Novel mutations in the parathyroid hormone (PTH)/PTH-related peptide receptor type 1 causing Blomstrand osteochondrodysplasia types I and II.

CONTEXT: The PTH/PTHrP receptor type 1 (PTHR1) has a key role in endochondral ossification, which is emphasized by diseases resulting from mutations in the PTHR1 gene. Among these diseases is Blomstrand osteochondrodysplasia (BOCD). OBJECTIVE: BOCD can be divided into two types, depending on the severity of the skeletal abnormalities. The molecular basis for this heterogenic presentation is unknown. DESIGN AND PATIENTS: We performed mutation analysis in two families with type I and in three families with the less severe form of BOCD type II. RESULTS: In one of the type I BOCD cases, a homozygous nonsense mutation (R104X) was found, resulting in a truncated PTHR1. In the second type I BOCD case, no mutation was found. A homozygous nucleotide change (intron M4+27C>T) was demonstrated in one of the type II BOCD cases creating a novel splice site. In dermal fibroblasts of the patient, this novel splice site was preferentially used, resulting in an aberrant transcript. The wild-type transcript remained, however, present, albeit at low levels. In the other two families with type II BOCD, a previously identified homozygous missense mutation (P132L) was found. Functional analysis demonstrated that the P132L mutant had low residual activity. CONCLUSIONS: In combination with data presented in literature, we conclude that type I BOCD is caused by a complete inactivation of the PTHR1, whereas low levels of residual activity due to a near complete inactivation of the PTHR1 result in the relatively milder presentation of type II BOCD.

[Efficacy of agreements within the Enchede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation]. / Effectiviteit van afspraken binnen de Enschedese stroke-service om patiënten met een beroerte adequaat te verwijzen van de stroke-unit in het ziekenhuis naar een verpleeghuis voor kortdurende reactivering.

OBJECTIVE: To assess the efficacy of agreements within the Enschede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation. DESIGN: Prospective, partly retrospective. METHOD: All patients who were referred from the stroke unit at Medisch Spectrum Twente to the CVA Rehabilitation Unit (CRU) in the period 1 July 1999-31 July 2003 were included. Referral took place via an active multidisciplinary approach and specific referral agreements. The primary outcome was the number of patients that could be discharged home after rehabilitation. In addition, we assessed the influence on final discharge destination of age, the Barthel and Rankin scores at the time of admission to the CRU and the medical complications during the period of rehabilitation. RESULTS: 232 patients were included (133 women and 99 men, mean age 76.4 years). Within 3 months, 63% of the patients were discharged home. After 6 months, 82% had returned home. 8% of the patients died within 6 months and 9% had to stay in a nursing home permanently. Of the patient aged 80 years or older, 75% could return home within 6 months. Patients with poor Barthel and Rankin scores and medical complications had a smaller chance of being discharged home. CONCLUSION: Effective referral of patients from the stroke unit to a nursing home for short-term rehabilitation is possible. With adequate patient selection, the use of good referral agreements and multidisciplinary consultations, most patients could finally return home.

Accurate differentiation of parkinsonism and essential tremor using visual assessment of [123I]-FP-CIT SPECT imaging: the [123I]-FP-CIT study group.

OBJECTIVE: To evaluate whether visual assessment of [123I]-FP-CIT (DaTSCAN, Nycomed Amersham, plc) single photon emission computerized tomography (SPECT) images can differentiate between parkinsonism and essential tremor (ET). METHODS: [123I]-FP-CIT SPECT imaging was conducted in a six-center study of 158 patients with a clinical diagnosis of parkinsonism compared with 27 ET cases and 35 healthy volunteers. Striatal uptake of the radioligand was graded normal or abnormal, and abnormal images were further graded to three levels of severity. An institutional read whereby each center visually assessed the images blinded to the clinical data and a consensus blinded read by a panel of five was undertaken. RESULTS: The institutional reading scored 154 of 158 cases of parkinsonism abnormal, all 27 cases of ET as normal, and 34 of 35 healthy volunteers as normal compared with the consensus blinded read scoring 150 cases of parkinsonism as abnormal, 25 ET cases as normal, and 33 healthy volunteers as normal. Sensitivity for the clinical diagnosis of parkinsonism was 97% and specificity for ET was 100% for the institutional read, whereas sensitivity was 95% and specificity 93% for the consensus blinded read. Semiquantitative analysis of specific: nonspecific caudate and putamen uptake were consistent with the results of visual inspection. CONCLUSION: Visual assessment of [123I]-FP-CIT SPECT images is an easily applied diagnostic test which is helpful in the differential diagnosis of tremor disorders and in confirming a clinical diagnosis of a hypokinetic-rigid syndrome.

