Biomedical subjects
Publications and source records attributed to S Werner.
We have cloned and sequenced cDNAs coding for two subunits of the peripheral arm of Neurospora crassa complex I. The two polypeptides are synthesized as precursor proteins which are processed to mature forms with predicted molecular masses of 24331 and 20982 Da.
Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2 during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud epithelium in transgenic mice. Newborn mice expressing the transgene were completely normal except that instead of normally developed lungs they had two undifferentiated epithelial tubes that extended from the bifurcation of the trachea down to the diaphragm, a defect that resulted in perinatal death. Thus, the dominant negative FGF receptor completely blocked airway branching and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in branching morphogenesis of the mammalian lung.
Fibroblast growth factors (FGF) regulate the growth and differentiation of cells through complex combinatorial signaling pathways. There are nine ligands that interact with a family of four tyrosine kinase FGF receptors (FGFR). Diversity in FGF signaling is determined in part by the affinity of specific ligand-receptor pairs. Alternative splicing in the FGFR ligand binding domain generates additional receptor isoforms with novel ligand affinities. For example, splicing events in the ligand binding domain of FGFR2 dramatically increases its affinity for keratinocyte growth factor (KGF/FGF-7). We have identified an alternatively spliced form of the FGFR3 mRNA, corresponding to known splice variants of FGFRs 1 and 2. We demonstrate both by binding studies on genetically engineered soluble receptors and by the mitogenic response of growth factor-dependent cell lines that this splice variant of FGFR3 (FGFR3 IIIb), by binding only acidic FGF (aFGF/FGF-1), has the most restricted ligand binding properties of any FGFR thus far described. Furthermore, by constructing a chimeric receptor that contains the homologous exon from FGFR2, we demonstrate that this single domain from FGFR2 is sufficient to confer upon FGFR3 the ability to bind KGF/FGF-7. The uniquely limited repertoire of ligands that interact with this receptor suggests that a novel ligand for FGFR3 IIIb exists.
A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.
To investigate the potential role of drug therapy in preventing or exacerbating seizure-related brain injury in the prepubescent brain, we administered kainic acid to rats at postnatal day 35. Therapy with daily phenobarbital was started directly before or 1 day after kainic acid was administered, and was continued through postnatal day 153. Rats receiving phenobarbital had therapeutic concentrations during most of the 24-hour dosing period, but also experienced supratherapeutic peak concentrations. The animals were subsequently tested using the water maze (a measure of visuospatial memory), open field (a measure of activity level), and handling tests (a measure of emotionality). The frequency of spontaneous recurrent seizures was monitored during and after phenobarbital therapy. Kainic acid resulted in status epilepticus on postnatal day 35 in all the rats that received it but those receiving phenobarbital first manifested a shorter and less severe status epilepticus as compared to the rats given kainic acid alone. Rats starting phenobarbital immediately before kainic acid was administered did not differ from control rats on behavioral testing and had no subsequent spontaneous recurrent seizures and no histological lesions. Rats receiving kainic acid alone performed significantly poorer than did control rats in the water maze, were more aggressive, had histological lesions, and manifested spontaneous recurrent seizures. As compared to the group treated only with kainic acid, rats receiving kainic acid followed by phenobarbital at postnatal days 36 to 153 manifested similar aggressiveness and histological lesions, similar frequency of spontaneous recurrent seizures after phenobarbital taper, and even greater disturbances in memory, learning, and activity level.(ABSTRACT TRUNCATED AT 250 WORDS)
Extensive investigation including whole-body examinations with computed tomography and magnetic resonance imaging did not detect the suspected ectopic ACTH-producing tumour in a patient with advanced Cushing's syndrome and hypokalemic alkalosis. Gamma camera examination with technetium-99m methoxyisobutylisonitrile (MIBI) depicted the tumour, which was localized in the anterior neck and mediastinum. This was later verified by surgery. 99mTc-MIBI is normally used for myocardial scintigraphy. Its accumulation is unspecific and merely reflects metabolic activity. Despite this, the present case shows that examination with this agent can provide important information with regard to tumour localization in a given situation, thereby serving as a complement to other imaging modalities. The current literature on 99mTc-MIBI for tumour diagnosis is reviewed.
