A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein.

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.

Cloning and functional characterization of the chicken c-ros promoter.

Our previous study has shown that chicken c-ros is specifically expressed in certain epithelial cells of kidney, intestine, lung, bursa, thymus, and testis, and the expression is regulated temporally and spatially. To explore the molecular basis for the regulation of c-ros expression, we have cloned and characterized the chicken c-ros promoter. The most 5' c-ros cDNA was isolated and sequenced. Using the 5' cDNA as a probe, three genomic DNA clones containing the 5' c-ros cDNA sequence were isolated. Primer extension and RNase protection analysis were used to map the transcription initiation site for the c-ros mRNA in kidney and intestine. The sequence of the 1.3-kb region upstream of the initiation site contains TATA and CAAT boxes at 26 and 54 nucleotides, respectively, upstream of the initiation site. In addition, transcription factor binding sites for AP1, AP2, and Oct1 and several direct and inverted repeats are present within 1 kb upstream of the initiation site. The 1.3-kb DNA, when placed upstream of the chloramphenicol acetyltransferase gene, was shown to be functionally active. Serial deletions of this putative c-ros promoter allowed us to define a minimum c-ros promoter and to identify positive and negative regulatory regions. Using two oligonucleotides corresponding to a positive regulatory and potential factor binding region, we have demonstrated, by gel mobility shift experiments, their specific binding to nuclear extracts from kidney, intestine, and thymus. The binding pattern corresponds to the tissue specificity and temporal control of c-ros mRNA expression.

Effects of insulin-like growth factor I or human growth hormone in fasted rats.

To study the effect of insulin-like growth factor I (IGF-I) treatment on growth and metabolism in fasted rats and compare it with the effect of human growth hormone (hGH), we infused 120 micrograms/ml IGF-I continuously or injected 200 micrograms hGH twice a day in fasted rats. After a 3 1/2-day administration of IGF-I in fasted rats, the body weights, kidney, spleen and adrenal gland weights were greater than those for untreated fasted rats (control). The body weights and the organ weights in hGH treated rats did not differ from those in control rats. Serum IGF-I levels in control, hGH treated and IGF-I treated rats were 64.0 +/- 6.1, 107.5 +/- 6.9 and 129.8 +/- 6.3 ng/ml, respectively, which were significantly different from each other. Blood urea nitrogen (BUN) levels were 13.9 +/- 1.1 ng/ml in IGF-I treated rats, which were significantly lower than those of control rats. Human GH treatment did not change BUN but affected nonesterified fatty acid (NEFA) and triglyceride. In IGF-I treated rats three-day urinary excretion of nitrogen and creatine were 163.5 +/- 14.6 mg and 9.53 +/- 1.53 mg, which were significantly less than those in control rats. These data indicate that IGF-I infusion inhibits body weight loss and catabolism in fasted rats and might be a useful therapy in catabolic conditions.

Measurement of free form of insulin-like growth factor I in human plasma.

A method to measure free form of insulin-like growth factor I (IGF-I) in human plasma using octadecylsilyl silica (Sep-Pak C18) cartridge has been developed. IGF-I was adsorbed by Sep-Pak C18 cartridge and eluted with 75% ethanol--0.01 M HCl. Labeled and non-labeled IGF-I were recovered in yields 92.5 +/- 2.1% (Mean +/- SEM) and 94.4 +/- 6.3% after adsorption to and elution from the Sep-Pak, respectively. When EDTA plasma was applied to the Sep-Pak, less than 5% of total IGF-I was recovered in the eluate. However, when acid-ethanol extracted plasma was applied to the Sep-Pak, IGF-I was recovered in yields greater than 75% of total IGF-I. When the Sep-Pak eluate was gel filtered, 88.4 +/- 4.0% of immunoreactive IGF-I eluted in the same fraction as synthetic IGF-I did, but the fraction passed through the Sep-Pak was observed as a high molecular weight form (bound form) of IGF-I. These data indicate that this Sep-Pak method does not extract all of the IGF-I in plasma, but extracts mainly the free form IGF-I. Using this method, IGF-I values of free form (fIGF-I) in EDTA plasma were measured. The fIGF-I values in normal adults, patients with acromegaly, and patients with growth hormone (GH)-deficiency were 2.4 +/- 0.1, 13.8 +/- 1.6, and 1.1 +/- 0.1 ng/ml, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Single sc administration of insulin-like growth factor I (IGF-I) in normal men.