A frame-shift mutation in the type I parathyroid hormone (PTH)/PTH-related peptide receptor causing Blomstrand lethal osteochondrodysplasia.

Blomstrand osteochondrodysplasia (BOCD) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. Comparison of the characteristics of BOCD with type I PTH/PTH-related peptide (PTHrP) receptor-ablated mice reveals striking similarities that are most prominent in the growth plate. In both cases, the growth plate is reduced in size due to a strongly diminished zone of resting cartilage and the near absence of columnar arrangement of proliferating chondrocytes. This overall similarity suggested that an inactivating mutation of the PTH/PTHrP receptor might be the underlying genetic defect causing BOCD. Indeed, inactivating mutations of the PTH/PTHrP receptor have been recently identified in two cases of BOCD. We describe here a novel inactivating mutation in the PTH/PTHrP receptor. Sequence analysis of all coding exons of the type I PTH/ PTHrP receptor gene and complementary DNA of a case with BOCD identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The mutation was inherited from both parents, supporting the autosomal recessive nature of the disease. The missense mutation resulted in a shift in the open reading frame, leading to a truncated protein that completely diverged from the wild-type sequence after amino acid 364. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7; the connecting intra- and extracellular loops; and the cytoplasmic tail. Functional analysis of the mutant receptor in COS-7 cells and of dermal fibroblasts obtained from the case proved that the mutation was indeed inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts responded to a challenge with PTH or PTHrP with a rise in intracellular cAMP levels, in sharp contrast to control cells. Our results provide further evidence that BOCD is caused by inactivating mutations of the type I PTH/PTHrP receptor and underscore the importance of this receptor in mammalian skeletal development.

Interleukin-17: A new bone acting cytokine in vitro.

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

IL-1alpha, IL-1beta, IL-6, and TNF-alpha steady-state mRNA levels analyzed by reverse transcription-competitive PCR in bone marrow of gonadectomized mice.

Loss of gonadal function in both females and males is associated with increased rates of bone loss by a yet unidentified mechanism. There is ample evidence that cytokines that are produced in the bone microenvironment and stimulate the activity and/or formation of osteoclasts are involved. In the present study, we examined whether gonadectomy increases cytokine production via increased transcription in the bone marrow of mice. For this, the in vivo steady-state mRNA levels of multiple cytokines were determined in the central bone marrow compartment of mice at different time points following ovariectomy or orchidectomy by reverse transcription-competitive polymerase chain reaction. The limit of detectable differences in mRNA expression was approximately 2-fold. Bone marrow mRNA levels of the cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) were elevated up to 30-fold after treatment of mice with lipopolysaccharide. Following gonadectomy, there were no differences in the mRNA expression of these cytokines in bone marrow of female and male mice 4, 7, and 14 days after surgery. Gender steroid deficiency does not, therefore, increase steady-state mRNA levels of IL-1alpha, IL-1beta, IL-6, and TNF-alpha in cells of the central bone marrow compartment in mice. If changes have occurred these should have been less than 2-fold or in a small cell population. These results do not preclude an important role of these cytokines in the induction of bone loss after gonadectomy. For example, bone marrow cells situated close to the bone surface or bone cells may be responsible for increased cytokine synthesis. Alternatively, the loss of gender steroids may alter post-transcriptional events in cytokine synthesis and activity or may modify the responsiveness of target cells.

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Pulsatile intravenous insulin replacement in streptozotocin diabetic rats is more efficient than continuous delivery: effects on glycaemic control, insulin-mediated glucose metabolism and lipolysis.