OBJECTIVE: To investigate secretory leukocyte protease inhibitor (SLPI) concentrations in tracheal lavage fluids of neonates with an endotracheal tube in place during the first month of life, and to evaluate the relationship of SLPI to neutrophil counts and elastase activity in patients in whom bronchopulmonary dysplasia (BPD) developed versus those in whom it did not. DESIGN: A prospective, inception cohort study. SETTING: University children's hospital neonatal intensive care unit. PATIENTS: Fifty-three neonates who weighed < 2000 gm at birth, and who had an endotracheal tube in place, were enrolled. Forth-one patients survived to 28 days; BPD developed in 24 but not in 17 patients. MAIN OUTCOME MEASURES: Tracheal lavage was performed on days 1, 2, 4, 7, 14, 21, and 28, and analyzed for neutrophils, elastase activity, and SLPI. Results were evaluated longitudinally for 28 days, and were compared between BPD and no-BPD groups during the first week. RESULTS: SLPI concentrations increased significantly for all patients during the study period. During the first week, SLPI concentrations were similar between BPD and no-BPD groups; neutrophil counts and elastase activity were higher in the BPD group. CONCLUSIONS: Patients in whom BPD ultimately developed had early evidence of increased pulmonary inflammation and a significantly less favorable protease-antiprotease balance. If elastase-induced injury contributes to the development of BPD, early therapy with recombinant SLPI might be beneficial by increasing the antielastase capacity of epithelial lining fluid.
We have recently demonstrated induction of expression of several members of the fibroblast growth factor family during wound healing, particularly for keratinocyte growth factor, which was more than 150-fold induced within 24 h after injury. To assess whether wound-healing disorders are associated with a defect in fibroblast growth factor regulation, we have now investigated the expression of these mitogens as well as their receptors in normal and wounded skin of genetically diabetic db/db mice, which are characterized by their impaired wound healing. We demonstrate that induction of keratinocyte growth factor expression in these mice is significantly reduced and delayed compared to normal mice. Induction of acidic fibroblast growth factor (FGF) and basic FGF expression was earlier in diabetic mice than in normal mice, but by 3 d after injury expression of these mitogens had already returned to the basal levels. In contrast, elevated levels of acidic FGF and basic FGF transcripts were detected within the first 5 d in wounds from normal mice. Thus, FGFs seem to be expressed in a limited fashion in the wound tissue of db/db mice during the period when re-epithelialization and granulation tissue formation normally occur. These findings provide an explanation for the beneficial effect of exogenous FGF in the treatment of impaired wound healing in these animals and suggest that induction of KGF early in repair may be critical for the rapid re-epithelialization in normal wound healing.
OBJECTIVE: Previous reports have shown an inverse relation between IGFBP-1 and insulin levels in healthy men, patients with insulin dependent diabetes mellitus, and insulinoma. We have investigated whether this inverse relation also exists in acromegaly, before and during treatment with octreotide, and whether changes in IGFBP-1 levels relate to GH and IGF-I levels. DESIGN: We studied short-term treatment with octreotide in a double-blind placebo-controlled 14-day clinical trial. PATIENTS: Eighteen patients with acromegaly were studied. MEASUREMENTS: Plasma GH and serum IGFBP-1 levels were measured at hourly intervals from 0700 to 1800 h before randomization (day 0) and on days 4, 6, 8, 14 and 20. Serum insulin was determined at 0700, 0800 and 0900 h, and serum IGF-I at 0700 h. RESULTS: Octreotide increased the daily mean IGFBP-1 level by 43% on day 8 and by 35% on day 14. The IGFBP-1 levels during octreotide were significantly higher (P < 0.05) compared to placebo, 29.9 +/- 3.9 vs 19.9 +/- 1.7 micrograms/l (mean +/- SEM) on day 8, and 28.3 +/- 3.2 vs 19.9 +/- 1.6 micrograms/l on day 14. Octreotide treatment significantly suppressed the insulin levels on all observation days by 40-48% compared to placebo. There was a significant inverse correlation between IGFBP-1 levels and insulin levels, both before treatment and on the last day of treatment (r = 0.79, P = 0.04; r = -0.90, P = 0.02, respectively). GH and IGF-I were significantly decreased in the octreotide group compared to the placebo group during the entire treatment period. The mean of age related standard deviation scores of IGF-I in the octreotide group decreased from a pretreatment value of 6.47 +/- 0.74 to 3.60 +/- 1.20 on day 14. There was no significant correlation between IGFBP-1 levels and levels of GH and IGF-I, either before or during treatment. CONCLUSIONS: Octreotide treatment, in addition to reducing GH, IGF-I and insulin levels, is associated with an increase in IGFBP-1 concentrations in patients with acromegaly, and it is suggested that the rise in serum IGFBP-1 is a consequence of the decrease in insulin secretion.