Total and free form of IGF-I in plasma increased in a dose dependent manner after sc IGF-I administration. Peak values of total IGF-I were obtained at 3-4 h after the administration, and then the values decreased gradually. However, peak values of free form of IGF-I were obtained at 2 h, and then rapidly decreased thereafter. The blood glucose, serum insulin and C-peptide levels decreased until 4 h after IGF-I administration in a dose dependent manner. Plasma IGF-II values significantly decreased at 4-12 h after IGF-I administration. Urinary urea nitrogen and sodium excretion decreased after IGF-I administration. Urinary GH excretion also decreased after 0.06 mg/kg IGF-I administration. These data demonstrate that IGF-I may play a role in glucose, protein and electrolyte metabolism, and plasma IGF-II levels and GH secretion might be regulated by IGF-I in man.

Radioimmunoassay for insulin-like growth factor II (IGF-II).

Insulin-like growth factor II (IGF-II) levels in human plasma were measured in physiological and pathological conditions by radioimmunoassay (RIA) with biosynthetic IGF-II. This RIA was specific for IGF-II and cross-reactivity with IGF-I was 1%. The sensitivity was 15 pg/tube with 50% displacement at 50 pg/tube. The intra- and inter-assay coefficients of variation for IGF-II were 6.3 and 9.3%, respectively. The plasma IGF-II levels in normal adults, patients with hypopituitarism and patients with active acromegaly were 589.6 +/- 15.8, 800.9 +/- 45.6 and 330.3 +/- 24.3 ng/ml, respectively. After human growth hormone (hGH) treatment in hypopituitarism, IGF-II slightly increased, but not significantly. After adenomectomy in patients with acromegaly, IGF-II significantly decreased. These data indicate that IGF-II concentrations in plasma were partially GH dependent. This GH dependency was less than that of IGF-I. IGF-II was low in patients with anorexia nervosa and with liver cirrhosis and high in patients with renal failure. In two cases with extrapancreatic tumor-associated hypoglycemia, plasma IGF-II was increased to 1123.8 and 843.5 ng/ml, and returned to normal after tumor resection. These data showed that IGF-II was partly dependent on GH and nutritional conditions and that IGF-II was the most likely cause of some cases of hypoglycemia with extrapancreatic tumor. This specific and sensitive RIA of IGF-II would be useful in evaluating its physiological and pathological role in plasma and tissue.

Effects of sc administration of recombinant human insulin-like growth factor I (IGF-I) on normal human subjects.

Recombinant human insulin-like growth factor I (IGF-I) was administered subcutaneously to each of 5 normal human subjects at doses of 0 mg/kg (control), 0.06 mg/kg, or 0.12 mg/kg successively at one week intervals. After 0.06 mg/kg or 0.12 mg/kg IGF-I injections, plasma IGF-I levels increased from 185 +/- 17 ng/ml (mean +/- SEM) to maximal levels of 396 +/- 21 ng/ml at 3 hours and from 169 +/- 14 ng/ml to 480 +/- 27 ng/ml at 4 hours, respectively. These two peak values were statistically different (p less than 0.05). After 0.06 mg/kg and 0.12 mg/kg IGF-I administration, blood glucose levels decreased from 85 +/- 2 mg/dl to minimal levels of 73 +/- 3 mg/dl at 3 hours and from 83 +/- 1 mg/dl to 50 +/- 4 mg/dl at 2 hours, respectively. These two minimal values were statistically different (p less than 0.001). Serum insulin and C-peptide levels were decreased in a dose dependent manner after IGF-I administration. There were no changes between blood urea nitrogen levels before and 4 hours after IGF-I administration. The urinary GH concentration decreased after 0.06 mg/kg IGF-I administration, but increased and maintained normal values after 0.12 mg/kg IGF-I administration.

Effects of short-term growth hormone therapy in short children without growth hormone deficiency.

Evaluation of 24-hour endogenous growth hormone (GH) secretion was carried out in 62 children, aged 7-16 years, who did not have classic GH deficiency (GHD). The mean 24-hour GH concentration, determined at 20-minute intervals over 24 hours, was variable, ranging from 1.28 to 11.39 micrograms/l with a mean of 4.95 +/- 2.55 micrograms/l (+/- SD). There was a positive correlation between mean 24-hour GH concentration and plasma insulin-like growth factor I (IGF-I) values (r = 0.54; p less than 0.01). Recombinant human GH, 0.1 IU/kg/day was administered to 30 of the 62 children for 6 months followed by 6 months' observation without treatment. Thereafter, GH was administered at the same dose for a further 6 months to 16 children. The mean height velocities before, during, and after the first treatment period were 4.3 +/- 0.9, 7.3 +/- 1.9 and 4.9 +/- 2.0 cm/year (mean +/- SD), respectively. The height velocity during treatment was greater than pre- and post-treatment values (p less than 0.001). The height velocity increased again during the second treatment period to a mean of 8.5 +/- 2.0 cm/year (p less than 0.001). Nine other children were treated continuously in a similar manner for 1 year and their height velocity increased significantly from 4.1 +/- 1.4 to 6.0 +/- 1.9 cm/year (p less than 0.001). According to our criteria, 29 of the 39 children (74.4%) who were treated for 6-12 months showed a GH-dependent height increase during therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of nocturnal urinary growth hormone values.

