. Their coexistence with a calcitonin receptor and biological response in some breast cancers offers the opportunity to investigate new aspects of breast cancer endocrinology.

Normal and malignant breast tissue is a target organ for 1,25-(0H)2 vitamin D3.

Malignant and benign human breast tumours as well as rabbit breast tissue were examined for specific receptors for 1,25-dihydroxyvitamin D3 using competitive binding studies and sucrose density gradient analysis. Classical high affinity, low capacity receptors for 1,25-dihydroxyvitamin D3 were found in breast and node tissue in seven of ten patients with breast cancer and in all three patients with benign neoplasms. An inflammatory breast mass showed no binding. Similar receptors were found in breast tissue from pregnant and lactating rabbits. Taken with other recent data, these results suggest that 1,25-dihydroxyvitamin D3 may activate calcium transport in the malignant as well as in the normal lactating breast.