OBJECTIVE: Several studies suggest that the combination of octreotide and bromocriptine is more effective than octreotide alone in reducing GH levels in patients with acromegaly. However, these studies have evaluated either the acute effects of single doses of octreotide and bromocriptine, or the effects of long-term combination therapy, in which octreotide was given only twice a day. The aim of the study was to evaluate the effects of the combination of octreotide and bromocriptine compared to octreotide alone, using a treatment scheme of three daily injections of octreotide during a period long enough to ensure a pharmacokinetic and pharmacodynamic steady state. PATIENTS: Eleven patients with acromegaly. DESIGN AND MEASUREMENTS: Different treatment regimes were performed during three periods. During the first period there was no administration of octreotide or bromocriptine, during the second period 100 micrograms octreotide was given subcutaneously three times a day, and during the third period 100 micrograms octreotide was given subcutaneously three times a day in combination with bromocriptine, given orally 5 mg twice daily. When the patients were without any treatment a single oral dose of 5 mg bromocriptine was given at 0730 h. Individual GH levels were assessed as the mean value of 11 observations during the day, at hourly intervals from 0730 to 1530 h, and at 1730 and 1930 h, and the GH levels for the whole group were calculated as the mean of the individual mean values. Serum IGF-I and PRL were measured in fasting samples at 0730 h on the same days as GH, GH, IGF-I, and PRL were evaluated at the end of each treatment regime. RESULTS: Basal mean GH value for all 11 patients was 54.2 +/- 17.4 mU/l. During octreotide therapy mean GH value was significantly reduced compared to basal mean GH level; 19.86 +/- 6.82 mU/l (P < 0.05). The reduction of mean GH during combination therapy was also significant compared to basal, 18.70 +/- 6.72 mU/l (P < 0.05). Basal mean IGF-I value for all 11 patients was 716 +/- 96 micrograms/l. During octreotide therapy mean IGF-I value was significantly reduced compared to basal mean IGF-I level; 458 +/- 100 micrograms/l (P < 0.05). The reduction of mean IGF-I during combination therapy was also significant compared to basal, 456 +/- 93 micrograms/l (P < 0.05). There was no difference between the levels of mean IGF-I during the two treatment periods. One patient, the only patient with hyperprolactinaemia, showed an additional reduction of GH levels of > 50% during combination therapy. This was also the patient showing the most pronounced reduction of GH levels after the administration of a single dose of bromocriptine. CONCLUSION: When octreotide is administered three times a day, the additive effect of bromocriptine on GH and IGF-I suppression seems to be negligible in most patients with acromegaly.
Human growth hormone (hGH) was analyzed by six monoclonal antibodies (Mabs) and a polyclonal antiserum (Pas) before and after molecular sieve chromatography of sera from healthy subjects. Their hGH levels were between < 0.2 and 0.4 ng/ml as determined with Pas. The six Mabs reacted with five distinct epitopes and bound to a hGH fragment corresponding to the amino acid sequence 15-125. Two of the Mabs showed reduced binding to 20-kD hGH. The binding of Mabs to dimeric forms of hGH varied. Human GH levels in unfractionated sera as determined with Mabs were < 3.1-390 ng/ml. After molecular sieve chromatography of the sera, one peak of hGH-immunoreactive material of high molecular weight (> 160 kD) and one at the elution volume of monomeric hGH were determined with Pas and Mabs. The major part of the high molecular weight hGH (> 160 kD) seemed to consist of 22-kD hGH molecules, since Pas and all Mabs detected the hGH immunoreactivity (> 160 kD) in a similar manner. This high molecular weight hGH (> 160 kD) was distinguishable from the identified, receptor-like hGH-binding protein in serum.
Pituitary growth hormone (GH), prolactin (PRL), placental lactogen (PL), insulin-like growth factor 1 (IGF-1) and GH-binding protein (GHBP) in plasma were determined in 12 women with gestational diabetes mellitus (GDM) and in 12 healthy pregnant women during a breakfast meal tolerance test. The women with GDM showed higher prepregnancy weight, body mass index (BMI), basal levels of glucose, insulin, and C peptide compared to the pregnant controls. No difference was found between the two groups in pituitary GH, PRL, PL and IGF-1 levels. Plasma levels of GHBP were higher in the women with GDM compared to pregnant controls. In all women there was an inverse correlation between PL and pituitary GH as well as between PL and GHBP, suggesting that PL inhibits pituitary GH secretion. A positive correlation between GHBP and BMI was found in all women, and the higher BMI in the GDM women seemed to be the cause of the higher GHBP levels in this group. In all women IGF-1, an indicator of the secretory activity of lactogenic hormones as well as of nutritional state, showed a positive correlation with the birth weights of the infants and was equally indicative in both groups.