Short-term exposure of tissues to pulses of insulin generally leads to an enhancement of insulin action. We have investigated the possible beneficial effects of long-term near-physiological continuous vs pulsatile intravenous insulin treatment of insulin-deficient streptozotocin (70 mg/kg) diabetic rats on blood glucose control, in vivo insulin action and in vitro insulin action in isolated adipocytes. First, we determined the 24-h peripheral plasma insulin profiles in normal rats under precisely controlled mealfeeding conditions. Basal plasma insulin levels (40 +/- 9 microU/ml) oscillate with a periodicity of 11.9 +/- 0.9 min (p < 0.05), and an amplitude of 60 +/- 10%. Subsequently, the 24-h insulin profile was mimicked in diabetic (D) rats by a continuous (c) or pulsatile (p) (6-min double, 6-min off) insulin infusion rate for 2 weeks, using a programmable pumpswivel unit. Control (C) rats received vehicle treatment. In Cc, Dc, Cp and Dp daily urinary glucose loss and average plasma glucose levels were 0 +/- 0, 7.5 +/- 4.4, 0 +/- 0, 0.8 +/- 0.4 mmol and 6.7 +/- 0.2, 11.5 +/- 2.7, 6.6 +/- 0.1, 5.9 +/- 1.4 mmol/l, respectively. Hypoglycaemia (< 3 mmol/l) was observed in 10 and 20% of the blood samples collected from Dc and Dp rats, respectively. After 2 weeks of treatment, in vivo peripheral and hepatic insulin action was measured by the hyperinsulinaemic euglycaemic (6 mmol/l) clamp with [3-3H]-glucose infusion. Pre-clamp counter-regulatory hormone levels were similar among rats. Compared to Cc and Cp, Dc showed a reduction in insulin sensitivity and responsiveness for peripheral glucose uptake whereas Dp only showed a reduction in insulin sensitivity. Suppression of hepatic glucose production by insulin was similar among rats. After 2.5 weeks of treatment, epididymal adipocytes were isolated. Specific [125I]-insulin binding, basal and insulin-stimulated [U-14C]-glucose uptake and isoproterenol-stimulated glycerol output were comparable among rat adipocytes. The inhibition of glycerol output by insulin was identical in Cp and Dp (V(max) = 48.6 +/- 6.1 and 42.3 +/- 4.6%) but blunted in Dc vs Cc (V(max) = 8.2 +/- 4.6 vs 44.0 +/- 7.2%, p < 0.01) adipocytes, suggesting a post-binding defect in the antilipolytic action of insulin in Dc rats. In conclusion, long-term near-physiological pulsatile intravenous insulin replacement in insulin-deficient diabetic rats is more efficient than continuous delivery in reducing blood glucose, lowering glucosuria, increasing insulin sensitivity and inhibiting lipolysis.

Tissue-related changes in insulin receptor number and autophosphorylation induced by starvation and diabetes in rats.

Insulin action is subject to regulation at the level of the insulin receptor and at postreceptor levels. Starvation and diabetes are often associated with insulin resistance for glucose metabolism in various tissues. In muscle, fat, and liver, we examined whether changes in the functionality of the insulin receptor correlated with changes in insulin action in the starved and diabetic state. Insulin-stimulated receptor autophosphorylation reflects an early physiologic step in transmission of the insulin signal, and for that reason, changes in autophosphorylation activity of the insulin receptor were used as a marker to determine the functionality of the insulin receptor. Glycoprotein fractions prepared from skeletal muscle, diaphragm, epididymal fat, and liver of control, 3-day starved, short-term 3-day (S) diabetic (streptozotocin, 70 mg/kg intravenously), and long-term 6-month (L) diabetic (neonatal streptozotocin 100 micrograms/g intraperitoneally) rats were used in this study. Receptor activity was monitored by measuring insulin-stimulated [gamma-32P]adenosine triphosphate (ATP) receptor autophosphorylation. In addition, to obtain information about whether changes in receptor autophosphorylation are related to changes in receptor number, relative numbers of high-affinity insulin receptors were determined by affinity cross-linking of [125I]insulin to the receptor alpha-chain and quantitation of the yield of labeled receptor alpha-chain. Control, starved, S diabetic, and L diabetic rats had plasma insulin and glucose levels of 294 +/- 42, 90 +/- 24, 48 +/- 12, and 216 +/- 30 pmol/L and 6.7 +/- 0.2, 4.1 +/- 0.2, 23.3 +/- 0.7, and 21.6 +/- 2.9 mmol/L, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Measuring right-hemisphere dysfunction in children: validity of two new computer tests.

The validity of two new computer-mediated tests for the detection of right-cerebral hemisphere lesions in children--the Right-hemisphere Dysfunction Test and the Visual Perception Test--was evaluated. Normative data were drawn from a group of 91 children (aged five to 14 years) and 14 young adults. The tests were also administered to 14 children with acquired lesions of either right- or left-cerebral hemisphere. The results demonstrate that the Right-hemisphere Dysfunction Test and the Visual Perception Test, with predictive values of 71 per cent and 88 per cent, respectively, are useful in clinical practice for detection of right-hemisphere dysfunction in children.