Nocturnal urinary growth hormone values were measured by a sensitive enzyme immunoassay in normal adults, patients with GH deficiency, patients with Turner's syndrome, normal but short children who had normal plasma GH responses to provocative tests, and patients with acromegaly. The mean nocturnal urinary GH values in patients with acromegaly were significantly greater than those in normal adults (1582.3 +/- 579.8 vs 53.5 +/- 8.6 pmol/mmol creatinine (+/- SEM); p less than 0.05). In the normal but short children and patients with Turner's syndrome, the mean nocturnal urinary GH values were 83.1 +/- 5.2 and 79.8 +/- 29.5 pmol/mmol creatinine, respectively. In patients with GH deficiency, the nocturnal urinary GH values were undetectable (less than 5.3 pmol/mmol creatinine) except in one patient where the value was 6.3 pmol/mmol creatinine. The nocturnal urinary GH values of the patients with GH deficiency were significantly lower than those of the other groups (p less than 0.05). In normal but short children, the nocturnal urinary GH values correlated significantly with mean plasma nocturnal GH concentrations (r = 0.76, p less than 0.001), and 24-hour urinary GH values (r = 0.84, p less than 0.001), respectively. In 4 patients with GH deficiency who had circulating anti-hGH antibody, the urinary GH values were also undetectable. These data indicate that nocturnal urinary GH value reflects endogenous GH secretion during collection time, and that measurement of the nocturnal urinary GH values is a useful method for screening of patients with GH deficiency and acromegaly.

Human growth hormone stimulates liver regeneration in rats.

To study the effect of human growth hormone (hGH) on liver regeneration in rats, 200 micrograms hGH was administered to partial hepatectomized rats twice a day for three days. The bw of hGH-treated rats was higher than that in untreated rats. After three day administration, the liver weight was 3.18 +/- 0.13 g, significant higher than that of untreated rats (2.68 +/- 0.17 g). Human GH also stimulated the mitosis in the liver. Serum insulin-like growth factor I (IGF-I) and albumin levels were significantly increased and urea nitrogen levels were significantly decreased in hGH-treated rats compared with those in untreated rats. When 120 micrograms/day IGF-I was continuously administered to partial hepatectomized rats for three days, the bw and the liver weight were not higher than those of controls. These data indicate that hGH directly stimulates liver regeneration and recover liver dysfunction in rats.

Treatment of acromegaly with long acting somatostatin analogue SMS 201-995.

Ten acromegalic patients were treated with the somatostatin analogue SMS 201-995 (SMS) for 3-38 weeks in various doses and by different administration routines (thrice daily or multiple sc injection). Plasma GH daily profiles, plasma IGF-I, urinary GH, serum TSH, IRI and fasting blood glucose (FBG) concentrations were measured before and during SMS treatment. Plasma GH rapidly decreased within one hour in all patients and was suppressed for at least 4 h after a 50 micrograms sc injection of SMS in 8 patients. Multiple injections of 300-600 micrograms/day SMS (25-50 micrograms X 12) suppressed GH throughout the day. Plasma IGF-I was completely normalized in 4 patients, and, in all but one of the others, decreased markedly. Urinary GH decreased within the first week of treatment in all patients and normalization was obtained in 3 patients. Shrinkage of the pituitary tumor, as determined by CT or MRI, was observed in 7 of 9 patients. Other clinical improvements, such as diminution or complete disappearance of swelling of soft tissues, excessive perspiration, and headache, were observed in 7 of 8 patients. Changes in serum TSH, IRI and FBG were seen in 3-4 patients, but without any apparent clinical problems. In conclusion, SMS is a useful clinical tool for treatment of acromegaly, and a multiple sc injection method seems to be preferable.

Urinary growth hormone (GH) measurements are useful for evaluating endogenous GH secretion.