Dopamine, acting via its specific receptor (DRD2) in the anterior pituitary, tonically inhibits pituitary prolactin secretion and lactotroph proliferation. In addition, dopamine agonist therapy for pituitary prolactinomas results in reduction of prolactin secretion and tumor regression. These observations lead to the speculation that functional dopamine uncoupling may release lactotrophs from the inhibitory effects of dopamine and contribute to the development of prolactin (PRL)-secreting pituitary tumors. We hypothesized that such an uncoupling may occur by inactivating mutation(s) of the DRD2. To test our hypothesis, we examined 79 pituitary tumors, mostly prolactinomas and mixed GH/PRL-secreting, for mutations in the coding exons of the DRD2 gene. We used the polymerase chain reaction and analyzed the fragments for migration abnormalities on denaturing gradient gel electrophoresis, complemented by direct DNA sequencing. No mutations were demonstrated, and all migration abnormalities detected by denaturing gradient gel electrophoresis were due to polymorphisms within the DRD2 gene. In addition, allelic losses in the multiple endocrine neoplasia type 1 region in 11q13 could not be demonstrated in all five informative prolactinomas. We conclude that mutations in the DRD2 gene do not occur in PRL or GH/PRL-secreting pituitary tumors and that allelic loss of 11q13 is uncommon in prolactinomas.
A new test apparatus for the testing of artificial heart valves makes it possible to realize both physiological flow through the valve during the flow period, and physiological pressure differences across the artificial valve during the closing phase, both for aortic and mitral valve positions, with freely selectable stroke volume and heart frequency. The major hydraulic parameters of the valve are measured directly (pressure drop, closing volume and closing time, leakage volume) or are calculated (e. g. energy loss). In association with the demonstration of the suitability of the HKP+ test apparatus for the measurement of hydraulic parameters in accordance with ISO 5840, the hydraulic properties of various types and sizes of valve with respect to pressure drop (resistance), together with their closing behaviour and leakage flow, are described for different fluids, and the correlations shown.
We have recently demonstrated a large induction of keratinocyte growth factor expression in dermal fibroblasts during wound healing. To identify possible mediators of KGF induction, we have now analysed the regulation of KGF expression in vitro in cultured murine and human fibroblasts. Here we demonstrate that KGF mRNA and protein expression is low in quiescent fibroblasts but is strongly induced upon serum stimulation. This induction can be mediated by at least two different intracellular pathways involving protein kinase C or cAMP-dependent kinases. Our finding that induction of KGF expression by serum is independent of de novo protein synthesis demonstrates, that KGF is the product of a primary response gene. The stimulatory effect of serum on KGF expression is likely to be a combinatorial effect of different mitogens, since several purified serum growth factors also stimulated KGF expression but to a lesser extent compared to serum. Furthermore, we also found a strong KGF induction by interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6, cytokines which are released by polymorphonuclear leukocytes and activated macrophages during wound healing. These data suggest that serum which is released upon hemorrhage as well as pro-inflammatory cytokines might be responsible for the KGF induction in vivo during skin repair.
We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as the endogenous subunit, indicating that correct assembly in the mitochondrial inner membrane did occur.
The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) is a nuclear-coded protein of the hydrophobic fragment of the enzyme. We have isolated and sequenced a full-length cDNA clone coding for this polypeptide. The deduced protein is 104 amino acid residues long with a molecular mass of 12305 Da. This particular subunit of complex I lacks a cleavable mitochondrial targeting sequence. In agreement with its localization within complex I, we have found that this subunit behaves like an intrinsic membrane protein. Nevertheless, the deduced protein is rather hydrophilic, exhibiting no hydrophobic domain long enough to traverse a membrane in an alpha-helical conformation. The 12.3 kDa subunit shows a significant similarity to the hinge protein of complex III, suggesting that these two polypeptides may be involved in identical functions. This complex I subunit is coded for by a single gene. Applying restriction-fragment-length-polymorphism mapping, we located the gene on the right side of the centromere in linkage group I, linked to the lys-4 locus.
In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.