Whole body and hepatic insulin action in normal, starved, and diabetic rats.

In normal (N), 3-days starved (S), and streptozotocin-treated (65 mg/kg) 3-days diabetic (D) rats we examined the in vivo dose-response relationship between plasma insulin levels vs. whole body glucose uptake (BGU) and inhibition of hepatic glucose production (HGP) in conscious rats, as determined with the four-step sequential hyperinsulinemic euglycemic clamp technique, combined with [3-3H]glucose infusion. Twelve-hour fasting (basal) HGP was 3.0 +/- 0.2, 2.1 +/- 0.2, and 5.4 +/- 0.5 mg/min in N, S, and D rats, respectively. Next, all rats were clamped at matched glycemia (6 mM). Lowering plasma glucose in D rats from +/- 20 to 6.0 mM did not increase plasma norepinephrine, epinephrine, glucagon, and corticosterone levels. For BGU, insulin sensitivity was increased (70 +/- 11 microU/ml) in S and unchanged (113 +/- 21 microU/ml) in D compared with N rats (105 +/- 10 microU/ml). Insulin responsiveness was unchanged (12.4 +/- 0.8 mg/min) in S and decreased (8.5 +/- 0.8 mg/min) in D compared with N rats (12.3 +/- 0.7 mg/min). For HGP, insulin sensitivity was unchanged (68 +/- 10 microU/ml) in S and decreased (157 +/- 21 microU/ml) in D compared with N rats (71 +/- 5 microU/ml). Insulin responsiveness was identical among N, S, and D rats (complete suppression of HGP). In summary, 1) insulin resistance in D rats is caused by hepatic insensitivity and by a reduction in BGU responsiveness. 2) S rats show normal hepatic insulin action, but insulin sensitivity for BGU is increased. Therefore, S and D rats both suffering from a comparable catabolic state (10-15% body wt loss in 3 days) show opposite effects on in vivo insulin action. This indicates that in vivo insulin resistance in D rats is not caused by the catabolic state per se.

Antilipolytic action of insulin in adipocytes from starved and diabetic rats during adenosine-controlled incubations.

Insulin action on adipocytes induces two major metabolic effects: stimulation of glucose transport and inhibition of lipolysis. Previously, we have shown that incubated isolated adipocytes from starved (S), and streptozotocin-treated diabetic (D) rats show insulin resistance on glucose transport. It is not known whether insulin resistance is also present on antilipolysis. In this study the antilipolytic action of insulin was investigated. Since basal lipolysis was low, lipolysis was first stimulated by isoproterenol (ISO). This showed that differences existed in sensitivity for ISO among control (C), S, and D adipocytes. We investigated whether changes in adenosine accumulation could attribute to the differences in ISO action and thereby influence insulin action. When endogenous accumulating adenosine was removed by adenosine deaminase and replaced by a fixed concentration (200 nM) of the nonhydrolyzable adenosine analog phenylisopropyladenosine, the differences in ISO action disappeared. This indicates that the sensitivity of C, S, and D adipocytes for ISO is strongly influenced by endogenous adenosine release. The dose-response relationship between insulin and inhibition of ISO-stimulated lipolysis showed that insulin sensitivity was increased and responsiveness unaltered in S and D compared to C adipocytes for incubations with both uncontrolled and controlled adenosine concentrations. This indicates that during S and D states, endogenous adenosine release has no major effect on insulin action. The increased sensitivity for insulin of S and D adipocytes was paralleled by an increased binding of [125I]iodoinsulin. The unaltered responsiveness for insulin indicates that there is no insulin resistance at the postbinding level for antilipolysis, i.e. intracellular processes for antilipolysis are intact. This is in contrast to glucose transport, where insulin resistance exists at the postbinding level during S and D. Thus, insulin resistance is no general phenomenon, but is confined to specific effector systems.

Enhanced plasminogen activator production of Syrian hamster embryo cells transformed by chemicals or the c-Ha-ras oncogene: type of plasminogen activators involved and their contribution to the transformed phenotype.