Daily (24-h) urinary GH excretion was measured using a highly sensitive sandwich enzyme immunoassay in 10 normal adults, 6 patients with hypopituitarism, 25 normal but short children who had normal plasma GH responses (peak plasma GH level, greater than 10 micrograms/L) to provocative tests, and 8 patients with acromegaly. The mean urinary GH values in the normal adults, patients with acromegaly, and patients with hypopituitarism were 13.8 +/- 4.0 (+/- SE) and 431.1 +/- 149.1 ng/g creatinine (Cr) (1.56 +/- 0.45 and 48.77 +/- 16.87 ng/mmol Cr) and undetectable, respectively; these mean values were significantly different from each other. In the normal but short children the urinary values ranged from undetectable to 55.8 ng/g Cr (6.31 ng/mmol Cr). All of the normal but short children and 4 patients with hypopituitarism participated in a 24-h endogenous GH secretion study. The urinary GH values correlated significantly with the mean 24-h plasma GH concentrations as an index of endogenous GH secretion (r = 0.81; P less than 0.001) and plasma somatomedin-C levels (r = 0.67; P less than 0.001), respectively. In 6 patients with acromegaly whose plasma GH levels were constant throughout a 4-h period, the urinary GH values also significantly correlated with the mean plasma GH levels (r = 0.95; P less than 0.01). These data indicate that urinary GH measurements reflect endogenous GH secretion and that measurement of urinary GH excretion is a useful, simple, and practical method for evaluating endogenous GH secretion.

Twenty-four hour plasma GH, FSH and LH profiles in patients with Turner's syndrome.

We studied the plasma GH profiles in 6 patients with Turner's syndrome and 6 normal girls of short stature by sampling every 20 min for 24 hours. We observed episodic secretion of GH in these subjects. The mean plasma 24 h GH level in patients with Turner's syndrome was 3.6 +/- 1.4 (SD) ng/ml which was significantly lower than that of normal short girls (7.1 +/- 2.2 ng/ml, p less than 0.01). The GH secretion during both nighttime and daytime was decreased in the patients with Turner's syndrome, however the number of pulses did not differ significantly. There were no correlations between the mean plasma 24 h GH level on one hand and peak GH level obtained after GH provocative test and plasma somatomedin C on the other. Plasma FSH and LH levels were also measured in 4 patients with Turner's syndrome. Both levels were elevated and there observed no clear pulsatile secretion of FSH, but, some pulsatile secretion of LH was observed in two patients. These data indicate that patients with Turner's syndrome have decreased endogenous GH secretion, even though they show normal GH responses to GH provocative tests.

Urinary IGF-I measurement and its clinical application.

Immunoreactive and receptor-reactive IGF-I was found to be present in human urine; 30% of the IGF-I immunoreactivity in urine was in its free form and the remainder was a high molecular weight form (approximately 40,000 MW). Urinary IGF-I was quantified by radioimmunoassay after extraction by Sep-Pak C18 cartridge, a method that measures only the free form of IGF-I. Daily (24-hour) urinary IGF-I excretion was measured in 3 hypopituitary children and 16 short normal children. The IGF-I level in the 24-hour urine samples correlated with the plasma IGF-I level and the mean 24-hour plasma GH concentration. The mean 24-hour plasma GH concentration, however, correlated better with the GH level in the 24-hour urine samples and the plasma IGF-I level than with the urinary IGF-I value. The mean IGF-I levels in single urine samples from normal subjects lay between those from patients with acromegaly (which were high) and those from patients with hypopituitarism (which were low). There were overlaps, however, in individual values between the normal and hypopituitary patients. These data indicate that urinary IGF-I values are altered by the GH secretion state, though the clinical application of urinary IGF-I measurement may be limited.

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation.

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.

Characterization of insulin-like growth factor I receptors on Madin-Darby canine kidney (MDCK) cell line.

Specific insulin-like growth factor I (IGF-I) receptors on the Madin-Darby canine kidney (MDCK) cell line were identified and characterized. [125I]IGF-I specifically bound to the cells, but [125I]insulin bindings to the cells was minimal. Unlabeled IGF-I displaced both the IGF-I and insulin bindings with potencies that were 100 and 10 times as great as insulin. By an affinity labeling technique, IGF type I receptors were present in the MDCK cells. IGF-I stimulated DNA synthesis and cell proliferation at physiological concentrations. On the other hand, insulin had a little effect on DNA synthesis. These data suggest that IGF type I receptors as demonstrated in MDCK cells are involved in DNA synthesis and cell proliferation.

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells).

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.