The plasminogen activator (PA) activity produced by Syrian hamster embryo (SHE) cells in different stages of neoplastic conversion was analysed. PA activity was characterized immunologically and by SDS-PAGE. Normal SHE cells had a very low PA activity. Although activity of either the tissue type of PA (t-PA) or the urokinase type (u-PA) or both were found to be increased in most immortal or transformed SHE cells, there was no correlation between enhanced production of a particular PA type and the development of the immortal or transformed phenotype. However, within a group of cell lines clonally derived from a culture of immortal cells, a positive correlation was found between extracellular t-PA, but not u-PA, activity and cellular growth rate. For the Syrian hamster PA species, crossreacting with anti-human u-PA, a mol. wt of 39 kd was observed. For the Syrian hamster PA species, crossreacting with anti-human t-PA, multiple species were found with mol. wts of 98, 72 and 59 kd respectively. Evidence was obtained that the 72-kd species represents the intact enzyme, the 59-kd species a partial digestion product thereof and the 98 kd species, which often appears as a doublet, a complex of either of these species with an inhibitor, likely to be secreted by the same cells. Finally, our data suggest a novel mechanism for the enhancement of t-PA activity of transformed cells, namely by a decrease in the effective extracellular amount of putative inhibitor.

Prolonged in vitro exposure of Syrian hamster embryo cells to 3-aminobenzamide induces transformation and chromosomal alterations but not gene mutations.

To investigate the genetic mechanisms involved in morphological transformation of Syrian hamster embryo (SHE) cells, we have studied the transforming potential of 3-aminobenzamide (3-AB). It was found that prolonged exposure to 3-AB induced morphological transformation of SHE cells as well as C3H10T 1/2 cells. At similar doses, 3-AB induced SCEs and chromosomal alterations (gaps, breaks and exchanges) in SHE cells, but no mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. These data strongly suggest morphological transformation can result from genetic alterations other than gene mutations. The possible nature of these genetic alterations involved will be discussed.

Fibroblasts from a leprechaun patient have defects in insulin binding and insulin receptor autophosphorylation.

Leprechaunism is an inherited human disorder associated with an extreme resistance of the target cells towards the action of insulin. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient (Geldermalsen, the Netherlands). In vitro, severe insulin resistance is reflected by a low level of insulin stimulated uptake of 2-deoxyglucose by these fibroblasts. This defect seems to be caused by a combination of two factors: a low level of insulin binding to intact cells and a strong decrease of insulin stimulated autophosphorylation of the receptor. The stimulation of autophosphorylation by insulin was approximately six-fold in control subjects and less than two-fold in the patient. No abnormalities were observed in the total number of insulin receptors in these cells and the molecular weights of the receptor subunits. In addition, the insulin concentration required for half maximal autophosphorylation is similar for the solubilised receptor from control and patient fibroblasts.

Epidermal growth factor enhances N-ethyl-N-nitrosourea-induced morphological transformation of Syrian hamster embryo cells.

To improve the usefulness of the Syrian hamster embryo (SHE) cell transformation system as a short-term test, it was investigated whether the variation in results due to serum variability could be reduced by the addition of epidermal growth factor (EGF). It was found that EGF-significantly (3-7-fold) enhanced the frequency of morphological transformation induced by N-ethyl-N-nitrosourea or benzo[a]pyrene if added to growth medium supplemented with a batch of serum which had a low ability to support transformation. Furthermore, addition of EGF to the assay medium enabled the demonstration of dose dependence of transformation with relatively small group sizes (up to 2000 colonies). Finally, it was observed that the transformed phenotype was easier to recognize in the presence of EGF. These data suggest that routine addition of EGF to the assay medium might reduce variability and enhance sensitivity of the SHE transformation assay.

Improper expression of insulin receptors on fibroblasts from a leprechaun patient.

Leprechaunism is an inherited human disorder characterized by severe insulin resistance. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient. In vitro, severe insulin resistance is reflected by a low level of insulin binding to the patients fibroblasts and impaired insulin-mediated uptake of 2-deoxyglucose. Quantification of the receptor in detergent-solubilized total glycoprotein indicates a normal receptor number, in agreement with the observed normal level of insulin receptor mRNA on northern blots. The insulin-stimulated autophosphorylation of the patient's receptor shows a normal profile. The insulin receptor is present on the plasma membrane as indicated by cell-surface iodination experiments. No abnormalities in the molecular masses of the receptor's alpha and beta chains were observed. The results indicate that an apparently normal receptor is synthesized in sufficient amounts but functional expression of the receptor on the plasma membrane is impaired.